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Book of poster abstracts

1A

Probing the P450 3A4 allosteric site via bioconjugation of ligand analogues Julie Ducharme1, Vanja Polic1, Karine Auclair1 1 Department of Chemistry, McGill University, Montreal, Canada. Introduction. P450 3A4 is the most abundant human P450 and is well-known for its wide substrate promiscuity, making it the most important drug-metabolizing enzyme. This enzyme has the particularity of binding multiple ligands simultaneously, which is associated with heterotropic or homotropic, positive or negative, cooperativity (1). Solving the kinetics of such complex systems remains challenging, and so is identifying the binding pockets involved. Many substrates are also known to be allosteric activators of P450 3A4. For instance, progesterone (PRG) is an activator of P450 3A4-catalyzed 7-benzyloxy-4-trifluoromethylcoumarin (BFC) debenzylation (2). To our knowledge, the location of the allosteric site is still debated and is also likely to depend on the specific effector involved. Aims. The aims are to define the location of the P450 3A4 PRG allosteric site and investigate how sensitive the allosteric activation is to the binding orientation of the effector. Methods. To probe the location of the allosteric site, a progesterone analogue (PGM) was covalently attached, separately at several locations near a peripheral binding pocket (3). The impact of the PGM label was evaluated by monitoring the changes in enzyme kinetics before and after labeling in the presence and absence of PRG effector. Results. A total of six different PGM bioconjugates were successfully generated. The kinetics studies of those bioconjugates, indicate that two PGM-labeled mutants are efficiently mimicking PRG allosteric activation. Interestingly, PGM-bioconjugate which better mimicked the PRG molecule in one of the crystal structure of P450 3A4 gave rise to higher permanent activation than other bioconjugates. This suggests that the orientation of the PRG effector in the allosteric site matters but is not crucial for activation. Discussion. Our method allowed us to narrow down the location of the P450 3A4 allosteric site. PGM labeling at positions that were not mimicking allostery led to different, yet still interesting results. In one case, the PGM labeled showed an antagonistic behavior while in other cases, it activated the BFC metabolism without occupying the allosteric site, suggesting that different mechanisms of activation exist. Conclusion. This work creates further opportunities to study other systems showing heterotropic activation. Our results are of considerable interest not only in the fields of biocatalysis and enzymology, but also in the area of drug metabolism and for the prediction of drug interactions. 1. Guengerich, F. P. (1999) Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu. Rev.

Pharmacol. Toxicol., 39, 1-17. 2. Domanski, T. L., He, Y.-A., et al. (2001) Phenylalanine and tryptophan scanning mutagenesis of CYP3A4

substrate recognition site residues and effect on substrate oxidation and cooperativity. Biochem., 40, 10150-10160.

3. Williams, P. A., Cosme, J., et al. (2004). Crystal structure of human cytochrome P450 3A4 bound to metyrapone and progesterone. Science, 305, 683-686


2B

Tryptophan-75 is a potential gating residue of cytochrome P450 2D6 Laura Lowe Furge1, Kevin D. McCarty1 1Department of Chemistry, Kalamazoo College, Kalamazoo, USA. Introduction. The active site of CYP2D6 is buried, and both access to the active site and resulting metabolism are influenced by amino acid side chains, a phenomenon referred to as tunnel gating. This investigation hypothesizes that tunnel gating exists in CYP2D6, and that it is mediated by tryptophan-75 residue. Aims. This investigation aims to determine how tryptophan-75 might serve as a gating residue and impact the kinetics of CYP2D6, to gain a deeper understanding of tunnel gating in CYP2D6, and to understand the role of plasticity in enzyme mechanisms. Methods. To study the influence of tryptophan-75 on ligand metabolism rates, a tryptophan-75 to alanine mutant (CYP2D6*W75A) along with CYP2D6*1 were expressed and purified in E. coli host cells. All experimentation conducted with the 2D6*W75A mutant was replicated with CYP2D6*1 for comparison. Results. The interaction of CYP2D6*W75A and the *1 control with the substrates dextromethorphan and bufuralol was characterized in terms of spectral binding properties and Michaelis Menten kinetics. Discussion. Molecular Dynamics studies have shown that tryptophan-75 has the ability to swing out from the 2b channel to discharge a ligand. Also, visualization of 2D6 crystal structures by molecular imaging software showed the presence of several distinct conformations of the residue, some of which were observed to obstruct the opening of the 2b tunnel and thus inhibit access and egress of substrates from the active site. Based on the mobility of the residue in 3D space, we theorize that a gating mechanism of tryptophan-75 impacts ligand metabolism rates of CYP2D6. Conclusion. This investigation provides greater understanding of gating mechanisms in CYP2D6. Understanding of channel gating in CYPs has clinical importance as many questions remain as to its role in the process of drug metabolism. (Support: NIH 2R15GM086767-03).

Figure 1. Tryptophan-75 (in yellow, at

left) visualized in ball-and-stick form in

two conformations observed in the P450

crystal structure.


3C

The effect of ageing and tryptophan hydroxylase 2 (TPH2) deficit on the CYP2D activity in rat brain and liver Anna Haduch1, Natalia Alenina2, Agnieszka Nikiforuk3, Piotr Popik3, Michael Bader2, Władysława A Daniel1 1Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland; 2Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; 3Department of Behavioral Neuroscience and Drug Development, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland. Introduction. Liver cytochrome P450 2D contributes to the metabolism of drugs, carcinogens and neurotoxins, while brain CYP2D plays an important role in the local metabolism of drugs and endogenous neuroactive substrates. Studies in rodents indicate that brain CYP2D mediates 5-methoxytryptamine O-demethylation to serotonin. This alternative pathway can gain significance in deficit of the main pathway of serotonin synthesis. Recent studies indicate a decreased CYP2D activity in aging rats and an increased CYP2D6 expression in human brain (the frontal cortex, substantia nigra, cerebellum) in elderly. But the activity of brain CYP2D has not been investigated. Aims. The aim of the present study was to ascertain whether the level of CYP2D activity changes with increasing age and in conditions of serotonin deficiency in the rat brain. Methods. The experiment was carried out on male Dark Agouti and Wistar Han rats. Livers and selected brain structures (the frontal cortex, hippocampus, hypothalamus, thalamus, brain stem, cortex, striatum and cerebellum) were isolated. Kinetic parameters of 5-methoxytryptamine O-demethylation (HPLC) were estimated in liver and brain microsomes from both strains. The activity of CYP2D was studied in Dark Agouti wild type (wt) rats (mature 3-month-old and senescent 21-month-old rats) and in tryptophan hydroxylase 2 (TPH2) deficient senescent rats by measuring the rate of bufuralol 1’-hydroxylation in liver or brain microsomes (HPLC). Results. Liver microsomes of Dark Agouti rats catalyzed the O-demethylation of 5-methoxytryptamine to serotonin with lower efficiency than those of Wistar Han. However, brain microsomes of both strains showed a similar efficiency of catalyzing this reaction, though lower than did liver microsomes. The activity of CYP2D in liver microsomes was significantly lower in senescent Dark Agouti wt rats than in the mature animals and further decreased in senescent TPH2-deficient rats. The CYP2D activity in the frontal cortex decreased in senescent wt rats, but increased in senescent TPH2-deficient rats (compared to senescent wt). However, the CYP2D activity in the hippocampus, hypothalamus and striatum increased with ageing in Dark Agouti wt rats and was not changed in senescent TPH2-deficient rats (compared to senescent wt). Discussion. The obtained results indicate that ageing negatively affects the liver CYP2D activity which can lead to the inhibition of drug metabolism. The brain metabolism of CYP2D substrates may be changed in regio-dependent way: elevated in the hippocampus, hypothalamus and striatum and diminished in the frontal cortex. The increased CYP2D activity in the frontal cortex at TPH2 deficit suggest activation of the alternative pathway of serotonin synthesis via CYP2D. Conclusion. Ageing and tryptophan hydroxylase deficit affect liver and brain CYP2D, which may have an impact on the metabolism of endogenous substrates and drugs catalyzed by this enzyme. (This study was financially supported by the Grant ERA-NET Neuron II JTC 2015 Respond and statutory funds from the Institute of Pharmacology, PAS, Kraków, Poland.)


4A

Determination of the distal ligand coordination to resting state cytochrome P450 CYP199A4 and its correlation to activity Joshua S. Harbort1, Tom Coleman2, Matthew N. Podgorski2, Rebecca R. Chao2, Jeanette E. Stok3, John B. Brunning4, James J. De Voss3, Jeffrey R. Harmer1, Stephen G. Bell2 1Centre for Advanced Imaging, University of Queensland, St Lucia, Australia; 2Department of Chemistry, University of Adelaide, Adelaide, Australia; 3School of Chemistry and Molecular Bioscience, University of Queensland, St Lucia, Australia; 4School of Biological Sciences, University of Adelaide, Adelaide, Australia. The crystal structure of 4-vinylbenzoic acid-bound CYP199A4 displayed a heme-bound water despite an 80% spin state shift to the high spin form as measured by UV-Vis spectroscopy. The activity of CYP199A4-catalysed 4-vinylbenzoic acid epoxidation was significantly reduced compared with 4-methoxybenzoic acid (O-demethylation) and 4-ethylbenzoic (hydroxylation and desaturation). This was in line with the observation of the heme-bound water in the presence of 4-vinylbenzoic acid substrate and was contrary to the more reactive nature of the vinyl group. The inhibitors 4-pyridin-3-yl- and 4-pyridin-2-yl-benzoic acid induced different Type II substrate bound UV-vis spectra, however the former induced a greater red shift in the absolute spectrum (424 versus 422 nm) as well as a greater absorbance change in the peak to trough ratio of the difference spectrum along with other changes. The crystal structures of CYP199A4, with both pyridinyl compounds bound in the active site, were solved. These revealed that while the nitrogen of 4-pyridin-3-yl-benzoic acid bound directly to the heme iron that of 4-pyridin-2-yl-benzoic acid coordinates an iron-bound water ligand providing an explanation for the different UV-vis spectra that are observed. EPR data in frozen-solution however showed that a large proportion of CYP199A4 with both pyridinyl compounds bound in the active site still have a coordinated water molecules.


5B

Investigating the catalytic cycle and redox partner protein dependent dynamics of cytochrome P450 (CYP450) by neutron scattering Gabriela C Schröder1,2 and Flora Meilleur1,2 1Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA and 2Neutron Scattering Division, Oak Ridge National Laboratory (ORNL), Oak Ridge, TN 37831, USA. Introduction: Cytochrome P450s most commonly catalyze the highly regio- and stereoselective hydroxylation of sp3-hybridised carbons with the concomitant addition of molecular oxygen and two electrons derived from reduced cofactors.1,2 The cleavage of the dioxygen bond requires the leaving oxygen atom to be protonated and multiple pathways have been proposed.3,4 To achieve this hydroxylation, P450cam derives the catalytically essential electrons from the redox partner proteins, Putidaredoxin (Pdx) and Putidaredoxin reductase (Pdr). P450 undergoes a number of conformational changes to achieve efficient catalysis when interacting with these partner proteins.5 Aims: Neutron diffraction provides a unique opportunity to visualize the protons in the enzyme active site.6 To investigate the protonation pathway of P450cam from Pseudomonas putida, we aim to perform neutron diffraction of the camphor bound and free enzyme. We also aim to investigate the conformational dynamics of P450cam when in complex with its redox partner, Pdx, by quasi-elastic neutron scattering, by masking Pdx and measuring P450cam conformational changes. Methods: We will attempt to grow P450cam crystals sufficiently large for neutron diffraction. We are expressing and purifying P450cam and crystallizing this in large volume drops. To investigate the conformational dynamics of P450cam, a covalent complex will be formed with Pdx. The Pdx will be perdeuterated to mask its signal by contrast matching. Neutron diffraction and spectroscopy data will be collected on suitable samples on IMAGINE and BASIS at ORNL. Results: P450cam has been successfully expressed and purified in large quantities and crystallization has yielded crystals in the form of needles. The P450cam-Pdx complex has been formed and large-scale expression and perdeuteration are planned for neutron scattering analysis. Discussion: Crystallization trials of the expressed P450cam protein have yielded crystals, however they are in the form of needles which have insufficient volume for neutron diffraction due to the low neutron flux during data collection. Mutagenesis of P450cam and Pdx has enabled us to form the covalent complex, and it will now be necessary to optimise formation of this complex to ensure that the large amounts of protein necessary for neutron scattering can be generated. Conclusion: Neutron scattering provides a powerful technique to investigate the mechanism of enzymes at an atomic level as well as probe their conformational dynamics. We aim to use this unique technique to further understand the reaction mechanism of P450cam. 1 Ortiz de Montellano P.R. (2005) Cytochrome P450: Structure, Mechanism and Biochemistry, 3rd ed. (P.R. Ortiz de

Montellano, ed.) Springer US, Boston, MA. 2 Bernhardt R. (2006) Cytochromes P450 as versatile biocatalysts. J. Biotechnol. 124, 128–45. 3 Schlichting I. et al. (2000) The catalytic pathway of cytochrome p450cam at atomic resolution. Science 287, 1615–

22. 4 Nagano S. & Poulos T.L. (2005) Crystallographic study on the dioxygen complex of wild-type and mutant

cytochrome P450cam: Implications for the dioxygen activation mechanism. J. Biol. Chem. 280, 31659–31663. 5 Lee Y.T. et al. (2010) P450cam visits an open conformation in the absence of substrate. Biochemistry 49, 3412–

3419. 6 Schröder G.C. et al. (2018) IMAGINE: Neutrons reveal enzyme chemistry. Acta Crystallogr. Sect. D Struct. Biol. 74,

778–786.


6C

Molecular basis for caffeine oxidation by human cytochromes P450 1A1 and 1A2 Grazyna D. Szklarz1, Diaa Shakleya1,2 & Werner J. Geldenhuys1 1Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV, USA; 2Current address: US Food and Drug Administration, Center for Drug Evaluation and Research, Silver Spring, MD, USA. Introduction. Caffeine is metabolized in the body by several P450 enzymes. However, it has been routinely used as an in vivo marker substrate to detect human P450 1A2 (CYP1A2) activity. This P450 is closely related to another human enzyme, CYP1A1, with which it shares 72% amino acid sequence identity. Therefore, it would be of interest to compare the activities of these two enzymes towards caffeine as a substrate. Aims. The aim of these studies was to compare the specificity of caffeine oxidation by closely related P450s, CYP1A1 and CYP1A2. Kinetic parameters, Vmax and Km, and the stoichiometry of the reaction were evaluated and the results were explained using molecular modelling. Methods. Human CYP1A1 and CYP1A2 were expressed in E.coli and purified by Ni-NTA affinity chromatography. The enzymes were incubated with caffeine, and its oxidation products, paraxanthine, theophylline and theobromine, were separated by HPLC using UV detection at 274 nm. After quantifying products, kinetic parameters, Vmax and Km, were calculated. For stoichiometry studies, NADPH oxidation rates were measured at 340 nm, oxygen consumption was measured with a Strathkelvin oxygen meter, and hydrogen peroxide formation was determined using xylenol orange iron(III) colorimetric assay. Molecular modeling simulations were performed using Schrodinger software. Caffeine molecule was docked into the crystal structures of CYP1A1 and CYP1A2 using an induced fit method. Molecular dynamics was conducted with Amber program. Results. As expected, caffeine was oxidized much more efficiently by CYP1A2 than CYP1A1. Vmax (nmol/min/nmol) values for the total metabolites produced by CYP1A1 and CYP1A2 were 0.04 and 0.52, respectively, while the corresponding Km values were 3.73 and 2.24 mM. For both enzymes, the major product was paraxanthine, followed by theophylline and theobromine, but with different metabolite ratios: 72:14:14 for CYP1A1 and 94:4:2 for CYP1A2. Moreover, CYP1A2 showed 2% coupling to caffeine oxidation, with 0.3% coupling for CYP1A1. In both cases, hydrogen peroxide accounted for over 50% of product. The differences in specificity between the two enzymes were interpreted using molecular modeling, which indicated that caffeine binds preferentially in the CYP1A2 active site, where the substrate is stabilized with hydrogen bonds not present in the case of CYP1A1. Discussion. Conversion of caffeine to paraxanthine is the major pathway (80%) of its metabolism, with CYP1A2 mainly responsible for this reaction. Although CYP1A1 is also able to metabolize this substrate, it shows much lower activity. Our current study provides a detailed analysis of both kinetics and stoichiometry of caffeine oxidation by CYP1A1 and CYP1A2 enzymes. The experimental results were confirmed with docking and molecular dynamics simulations of caffeine in the enzyme crystal structures, providing a more complete picture of caffeine metabolism. Conclusion. In summary, our results provide firm evidence that caffeine is a much better substrate for CYP1A2 than for CYP1A1.


7A

Ordered chimerogenesis applied to CYP2B P450 enzymes Thomas Lautier1, Jacqueline Loeper2, Laetitia Jezequel3, Philippe Urban1, Denis Pompon1, and Gilles Truan1 1 LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France; 2 Université Pierre-et-Marie-Curie, 4 Place Jussieu, 75005 Paris, France; 3 Solvay, Product Compliance, 52 rue de la Haie-Coq, 93306 Aubervilliers, France. Introduction. How different P450 enzymes with similar 3D structures may exhibit different, although sometimes overlapping, broad substrates specificities? Structural studies on CYP2B enzymes identified some of the structural features that are related to their high plasticity. Aims. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking observed functional shifts to sequence element swaps between CYP2B6 and CYP2B11. Methods. A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast. Results & Discussion Most of the chimeras exhibit a high affinity for cyclophosphamide. A few exhibit an affinity similar to that of CYP2B6 without altered behaviour toward the other substrate assayed. The control of the affinity toward CPA in CYP2B enzymes was shown to depend mainly on the F’/G’ cassette and, to a lesser degree, on the A-helix located at the N-terminus part of the protein (1). None of the segment swapped that are localized at the core of the protein molecule resulted in a CPA affinity shift. Conclusion. Some short sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity. Expend the link between the F’/G’ cassette sequence with the metabolites families could help for isoforms choices in a metabolic engineering view. 1. Lautier T., Urban P., Loeper J., Jezequel L., Pompon D., Truan G. (2016) Ordered chimerogenesis applied to CYP2B P450 enzymes. Biochim Biophys Acta. (7):1395-403.


8B

Ligand-dependent dynamics of cytochrome P450 3A4 in nanodiscs Lorela Paço, Michelle Redhair, Nicholas A. Treuheit and William M. Atkins Department of Medicinal Chemistry, University of Washington, Seattle WA, United States. Introduction. Cytochrome P450 3A4 is the principal drug metabolizing enzyme in humans and often displays non-Michaelis-Menten ligand kinetics. Despite numerous studies and publication of over 40 crystal structures of complexes with different ligands, our understanding of the allosteric properties of CYP3A4 remains incomplete, making it difficult to predict drug interactions with novel substrates, effectors and inhibitors. Aims. Here we look at the effect of alpha-naphthoflavone (ANF), a synthetic flavone derivative and well-known allosteric effector, on CYP3A4 structural dynamics. Methods. Importantly, CYP3A4 was incorporated in MSP1D1 (membrane scaffold protein 1D1) nanodiscs to provide monomeric and monodisperse units of the membrane protein in a lipid bilayer environment. Using hydrogen deuterium exchange mass spectrometry (H/DX MS) we investigated the effect of 20 µM ANF on the solvent accessibility and dynamics of backbone amide protons upon incubation of 1 µM CYP3A4 nanodiscs in deuterated buffer for various times (7 sec to 100 min). Following pepsin digestion of the quenched samples, peptides were separated via LC-MS coupled with ion mobility mass spectrometry. H/DX was calculated by measuring the shift in the mass envelope of each peptide over time. Results and discussion. Analyzed peptides covered 85% of the protein sequence. Similar to previous work in our lab with the inhibitor ketoconazole (KTZ) and substrate midazolam (MDZ), H/DX analysis revealed modest but significant differences in the exchange profiles of the ligand free and ANF-bound CYP3A4 nanodiscs. Three regions are more protected from solvent deuterons in the ANF complex: the loop between the K helix and b1 sheet (residues 365-371), the end of the I helix and N-term half of the J helix (res 317-333), and the C-term of the L helix (res 457-463). The K-b1 loop is also protected in the H/DX analysis of CYP3A4 nanodiscs with KTZ and MDZ, presumably due to its proximity to the ligands in the active site. The peptide connecting the I helix to the J helix is uniquely protected in the CYP3A4-ANF complex, alluding to a modulatory ANF molecule binding at this site. Two regions showed increased H/DX upon ANF binding: the F helix and F-F’ loop (res 193-213), and the C helix (res 126-137), primarily in the longest time point in the study, marking a slow increase in solvent exposure and/or peptide dynamics over time. Interestingly, a similar trend was observed for the 193-213 peptide in the H/DX analysis of the CYP3A4 complex with MDZ, but not KTZ. This region contains residues 211-213, which are part of an allosteric pocket on top of the phenylalanine cluster that has been suggested to be a putative “effector” site in CYP3A43. Increased H/DX within the C helix is significant because it is involved in cross-linking of CYP3A4 to cyt-b5, supporting a previously proposed mechanism in which ANF promotes CYP3A4 metabolism by modulating its interaction with redox partners4. Similar increase in H/DX of the C helix is observed for the KTZ complex, but not the MDZ complex, the latter displaying a decrease in H/DX within the neighboring B-C loop instead. Finally, the G helix is protected in the presence of KTZ and MDZ, but showed no change in H/DX when CYP3A4 is bound to ANF. The F’-G’ motif located in the protein-membrane interface, could not be reliably analyzed. Conclusion. In conclusion, ANF induces modest differences in the conformational dynamics of CYP3A4 nanodiscs. Comparison of H/DX profiles of CYP3A4 with a typical inhibitor (KTZ) and substrate (MDZ) reveals both distinct differences unique to ANF, as well as shared effects on similar regions in the substrate and/or inhibitory complex. The results are consistent with ANF being an allosteric effector that exerts context-dependent effects on the metabolism of substrates and provide further evidence for the conformational plasticity of CYP3A4 and its ability to accommodate a wide range of ligands.


9C

Ongoing characterisation of the promising targets CYP121 and CYP141 from Mycobacterium tuberculosis H37Rv Irwin R Selvam1, Martyn Frederickson2, Richard B Tunnicliffe1, Harshwardan Poddar1, Colin Levy3 Kirsty J McLean1, Anthony G Coyne2, Chris Abell2, Andrew W Munro1 1School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom;2Department of Chemistry, University of Cambridge, Cambridge, United Kingdom;3Manchester Protein Structure Facility (MPSF), Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom. Introduction. Tuberculosis (TB) is the leading cause of death due to a single infectious agent worldwide and is endemic in numerous developing countries. The principal human pathogen is the rod-like, aerobic bacillus Mycobacterium tuberculosis (Mtb). The emergence and spread of drug- (and multidrug-) resistant strains of Mtb is threatening the progress made thus far in controlling the spread of TB. In addition to drug-resistance, there are a number of issues with current antitubercular regimens including drug toxicities, pharmacokinetic drug–drug interactions, particularly with antiretroviral drugs, and patient adherence given the lengthy treatment schedules. As such, there is an urgent need for novel antitubercular agents which are efficacious, well-tolerated and economical. The mycobacterial cytochrome P450s CYP121 (Rv2276) and CYP141 (Rv3121) were previously identified to be promising targets for pharmacological inhibition. Aim. This work outlines the structural and functional characterisation of a novel series of small molecule inhibitors against CYP121. In addition, ongoing work on elucidating the substrate, structure and physiological role of CYP141 is presented. Methods. Native CYP121 was expressed in Escherichia coli and crystallised in the presence of a range of inhibitors. UV-Vis binding titrations were conducted to determine the dissociation constants for select inhibitors. A His-tagged CYP141 construct was expressed in E. coli and successfully purified to a level suitable for crystallography. Preliminary UV-Vis binding titrations were conducted on a range of compounds. Results. Inhibitor-bound structures of CYP121 were successfully solved. Inhibitors were found to occupy a hydrophobic pocket 8-9 Å from the haem. UV-Vis binding titrations identified a number of low micromolar inhibitors. Crystallography trials for CYP141 are underway. Conclusion. The work presented herein contributes to the development of novel inhibitors for CYP121 and provides important insights into the properties of the CYP141 enzyme.


10A

Structural characterisation of CYP142A1 from Mycobacterium tuberculosis in complex with novel inhibitory compounds Matthew Snee1, Mona M Katariya2, Anthony G Coyne2, Richard B Tunnicliffe1, Colin W Levy3, Kirsty J McLean1, Chris Abell2, Andrew W Munro1 1Manchester Institute of Biotechnology, University of Manchester, Manchester, UK; 2Department of Chemistry, University of Cambridge, Cambridge, UK; 3Manchester Protein Structure Facility (MPSF), Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. Introduction. The human pathogen Mycobacterium tuberculosis (Mtb) is responsible for millions of deaths worldwide each year, and the continuing progression of antibiotic resistance makes the development of new antimicrobial compounds a pressing concern. As part of its pathogenic cycle, Mtb is able to metabolise host lipids which may provide a carbon source as well as a means for evasion of the immune response. The first step in degradation of lipids such as cholesterol is oxidation catalysed by cytochrome P450 enzymes. These enzymes have been shown to be vital for infectivity in Mtb and therefore represent an attractive target for inhibitory compounds. Aims. The aim of this project is to produce near-atomic resolution x-ray crystal structures of target cytochrome P450 enzymes (CYP142A1) in complex with candidate inhibitors provided by collaborators from the University of Cambridge2. This would allow for rational elaboration of the chemical structures in order to improve potency and specificity. Methods. CYP142A1 was purified to homogeneity using affinity and size exclusion chromatography. The enzymes were crystallised using the sitting drop method, and compounds were soaked into the crystals using liquid soaking techniques. Data were collected at the Diamond light source synchrotron facility and structures were solved by molecular replacement using the ligand-free structure as a search model. Results. Several cholesterol-oxidase co-crystal structures were solved to near-atomic resolution and will provide a good starting point for rational improvement of these compounds. The compounds exploit the shape of the active site effectively, but there is scope for growing the compounds to target a side pocket further away from the haem. Discussion. The co-crystal structures produced during this project will help to aid elaboration of the compounds in order to make them more potent and specific. This is important as the current first-line anti-tuberculosis compounds (e.g. econazole), whilst effective, are known to inhibit human cytochrome P450 enzymes, which can lead to serious side effects and drug-drug interactions. Conclusion. This work represents a progression of previous chemistry and should provide an insight that will be valuable in improving the properties of the compounds with a view to creating effective antimicrobials. Compounds that display specificity within Mtb cytochrome P450s can be employed to gain more information on the specific role of each enzyme in the infectious cycle.


11B

Investigation of different cytochrome P450 substrate binding modes using CYP199A4 Matthew N. Podgorski1, Tom Coleman1, Joshua S. Harbort3, John B. Bruning2, Jeffrey Harmer3 & Stephen G. Bell1 1Department of Chemistry, University of Adelaide, Adelaide, Australia; 2School of Biological Sciences, University of Adelaide, Adelaide, Australia; 3Centre for Advanced Imaging, University of Queensland, Brisbane, Australia. Introduction. CYP199A4 from the bacterium Rhodopseudomonas palustris HaA2 can tightly bind and rapidly oxidise para-substituted benzoic acids. However, it is unable to demethylate 3-methoxybenzoic acid, implying that the meta substituent is positioned too far from the heme iron to react.1 Whereas 4-methoxybenzoic acid induces a type I spectrum, 4-pyridin-2-yl- and 4-pyridin-3-yl-benzoic acid induce different type II spectra upon binding to CYP199A4 (Figure 1). Aims. We set out to explain CYP199A4’s substantially higher activity towards para- compared to meta-substituted benzoic acids and to rationalise the UV-Vis spectra induced by the different benzoic acid substrates. Methods. In vitro NADH assays were performed and docking, X-ray crystallography and EPR were used to elucidate ligand binding modes. Results. Crystal structures revealed that 4-pyridin-3-ylbenzoic acid directly coordinates to the heme iron but that 4-pyridin-2-ylbenzoic acid forms a hydrogen bond to an iron-bound water molecule (Figure 1). Low-temperature EPR measurements indicated that an iron-bound water was retained to some extent in both samples. Crystal structures of 3-methoxy-, 3-methylthio- and 3-methylamino-benzoic acid-bound CYP199A4 unexpectedly showed that the meta substituent is held near the heme iron. Despite this, the iron-bound water is not displaced. The bulkier substrate 3-ethoxybenzoic acid did displace the water and was metabolised, albeit with low activity.2 Discussion. The inability of meta-substituted benzoic acids to efficiently displace the water ligand explains the enzyme’s low activity towards these substrates. The different type II spectra observed appear to be explained by the different binding modes of the ligands in the crystal structures. Conclusion. This study highlights the complexity of substrate binding phenomenon in cytochrome P450 monooxygenase systems. .

Figure 1. UV-Vis difference spectra induced by binding of 4-pyridin-2-yl- (grey) and 4-pyridin-3-yl-benzoic acid (black) to CYP199A4 and crystal structures of the enzyme-ligand complexes. 1. Coleman et al. (2015). CYP199A4 catalyses the efficient demethylation and demethenylation of para-substituted benzoic acid derivatives. RSC Adv., 5, 52007-52018. 2. Coleman et al. (2018). Cytochrome P450 CYP199A4 from Rhodopseudomonas palustris catalyzes heteroatom dealkylations, sulfoxidation, and amide and cyclic hemiacetal formation. ACS Catal., 8, 5915−5927.

300 400 500 600

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0.0

0.1

4-pyridin-2-yl-

benzoic acid

Ab

so

rba

nce

Wavelength (nm)

4-pyridin-3-ylbenzoic acid


12C

Regulation of cytochrome P450 oxidoreductase by small-molecules: Single molecule insights linking conformational sampling to function Simon B Jensen1,2, Matias E Moses1,2, Sara Thodberg3, Cecilie C Hansen3, Darui Li1,2, Amit V Pandey4, Birger L Møller3, Thomas Laursen3, Nikos S Hatzakis1,2 1Department of Chemistry & Nanoscience Centre, University of Copenhagen, Denmark; 2Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark; 3Department of Plant & Environmental Sciences, University of Copenhagen, Denmark; 4Pediatric Endocrinology, Diabetology and Metabolism, University Children’s Hospital, Inselspital, Bern, Switzerland. Cytochrome P450 oxidoreductase (POR) is a crucial redox partner to a wide spectrum of cytochrome P450 enzymes (CYPs). POR is a highly dynamic enzyme constantly fluctuating between “extended” and “compact” conformations. This conformational sampling is a consequence of the underlying energy landscape governing the dynamic exploration of space and has previously been linked to the function of POR (1–3). We, and others, recently showed that the interplay between POR and CYPs is substrate dependent and the formation of dynamic POR-CYP metabolons rely on factors such as lipid composition, ionic strength and substrate concentration (4, 5). Here, we show for the first time that small-molecule ligands may modulate the selectivity of POR in donating electrons and selectively activating a subset of downstream CYP partners. The effect, which is observed in both liposomal assays and cell studies, is dependent on the electron acceptor, indicating that ligands may affect the selectivity of POR towards certain CYPs. We speculate that the biased selectivity of POR towards downstream CYPs originates from an altered energy landscape and consequently altered conformational sampling. To prove this, we have established a single molecule FRET assay allowing us to directly observe and quantify POR conformational sampling at the single molecule level (1). The assay enables us to identify the existence of multiple conformational states and quantify their abundance and how their sampling is biased by ligands. This will ultimately aid our understanding of the links between conformational sampling and enzyme function, and potentially pave the way for novel pharmaceutics and biotechnological applications. 1. Bavishi, K. et al. (2018). Direct observation of multiple conformational states in Cytochrome P450

oxidoreductase and their modulation by membrane environment and ionic strength. Scientific Reports, 8:6817, 1-9.

2. Freeman, A. et al. (2017). Orchestrated Domain Movement in Catalysis by Cytochrome P450 Reductase. Scientific Reports, 7:9741, 1-11.

3. Laursen, T. et al. (2014). Single molecule activity measurements of cytochrome P450 oxidoreductase reveal the existence of two discrete functional states. ACS Chem. Biol., 9, 630–634.

4. Gentry, K. A. et al. (2018). Substrate mediated redox partner selectivity of cytochrome P450. Chem. Commun., 54, 5780–5783.

5. Laursen, T. et al. (2016). Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum. Science, 354, 890-893.


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Comparison of computational methods for site(s) of metabolism (SOM) prediction of protein kinase inhibitors metabolised by CYP3A4 Pramod C. Nair1,2, Ross A. McKinnon2 and John O. Miners1,2 Department of Clinical Pharmacology1 and 2Flinders Centre for Innovation in Cancer, College of Medicine and Public Health, Flinders University, Adelaide, SA, Australia. Introduction. Small molecule protein kinase inhibitors (KIs) are an effective targeted therapy for multiple types of cancers [1]. KIs are mainly biotransformed through oxidation reactions catalysed by CYP3A4. SOM prediction is a useful tool for identifying metabolically labile sites of KIs (and other drugs) in the drug discovery pipeline, and complements experimental data. Aims. This study sought to predict the SOM of KIs using a range of computational methods and to identify amino acids important for KI binding within the CYP3A4 active site. Methods. SOMs were collated for a dataset of 31 marketed KIs metabolised by human CYP3A4. A range of computational approaches were evaluated for SOM prediction: molecular docking (using three CYP3A4 X-ray crystal structure templates); molecular superpositioning (using 4 ligand templates); and Web-based methods (using three algorithms). Molecular docking additionally identified amino acids involved in KI binding within the CYP3A4 active site. Results. Since CYP3A4 is known to exhibit plasticity in the catalytic site [2], three X-ray crystal structures were investigated as templates for molecular docking. Docking in the bromoergocryptine-bound structure (3UA1) provided superior SOM prediction (77%) compared to the unliganded and ritonavir-bound structures (74% and 68%, respectively). Of the various scoring functions investigated, the PMF-score showed more consistent SOM prediction. The web-based SOM prediction algorithms provided marginally better predictivity (77%-87%), whereas the substrate superpositioning (molecular overlay) approach using 4 different compounds as templates was less effective (42%-71% prediction accuracy). Docking of the KIs in the CYP3A4 active sites identified Glu37 Phe57, Asp76, Arg105, Arg106, Ser119, Arg212, Phe215, Thr224, Arg372, and Glu374 as important residues for substrate binding. Discussion. The study demonstrated that SOM prediction of KIs was dependent on the CYP3A4 X-ray crystal structure employed as the template, consistent with the known plasticity of this protein. Web-based SOM algorithms provided the best predictivity. Hydrophobic, hydrogen-bonding, and charge interactions contribute to KI binding in the CYP3A4 active site. The approaches adopted here are likely to be applicable to SOM prediction of other CYP3A4 substrates. Conclusion. The data indicate that computational approaches may be used to predict the SOM of KIs, but predictivity is dependent on the approach adopted. Similar considerations would be expected to be important for other classes of CYP3A4 substrates. 1. Rowland A, van Dyk M, Mangoni AA, Miners JO, McKinnon RA, Wiese MD, Rowland A, Kichenadasse G, Gurney H, Sorich MJ. (2017). Kinase inhibitor pharmacokinetics: comprehensive summary and roadmap for addressing inter-individual variability in exposure. Expert Opin Drug Metab Toxicol., 13, 31-49. 2. Nair PC, McKinnon RA, Miners JO. (2016). Cytochrome P450 structure-function: insights from molecular dynamics simulations. Drug Metab Rev., 48, 434-52.


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Pathways and energetics of ligand unbinding in CYP2D6 Kyle A Furge1, Michael C Orwin1 & Laura L. Furge1 1Department of Chemistry, Kalamazoo College, Kalamazoo, USA. Introduction. CYP2D6 metabolizes ~15% of pharmaceutical compounds. Combined analysis of the CYP2D6 crystal structure and molecular dynamics simulations indicate that molecules enter and exit the active site by using a small number of solvent accessible tunnels and channels. We are interested in two channels, channel 2c and channel 2b, as these channels may be partially blocked by amino acid residues that narrow the channel and potentially act as "bottleneck" or "gating" residues which inhibit movement to and from the active site. Aims. The aim of this study is to identify if there is a preferred channel or path for CYP2D6-ligand binding/unbinding. In parallel, we intend to quantify the rate limiting steps as the ligands move along each channel from the active site to the protein surface. Methods. Two CYP2D6 ligands, bufuralol and rolapitant, were placed near the heme and molecular dynamics augmented with adaptive biasing potentials were used to generate unbinding trajectories. The energy landscape of the unbinding trajectories were aligned with structural features to identify potential rate limiting steps. Results. The large ligand, rolapitant only exited via the channel 2c. Preliminary analysis indicates that multiple steric clashes result in rate limiting steps that affect ligand unbinding. In contrast, the smaller ligand bufuralol, exited via multiple channels. Analysis of structural features indicate that breaking of hydrogen bond between the protein and ligand represent rate limiting steps in bufuralol unbinding. Discussion. Identification and quantification of the kinetics of ligand entrance and exit will enhance our understanding of how amino acid variation along these channels could affect protein function. Conclusion. We anticipate that use of adaptive biasing potentials enhanced molecular dynamics will be a productive method to probe binding/unbinding events for CYP2D6. (Support: NIH 2R15GM086767-03)


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Light-driven biocatalytic C-hydroxylation by CYP102A1 through direct transfer of photoinduced electrons Thien-Kim Le, Chul-Ho Yun Department of Biological Sciences and Biotechnology, School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea. Introduction. High regioselective and stereospecific oxygenation activities of the P450s toward a variety of substrates are of considerable importance in applications to drug development and the synthesis of fine chemicals. To overcome the dependence of P450 catalysis on cofactor NAD(P)H and the reductase, alternative approaches have been attempted. Despite the immense potential of P450s, the dependence on nicotinamide cofactor (NADPH) and NADPH-P450 reductase (CPR) limits their employment in the chemical industry.

Aims. Here, we present a visible light-driven platform for biocatalytic C-hydroxylation reactions using natural flavin molecules, especially flavin mononucleotide, as a photosensitizer.

Methods. We have devised and experimentally implemented a catalytic scheme for flavin-sensitized P450 catalytic reactions free of NADPH and a reductase domain. Using visible light as a source of energy, heme domains of bacterial CYP102A1 variants were employed for photobiocatalytic C-hydroxylation reactions of substrates (e.g. 4-nitrophenol and lauric acid) in the absence of NADPH.

Results. Employing visible light as a source of energy instead of nicotinamide cofactor, the bacterial CYP102A1 heme domain was successfully applied for photobiocatalytic C-hydroxylation of 4-nitrophenol and lauric acid—in the absence of NADPH and CPR.

Discussion. We present a proof of concept that the photoactivation of flavins is productively coupled with the direct transfer of photoinduced electrons to the P450 heme iron, achieving photobiocatalytic C-hydroxylation reactions. The light-driven, flavin-sensitized P450 catalysis could be performed in cost-effective and eco-friendly ways to create a promising discipline of green and sustainable chemistry with high potential for P450 applications.

Conclusion. The current study demonstrates that the need for reductase (i.e., CPR) and cofactor NADPH can be avoided using visible light as a source of energy and EDTA as an electron donor. We have shown that natural flavins mediate light-driven catalysis by P450s. The photoactivation of flavins was productively coupled with the direct transfer of photoinduced electrons to P450 heme iron to boost P450 monooxygenase, achieving photobiocatalytic C-hydroxylation reactions.

1. Le, T. K., Park, J. H., Choi, D. S., Lee, G. Y., Choi, W. S., Jeong, K. J., Park, C. B., Yun, C. H. (2019). Solar-driven biocatalytic C-hydroxylation through direct transfer of photoinduced electrons. Green Chem. 21, 515-525.


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Electron carrier engineering towards increased partitioning of photosynthetic reducing power to cytochrome P450 catalysis Silas B Mellor1, Marcos H Vinde1,2, Agnieszka Z Nielsen1, Guy T Hanke3, Kaltum Abdiaziz3, Maxie M Roessler3, Meike Burow1, Mohammed Saddik Motawia1, Birger Lindberg Møller1, Poul Erik Jensen1 1Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark, 2Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Australia (present address), 3School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom. Introduction. Photosynthetic organisms are promising chassis for metabolic engineering. Photosynthesis imparts high resource use efficiencies, and organisms like plants can be cultivated cheaply at the industrial scale. Heterologous P450s can be targeted to thylakoid membranes in plant chloroplasts and cyanobacteria, where the photosynthetic apparatus functionally substitutes native reductases via the endogenous electron carrier ferredoxin. But because ferredoxin acts as a distribution hub for photosynthetic reducing power electrons from this source are heavily contested, which limits photosynthesis-driven P450 output. Aims. This study aims to improve partitioning of photosynthetic reducing power towards P450-based catalysis. Methods. We compare the ability of four electron carriers to increase photosynthesis-driven P450 activity by characterizing their electron transfer kinetics with the model P450, CYP79A1. The electron carriers are also tested in vivo by fusing them to chloroplast-targeted CYP79A1 expressed transiently in tobacco. Results. The electron carriers show only modest differences in electron transfer rates towards CYP79A1 in vitro. Only one carrier, an engineered flavodoxin-like protein, supplies significant reducing power when an enzyme that competes for reduced ferredoxin is present, because its redox potential renders delivery of electrons to endogenous enzymes inefficient. In vivo investigation via transient expression of CYP79A1-carrier fusions in tobacco show improved sequestration of photosynthetic reducing power for all but one carrier. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison at nearly 25-fold on a per protein basis. Discussion. This study demonstrates that novel electron carriers can be used to construct electron transfer chains, which alleviate the problem of endogenous competition. Key factors such as redox potential and affinity for photosystem I governs the effectiveness of electron carriers for this purpose. This sets down concrete design goals for future electron carrier engineering. The fusion strategy reported here boosts photosynthesis-driven P450 activity and is applicable to metabolic engineering aimed at producing valuable natural products. Conclusion. Electron transfer to heterologous P450s from photosynthetic electron transport can be improved by using non-native electron carriers to construct bespoke electron transfer chains.


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Characterization of NADP+-specific glucose-6-phosphate dehydrogenase from Solanum lycopersicum and application to NADPH-generating system for P450-catalyzed reactions Chan Mi Park, Heon Jeong, Chul-Ho Yun Department of Biological Sciences and Biotechnology, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea. Introduction. Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) catalyzes oxidation of glucose-6-phosphate (G6P) to 6-phospho-δ-gluconolacton simultaneously with reduction of nicotinamide adenine dinucleotide phosphate (NADP+) to reduced form (NADPH). Cytochromes P450 can catalyze various oxidative metabolic reactions of endogenous and exogenous compounds. Their catalytic diversity and vast substrate range with regio‐ and stereo‐specificity have high potential in applications to drug metabolism and in the fine chemical synthesis. However, high-cost nicotinamide cofactor (NADPH) supply is essential for P450-catalyzed reactions. G6PDH have been used for NADPH-generating system (NGS) of P450-catalyzed reactions. In present, only two G6PDHs from Leuconostoc mesenteroides (LMG) and Saccharomyces cerevisiae (SCG) can be obtained commercially. G6PDH in addition to NADP+ are highly cost to generate NADPH. Aims. To overcome dependency on NADPH of P450 reaction and to supply G6PDH enzyme economically, here we purify recombinant G6PDH from S. lycopersicum (His-G6PDH) and optimize NGS with His-G6PDH for P450-catalyzed reactions. Methods. Cytosolic G6PDH gene was amplified by PCR using cDNA from S. lycopersicum as a template and cloned to expression vector pET-28a. His-G6PDH was expressed in Escherichia coli and purified using affinity chromatography. His-G6PDH activity was measured by monitoring production of NADPH. Ability of His-G6PDH as NGS was evaluated by CYP102A1-catalyzed reactions and was compared with commercial enzymes, LMG and SCG. The metabolites were analysed by HPLC. Results. Catalytic efficiency (Vmax/Km) of His-G6PDH for G6P and NADP+ was 2.26 min-1 μM-1 and 17.8 min-1 μM-1, respectively. His-G6PDH could not catalyze NAD+-linked reactions. His-G6PDH supported CYP102A1-catalyzed reaction to produce 5’-OH omeprazole by NADPH generation. Discussion. Although catalytic efficiency of His-G6PDH was lower than those of commercial enzymes, His-G6PDH could support NADPH to P450 reactions sufficiently. Since His-G6PDH can be easily purified using affinity chromatography, it can be provided as active cost-effective and enzyme source and it might replace commercial enzymes in the laboratory and related industry. Conclusion. We first reported application of plant NADP+-specific G6PDH to P450-catalyzed reaction. His-G6PDH could be a cost-effective alternative applies to NSG for P450-catalyzed bioconversions as well as other NADPH-needed reactions.

1. Kruger, N. J. & von Schaewen, A. (2003). The oxidative pentose phosphate pathway: structure and organisation. Curr. Opin. Plant Biol. 6(3), 236-246.


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Site-directed mutagenesis of P450BM3 to improve steroid hydroxylation Wenyu Chen1, Aaron Leung1, Luet Lok. Wong1 1Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, United Kingdom. Introduction. Androstenedione (AD) and dehydroepiandrosterone (DHEA) are the precursors of the androgen and estrogen gender hormones, respectively, in human steroidogenesis. AD is either hydroxylated at the C19 methyl followed by demethylation to aromatise the A-ring to form estrone (CYP19A1 aromatase) or converted to testosterone by reduction of the 17-ketone to the alcohol. Bacterial P450s have been reported to hydroxylate AD at the 1α, 15β and 16α positions.1, 2 The 7β-OH and 7-oxo- derivatives of DHEA have neuroprotective and anti-inflammatory properties. Other hydroxylated products could be precursors for pharmaceutical substances. Therefore, selective functionalisation of the steroid skeleton would be of great interest. Both rational design and directed evolution have been applied to engineer P450 enzymes for steroid oxidation.3, 4 Aims. The aim of the project is to engineer selectivity of dehydroepiandrosterone and androstenedione oxidation by mutagenesis, aided by docking studies. Methods. AD and DHEA were screened for oxidation with a library of CYP102A1 variants. Preparative-scale (10-100 mg) reactions enabled product purification by silica-gel chromatography and NMR characterisation. New variants were then generated to enhance activity and selectivity. Results. The initial variant library oxidised both AD and DHEA with high conversion and gave diverse products. AD gave 4 major products that were characterised as the 2β- (60%), 6β- (44%), 16β- (64%) alcohols and the hydroxylation-enolisation product 16-oxo-testosterone (35%). Further site-directed mutagenesis on the mutants shifted the hydroxylation to new sites at 7β (40%), 15β (40%) and 16α (31%) with high conversion. DHEA was mainly converted to 7β-hydroxy-DHEA (80%) along with several minor products including 4β-, 15β- (56%), and 16α-hydroxy-DHEA (50%) and 16-oxo-androstenediol (48%). Product selectivity was further optimised by protein engineering. Discussion. Both AD and DHEA were oxidised on the A-, B- and D- rings at unactivated C–H bonds generating multiple hydroxylated products, some with high selectivity. Based on F87A-containing variant, the I263, A264 residues on the I helix, the A328, P329, A330 residues on β1-sheet, are responsible for these significant selectivity shifts. Conclusion. Protein engineering offers the possibility of selective C–H bond functionalisation of steroids at activated and unactivated sites. 1. Bracco, P., Janssen, D.B., et al. (2013). Microbial Cell Factories, 12. 2. Khatri, Y., Ringle, M., et al. (2016). Chembiochem., 17, 90-101. 3. Nikolaus. J., Nguyen, K.T., et al. (2017). Steroids, 120, 41-48. 4. Acevedo-Rocha, C.G., Gamble, C.G., et al. (2018). ACS Catalysis, 8, 3395-3410.


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Ancestral reconstruction of the CYP2D subfamily, antecedents of human CYP2D6, an important drug metabolising enzyme Stephlina A D’Cunha1, Gabriel Foley1, M Boden1, Ann-Sofie Sandinge2, Ulrik Jurva2, Shalini Andersson3, Elizabeth M J Gillam1 1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia; 2DMPK, Early Cardiovascular, Renal and Metabolism, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden; 3Discovery Sciences, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden. Introduction: Cytochrome P450 enzymes present attractive candidates as biocatalysts in industrial processes owing to their ability to catalyse diverse stereo- and regio-selective reactions. However, most natural P450 enzymes are unstable and have poor efficiency, limiting their use. In humans, CYP2D6 is responsible for the oxidative clearance of ~13% of drugs and has potential applications in the biosynthesis of authentic metabolite standards and new lead compounds in drug discovery and development. However, it lacks the required thermostability to be employed as an industrial biocatalyst. Ancestral sequence reconstruction (ASR) has been used to generate proteins that are much more thermostable than their extant descendants, including cytochrome P450 enzymes. Aims: To resurrect the ancestors of the CYP2D subfamily, determine their functional and physical attributes and explore their ancestral functions and potential for biocatalytic applications. Methods: Six ancestors from the CYP2D subfamily were reconstructed using a maximum-likelihood, joint reconstruction approach. These were synthesized and expressed as recombinant proteins in E. coli. Whole cell preparations were used to determine the thermostability, while bacterial membranes were used for biochemical and catalytic studies. Results: In comparison to the corresponding extant forms, the ancestors demonstrated significantly higher thermostability with 10T50 values (the temperature at which half the protein remains folded after ten minutes) increased by between 5 and 25 C compared to the corresponding extant forms. The younger ancestors showed activity towards the extant CYP2D substrates; however limited to no activity was observed with the older forms when supported with the extant human reductase (hCPR). Preliminary studies suggest that activity towards certain substrates could be enhanced to some degree by coupling with a reconstructed ancestral CPR. Discussion: The parallel changes in thermostability and activity seen in these ancestors suggests that these enzymes may have acquired the ability to metabolise xenobiotic substrates at the cost of overall stability of the P450 structure. While the oldest ancestors showed generally poor activity towards drug-like substrates, when supported by the extant hCPR, it may be possible to use them as templates for further engineering. Conclusion: The increased stability of the ancestral CYP2D forms makes them robust scaffolds for further engineering as thermostable biocatalysts for industrial applications. The next phase of this investigation is to more fully elucidate the substrate range of these ancestral proteins towards both endo- and xenobiotic substrates to determine their historic function and thereby gain insights into their evolution. Any useful and novel activities exhibited by these enzymes can also be harnessed and further evolved for biocatalytic applications.


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Thermostability correlates with evolutionary age in ancestors of promiscuous xenobiotic-metabolizing enzymes Raine E. S. Thomson1, Yosephine Gumulya1, Gabriel Foley1, Julian Zaugg1, Mikael Boden1, Ulrik Jurva2, Shalini Andersson3, Elizabeth Gillam1

1School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia; 2DMPK, Early Cardiovascular, Renal and Metabolism, R&D BioPharmaceuticals, and 3Discovery Sciences, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden.

Introduction: Recently, the ancestors of CYP3 and CYP2D enzymes, that are predicted to have existed ~450 million years ago, have been resurrected. Both ancestors were found to be stable at temperatures ~30°C higher than their respective extant descendants, and the CYP3 ancestor was exhibited a similar substrate range and catalytic efficiency to the major human CYP3 form, CYP3A4. Since the vertebrates in which these ancestors would have been present are thought to have been mesophiles, we hypothesized that the thermostability of the ancestors has been carried over from more ancient P450s, that would have needed to have been able to tolerate much hotter ambient temperatures, and that in the absence of continued selection pressure, thermostability has been progressively lost in the subsequent evolution of these families.

Aims: The principal aim was to determine whether thermostability correlates with evolutionary age. Using the CYP2 family as a model, we sought to characterise the substrate spcificity of the ancestors to explore the evolutionary trajectory leading to the extant xenobiotic-metabolising forms. Methods: Twelve CYP2 family ancestors were resurrected from various points along the mammalian CYP2 lineage. The ancestral forms were expressed in E. coli, in parallel with their extant descendants, then assessed for thermostability by using the characteristic Soret peak in the Fe(II).CO vs Fe(II) difference spectrum as a measure of functional haemoprotein. Substrate promiscuity was assessed for each ancestor using a set of typical P450 probe substrates (testosterone, coumarin, alkoxyresorufins and luciferin derivatives). Ligand binding specificity was further defined by screening for inhibition of a marker activity using a set of 48 chemically diverse drug compounds. Results: All 12 resurrected ancestors could be expressed in E. coli, adopted the canonical P450 fold and were more stable than their direct descendants. A general trend of increasing thermostability was seen with increasing age, with the older ancestors able to withstand temperatures ~30°C higher than the extant forms. Furthermore, all ancestors showed typical monooxygenation activity towards one or more P450 probe substrates, and showed comparable promiscuity to the extant forms. Discussion: We hypothesise that rapid genetic diversification of the CYP2 family in response to animal-plant chemical warfare created the catalytically diverse and specialized forms we see today at the cost of introducing mutations that destabilized the proteins in the absence of continued purifying selection pressure to maintain thermostability. The comparable substrate promiscuity of the ancestral and extant forms suggests that the ancestral CYP2 forms studied here may have had a dominant role in xenobiotic metabolism rather than a specific physiological role in clearing a particular group of endobiotics. Our observations demonstrate that stability and function are not inherently incompatible and that proteins can be stabilised without sacrificing functional versatility. Conclusion: ASR has allowed us to explore the evolutionary trajectory of the CYP2 family and to identify robust P450s for use as biocatalysts or as templates for further engineering.


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A toolbox for selective -caryophyllene oxidation by engineered CYP102A1 Matthew Fisher1, Yang Cao1,2, George Woodward1 & Luet Lok Wong1,2 1 Department of Chemistry, University of Oxford, Oxford, United Kingdom; 2 Oxford-Suzhou Centre for Advanced Research, Suzhou, Jiangsu, China. Introduction. Oxyfunctionalised derivatives of plant-derived sesquiterpenes are of interest for their biological

properties, e.g. santalol and patchouli alcohol. One such sesquiterpene, -caryophyllene, is abundant in clove and basil essential oil and has analgesic properties as a selective CB2 receptor agonist. Its endocyclic epoxide

displays anti-tumour activity by reducing NF-B activation and the 14-alcohol has been observed in the flavour active zone of highly hopped beer via GC-O.1,2 However, little information is available for other derivatives since previous chemical and enzymatic oxidation studies have yielded limited numbers of products.3,4

Aims. Devise new routes to oxygenated -caryophyllene products and study their biological properties.

Methods. -Caryophyllene oxidation by CYP102A1 variants was performed in 24-well plates and the organic extracts were analyzed by GC. Products were then isolated from preparative scale reactions (100 – 200 mg) and characterised. Residues and mutations of interest were identified, and selectivity of oxidation explored via a hybrid molecular dynamics/docking approach to design new variants. Results. Negligible turnover was observed with the WT, whereas engineered variants displayed TON >5,000 at 200 mg scale. 15 oxidation products were observed and 8 major products comprising mono- and di-epoxides, diepoxide-alcohols and two allylic alcohols were characterised. All products had notably different aromas to the substrate. Engineered libraries were constructed on several active site residues, allowing increases in selectivity to 46% & 49% for diepoxide isomers. Oxidation at a non-activated C–H bond (diepoxide-2-ol, 24%) was also observed. Discussion. Epoxidation was the dominant mode of oxidation, with only two allylic alcohols being observed. Allylic oxidation was generally promoted by mutations at the F81, I263, and A328 positions whereas epoxidation was promoted by mutations at V78, A82 and F87. Conclusion. The results show the potential of engineered CYP102A1 variants as selective oxidation catalysts

for -caryophyllene. 1 K. Fidyt et al. (2016). β- caryophyllene and β- caryophyllene oxide—natural compounds of anticancer and

analgesic properties. Cancer Med., 5, 3007–3017. 2 T. Praet et al. (2016). Heat-Induced Changes in the Composition of Varietal Hop Essential Oils via Wort

Boiling on a Laboratory Scale. J. Am. Soc. Brew. Chem., 6, 265. 3 A. Schifrin et al. (2015). New Sesquiterpene Oxidations with CYP260A1 and CYP264B1 from Sorangium

cellulosum Soce56. ChemBioChem, 16, 2624–2632. 4 B. Steenackers et al. (2015). Chemical transformations of characteristic hop secondary metabolites in

relation to beer properties and the brewing process. Food Chem., 172, 742–756.


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Wood aroma synthesis by P450-catalysed sesquiterpene oxidation Xiao Juie Wong1, Yang Cao,1,2 and Luet Lok Wong1,2 1 Department of Chemistry, University of Oxford, Oxford, United Kingdom; 2 Oxford-Suzhou Centre for Advanced Research, Suzhou, Jiangsu, China. Introduction. Oxygenated sesquiterpenes often possess pleasant aromas and flavours that contribute to the desirability of wood essential oils such as sandalwood oil and cedarwood oil. Sandalwood oil has been used for millennia as an incense for worship and as a valuable fragrance.1 The derivatives of α-cedrene, the main component in cedarwood oil, has attracted interest because of their application as aromas and food preservatives. Excessive logging has endangered some species (e.g. sandalwood) and thus, the discovery of alternative ways to generate the aromatic components for the woody note has become increasingly important. Aims. The aim of this project is to explore the oxidation of sesquiterpenes from wood essential oils for the synthesis of compounds with woody notes. Methods. The commercially available α-cedrene was screened for oxidation with 24-well plates of CYP102A1 variants and products analysed by GC. Preparative scale reactions were carried out with mutants which showed high conversion and product selectivity for product isolation and characterisation. Small panels of variants with mutations at 2 or 3 residues were prepared in order to increase activity. Results. Numerous products were observed over a long GC retention time window across the variant library, indicating multiple oxidations. Six major products were purified from preparative scale reactions and identified. Oxidation was observed at the C8,C9 double bond, the allylic C–H bonds of C10 and the C15 methyl group, together with further oxidation products arising from these. A small library of variants with combinations of amino acid substitutions at F87, A328 and I263 was constructed and led to variants with high selectivity (>80%) for the oxide and the C10 and C15 alcohols, as well as products arising from oxidation at non-activated C–H bonds in the C5 ring. Further oxidation to the C15 aldehyde and carboxylic acid were also observed. Discussion. The tricyclic structure of α-cedrene presented challenges to selectivity engineering. More combinations of mutations at different active site residues are necessary for high selectivity of non-activated positions. Conclusion. Engineered CYP102A1 variants demonstrate the potential to catalyse the selective as well as varied oxidation of sesquiterpenes from wood essential oil. 1. Scartezzini, P. & Speroni, E. (2000). J. Ethnopharmacology., 71, 23-43.


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Engineering of geraniol oxidase for heterologous production of plant terpenoids in baker’s yeast Saccharomyces cerevisae

Konrad Viehrig1, Jie Zhang1, Michael K. Jensen1, Jay D. Keasling1,2,3,4

1 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark; 2 Joint BioEnergy Institute, Emeryville, CA, USA; 3 Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory; Berkeley, CA, USA; 4 Department of Chemical and Biomolecular Engineering & Department of Bioengineering; University of California, Berkeley, CA, USA. Introduction. Using baker’s yeast Saccharomyces cerevisiae as a cell factory for production of rare plant-derived natural products is an attractive yet challenging strategy to allow sustainable large-scale production of such compounds.1 Especially for refactoring the biosynthesis of plant secondary metabolites in yeast, multiple P450 enzymes often need to be functionally expressed, and properly interact with their respective plant P450 reductase. Overexpression of heterologous P450 may also lead to generation of reactive oxygen species (ROS), which can be toxic to the host at higher levels.2 Aims and Methods. Using Geraniol oxidase (G8O) as a model plant P450, we generated chimeric mutants by swapping the native transmembrane domain of G8O with domains from other P450s. Self-supplying P450-CPR fusion proteins of these chimeric P450s were also generated. In vivo substrate conversion of these enzymes was assayed via gas chromatography and the host cells were tested for ROS stress levels via a fluorescence-based assay.3 Results and Discussion. The generated chimeric G8O enzymes and G8O-CPR fusions are expressed as catalytically active proteins in the yeast host, yet with varying activities. In one case, low activity of a mutant was rescued by co-expression of cytochrome b5 gene from the same host plant. Self-sufficient G8O-CPR fusions did not lead to highly elevated ROS levels in the host cells in our case. Conclusion. Swapping transmembrane domains of heterologously expressed membrane-bound P450s and creating P450-CPR fusions can lead to functionally active enzymes with better performance than the native versions and is considered a useful approach to overcome compatibility issues in heterologous production systems in yeast. 1. Trenchard, I. J., & Smolke, C. D. (2015). Engineering strategies for the fermentative production of plant

alkaloids in yeast. Metabolic engineering, 30, 96-104. 2. Zangar, R. C., Davydov, D. R., & Verma, S. (2004). Mechanisms that regulate production of reactive

oxygen species by cytochrome P450. Toxicology and applied pharmacology, 199(3), 316-331. 3. Zhao, F., Bai, P., Liu, T., Li, D., Zhang, X., Lu, W., & Yuan, Y. (2016). Optimization of a cytochrome P450

oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae. Biotechnology and bioengineering, 113(8), 1787-1795.


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Selective remote functionalisation of azepane by engineered CYP102A1 Yuan Zhang1, Yushu Li1, Ellie Jaques1, Luet Lok Wong1 1Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, United Kingdom. Introduction. Balanol inhibits a number of protein kinase C (PKC) isozymes and is thus important for pharmaceutical uses. The total synthesis of balanol requires up to 20 steps, hindering the preparation of derivatives for further studies. A simpler route would be to functionalise azepane at the remote positions to the ring nitrogen.1 Such functionalisation of non-activated C–H bonds is difficult to achieve by chemical catalysts. Aims. This project aims to engineer CYP102A1 for the selective functionalisation of azepane at remote positions (β- and γ- to the nitrogen) to generate intermediates for the synthesis of balanol and related compounds which may have pharmacological effects. Methods. N-Boc, N-Tf, N-Ts, and N-Ms-azepane were synthesised and screened for oxidation with a library of 96 CYP102A1 variants. Active variants were used for preparative scale reactions which allowed product purification and characterisation. New variants were designed based on the results from the initial screening to optimise the selectivity for remote functionalisation products. Results. For N-Boc-azepane, 76 out of 96 variants gave ≥50% conversion. Variants containing the A330P and A330W mutations showed high activity and regio- and enantio-selectivity for γ-oxidation (94% conversion, 92% γ-alcohol, TON ≥ 2000, S:R = 91:9). The highest enantioselectivity for the R-isomer was 87% but the regioselectivity was low (20%). The regioselectivity for β-oxidation was also moderate (56%). The N-Tf and especially the N-Ts derivative gave more β-oxidation (63%) with variants containing the S72W and A330W mutations. Discussion. The CYP102A1 variant library is able to selectively functionalise azepane at remote positions, especially the γ-position. The mutations A330P and A330W played key roles in selectivity. The A330P mutation constricts the active site of CYP102A1 due to P329 being relocated into the substrate access channel,2 which potentially alters substrate binding and hence selectivity. A330W has similar effects though to different extent. Different derivatives of azepane showed varied selectivity pattern in CYP102A1 oxidation. Conclusion. Highly selective γ-oxidation by CYP102A1, especially for the S-enantiomer, was achieved for N-Boc-azepane. Further work is required for selective β-oxidation. 1. Saha, T., Maitra, R. and Chattopadhyay, S. K. (2013). Beilstein J. Org. Chem., 9, 2910-2915. 2. Whitehouse, C. J., Yang, W., et al. (2010). Chembiochem., 11, 2549-2556.

Balanol


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Exploring the activity and selectivity of the CYP101B1 and CYP101C1 enzymes from Novosphingobium aromaticivorans. Saurabh Kumar Ahirwara, Joel H.-Z. Leea, Stephen G. Bella aDepartment of Chemistry, The University of Adelaide, Adelaide, Australia. Introduction. CYP101B1 and CYP101C1 are P450 enzymes from the bacterium Novosphingobium aromaticivorans DSM12444. Both ustilise a common electron transfer system, consisting of a ferredoxin reductase (ArR) and a ferredoxin (Arx). These P450s are capable of oxidising a broad range of substrates including terpenes and norisoprenoids1-2. Aims. To understand the role that the position of the double bonds and the oxygen functional group (ketone versus alcohol) have on substrate oxidation by CYP101B1, CYP101C1 and its mutant (Met82Leu), using different ionones, damascones and ionols as substrates (Figure 1). Methods. In vitro turnovers were performed to check the oxidation of the substrates and whole-cell turnovers were used to obtain the oxidised products in a higher quantity. The metabolites were separated via HPLC and characterised by NMR. Results and Discussion. β-ionone and β-damascone were preferentially oxidised by CYP101B1 at C3 whereas the CYP101C1 and its mutant (Met82Leu) favoured the oxidation of these substrates at C4 (Figure 1). In certain instances, an epoxide metabolite at the C3 and C4 positions was observed, which must arise from a desaturation metabolite which is then epoxidised. With β-ionol, the CYP101B1 gave the 4-hydroxy metabolite as the major product whereas the CYP101C1 and its mutant (Met82Leu) favoured 3-hydroxylation (Figure 1). Both CYP101B1 and CYP101C1 favoured C3 hydroxylation with -ionone, ionol and damascone whereas -damascone was selectively epoxidised at the cyclic double bond.

1. Ma, M.; Bell, S. G.; Yang, W.; Hao, Y.; Rees, N. H.; Bartlam, M.; Zhou, W.; Wong, L. L.; Rao, Z., Structural analysis of CYP101C1 from Novosphingobium aromaticivorans DSM12444. Chembiochem 2011, 12 (1), 88-99. 2. Hall, E. A.; Bell, S. G., The efficient and selective biocatalytic oxidation of norisoprenoid and aromatic substrates by CYP101B1 from Novosphingobium aromaticivorans DSM12444. RSC Advances 2015, 5 (8), 5762-5773.

Figure 1. Substrates tested for oxidation with CYP101B1, CYP101C1 and its mutant (Met82Leu) and the crystal structure of β-Ionone-bound CYP101C1.1


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The vertebrate ancestor of cytochrome P450 family 3: Optimization of expression and reaction conditions for industrial application Jong-Min Baek1, Yosephine Gumulya1, Silja Strohmaier1, Ann-Sofie Sandinge2, Ulrik Jurva2, Shalini Andersson3, Elizabeth M.J. Gillam1 1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia; 2DMPK, Early Cardiovascular, Renal and Metabolism, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden; 3Discovery Sciences, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden. Cytochrome P450 family 3 (CYP3) enzymes play important roles in the metabolism of drugs and certain endobiotic compounds. In humans, CYP3A4 is the major contributor to the oxidative clearance of drugs. Its substrate promiscuity and varying degrees of regio- and stereo-selectivity towards drug substrates makes CYP3A4 potentially attractive as an industrial biocatalyst. However, poor stability, low catalytic rates and the requirement for a reducing cofactor (NADPH) and redox partner (human cytochrome P450 reductase, hCPR) limits the direct application of CYP3A4 in industrial applications. Ancestral sequence reconstruction has been used to resurrect the vertebrate ancestor of the CYP3 family, CYP3_N1, which showed a significant increase in thermostability over extant CYP3 forms1. CYP3_N1 also showed similar substrate promiscuity to CYP3A4, being active towards typical CYP3A4 substrates but with varying catalytic rates1. Despite the marked improvement in the stability of CYP3_N1 over CYP3A4, the requirement for CPR and NADPH still limits its industrial usefulness. In this study, we investigated ways to improve catalytic efficiency while reducing the total cost of the CYP3_N1 mediated reaction. CYP3_N1 expression was able to be expressed in a chemically defined medium and the optimised expression level was comparable to that obtained in a nutrient rich medium, Terrific Broth. CYP3_N1 also exhibited higher organic solvent tolerance compared to CYP3A4 (up to ~25 % (v/v) DMSO and ~15 % methanol or acetonitrile). Importantly, CYP3_N1 was found to be able to use the oxygen surrogate, cumene hydroperoxide (CuOOH) enabling the NADPH and CPR-requiring steps in the catalytic mechanism to be bypassed and making the overall reaction considerably more cost effective. The CYP3_N1-mediated hydroxylation of a typical CYP3A4 substrate, testosterone, can be supported by CuOOH at elevated temperatures, up to ~70 °C. CYP3_N1 exhibited an optimal reaction temperature of 50 °C, and the optimal concentration of CuOOH varied depending on the reaction temperature. Due to the inherent reactivity of CuOOH towards the haem prosthetic group, supplementation of a low concentration of CuOOH repeatedly over extended incubation times allowed extension of the reaction of up to 56 hrs, thereby improving the total substrate turnover. In summary, the superior thermostability, activity towards typical CYP3A4 substrates and improved yield of CYP3_N1 under optimal reaction conditions will enhance its usefulness as an industrial biocatalyst. In particular, the ability to express CYP3_N1 in chemically defined medium and support activity effectively with CuOOH, eliminating the need for NADPH and CPR in a reaction, will make CYP3_N1 a more cost-effective biocatalyst than extant CYP3 forms in the regio-selective functionalization of C-H bonds in pharmaceutical and fine chemical synthesis. 1. Gumulya Y, Baek JM, Wun SJ, Thomson RES, Harris KL, Hunter DJB, Behrendorff JBYH, Kulig J, Zheng S, Wu

X, Wu B, Stok JE, De Voss JJ, Schenk G, Jurva U, Andersson S, Isin EM, Bodén M, Guddat L, Gillam EMJ (2018). Engineering highly functional thermostable proteins using ancestral sequence reconstruction. Nature Catalysis, 1, 878-888.


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Biochemical studies of peroxygenase enzymes for applications in the fuel industry Alessia Andrews1, Sarah Matthews1, Harshwardhan Poddar1, Hazel M. Girvan1, Thomas A. Jowitt1, Kamila Pacholarz1 and Andrew W. Munro1 1Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, M1 7DN, Manchester, UK. Introduction P450 KR, from the soil bacterium Kocuria rhizophila, is a novel member of the CYP152 peroxygenase family. Peroxygenases are a distinct class of the P450 superfamily, which have evolved to use hydrogen peroxide efficiently as the sole oxygen and hydrogen donor during catalysis. On account of their natural alkene producing abilities, members of this P450 group are of significant interest to the petrochemical industry. Aims Express, characterise and analyse a selection of P450 peroxygenase enzymes, with a particular focus on assessing their suitability for industrial applications. Methods P450 KR was subjected to extensive biochemical and biophysical characterisation. Mass spectrometry techniques have been vital in analyses of the P450 KR product profile, as well as crystallographic studies to elucidate vital structural information. Results Spectroscopic studies on P450 KR displays have revealed an unusual Soret absorbance maximum at ~424 nm, unlike many other P450 family members. Substrate turnover reactions and subsequent metabolite analysis have affirmed the versatile abilities of P450 KR. Primarily, P450 KR functions as a mid-chain length fatty acid decarboxylase generating terminal alkenes. In addition, P450 can serve as a hydroxylase, producing to 2-OH and 3-OH hydroxylated fatty acids. Further studies have also revealed that the use of alternative substrates and novel oxidation methods can lead to a more varied peroxygenase product profiles. Via extensive crystallographic and Ultracentrifugation studies (AUC), the novel dimeric structure of KR has been revealed. An extended N-terminal helix from one monomer forms “zipper”-like interactions with the corresponding helix on the partner monomer. These interactions are stabilised by disulphide bonds formed between two cysteine residues located at the base of the N-terminal helix and in an adjacent beta sheet element within the same monomer. These unusual structural interactions are unprecedented for a P450 of this class. Discussion Biohydrocarbons such as alkenes provide an ideal alternative to current transportation fuels. They have similar physical characteristics and chemical compositions which allow them to be compatible with the existing fuel infrastructure. P450 KR looks like an ideal tool with its ability to produce alkenes from free fatty acids in a single enzymatic step. Conclusions Ongoing research includes the biophysical analysis of the dimeric interface in P450 KR and potentially other CYP152 enzymes. Further research is required to implement alkene-producing peroxygenases in whole-cell systems to provide sustainable routes to biofuels for the future.


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Regioselective hydroxylation of monacolin J by CYP102A1 to produce 6’-hydroxymethyl metabolite, an inhibitor of HMG-CoA reductase Ngoc-Tan Cao, Chan Mi Park, Chul-Ho Yun Department of Biological Sciences and Biotechnology, School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea. Introduction. Statins inhibit the enzyme HMG-CoA reductase which plays a central role in the production of cholesterol. As high cholesterol levels are associated with cardiovascular disease, they reduce cardiovascular disease and mortality in those who are at high risk of cardiovascular disease. Monacolin J is a statin which is made by red yeast rice. It is an intermediate biosynthetic precursor of lovastatin, which is used for the chemical synthesis of simvastatin. At present, limited information regarding monacolin J and its metabolites are available. Aims. In this study, we have tried to make novel metabolites from monacolin J, using P450 as a biocatalyst, with an inhibitory effect on the HMG-CoA reductase. Methods. A set of CYP102A1 variants was used to make metabolites of monacolin J. The metabolites were analyzed and purified by HPLC. Chemical structure of a major metabolite was identified by LC-MS and NMR. Results. Several CYP102A1 variants were able to make several metabolites of monacolin J. One major metabolite was purified by HPLC and its chemical structure was identified by LC-MS and NMR. The metabolite is 6’-hydroxymethyl monacolin J which has never been reported previously. It is made by regioselective hydroxylation at C-6’ methyl group. Enzymatic properties of CYP102A1 variants to produce 6’-hydroxymethyl monacolin J were studies by using highly active variants. The 6’-hydroxymethyl metabolite showed a higher inhibitory effect on HMG-CoA reductase than that of Monacolin J. These statin compounds have comparable inhibitory effects on the HMG-CoA reductase when compared to lovastatin and simvastatin. Discussion. The 6’-hydroxymethyl monacolin J itself can be used as an HMG-CoA reductase inhibitor and it also can be used as a lead compound to make other statin derivatives by further chemical and enzymatic modification of the hydroxyl groups. Conclusion. We report an efficient enzymatic strategy to make a novel metabolite of monacolin J by using bacterial CYP102A1. The metabolite is 6’-hydroxymethyl metabolite with an inhibitory effect on HMG-CoA reductase. 1. Huang, X. et al. (2017) Single-step production of the simvastatin precursor monacolin J by engineering of

an industrial strain of Aspergillus terreus. Metab. Eng. 42:109-114. 2. Liang, B. et al. (2018). Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an

Industrial Strain of Aspergillus terreus by Employing the Evolved Lovastatin Hydrolase. Biotechnol. J. 13, e1800094.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Liang%20B%5BAuthor%5D&cauthor=true&cauthor_uid=29637704

https://www.ncbi.nlm.nih.gov/pubmed/29637704

https://onlinelibrary.wiley.com/toc/18607314/2018/13/6


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Generation of metabolites of neohesperidin dihydrochalcone by bacterial CYP102A1 Heon Jeong, Chul-Ho Yun Department of Biological Sciences and Biotechnology, School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea. Introduction. Cytochrome P450 enzymes are used as biocatalysts for the production of various fine chemicals and pharmaceuticals. Neohesperidin dihydrochalcone (NHDC) is originally derived from neohesperidin, an artificial sweetener, which is abundant at citrus. NHDC is a glycoside of dihydrochalcone derivatives, which show several biological functions of antioxidant, anti-obesity, and anti-cancer. Especially, NHDC shows the antioxidant effect by inhibition of hypochlorous acid induced DNA strand breakage, protein degradation, and cell death. Aims. To generate novel metabolites of NHDC by oxidation reaction using bacterial CYP102A1 variants as a biocatalyst. Methods. CYP102A1 wild type and a set of its variants prepared by site-directed mutagenesis and random mutagenesis were used to produce metabolites of NHDC. A major metabolite was purified and characterized by HPLC and LC-MS analyses. Results. When 53 variants were tested for the ability to produce metabolites of NHDC, 6 variants showed an apparent activity to produce a major metabolite (>0.1 min-1). Among the activity variants, three highly active variants were selected for further experiments, such as identification of the metabolite, and determination of kinetic parameters and total turnover numbers. The structure of the major product was analyzed by LC-MS and molecular mass of the metabolite was -2 than that of substrate. Discussion. We found CYP102A1 can catalyze the oxidation reaction toward NHDC to produce an oxidation metabolite. The metabolite might result from dehydrogenation reaction as molecular mass of the metabolite was -2 than that of substrate. Although there is a steric hindrance of carbohydrate moiety in NHDC, a glycoside of dihydrochalcone, dihydrochalcone moiety of NHDC seems to be oxidized by P450 reaction. This metabolite can be used for several biotechnological applications, such as development of bioactive compounds and drug leads for several biotechnological applications. Conclusion. In this study, we found that a set of CYP102A1 variants can make a major metabolite of NHDC, which might be a product of dehydrogenation reaction. 1. Kang, J. Y. et al. (2014). Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium. Biotechnol. Bioeng. 111:1313-1322. 2. Choi, J. M. et al. (2007). Antioxidant Properties of Neohesperidin Dihydrochalcone: Inhibition of Hypochlorous Acid-Induced DNA Strand Breakage, Protein Degradation, and Cell Death. Biol. Pharm. Bull. 30:324-330.


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Regio- and enantio-selective chemo-enzymatic C-H-lactonization of decanoic acid to (S)-δ-decalactone Bethan S. Jones1, Jack Manning1, Michele Tavanti1, Joanne L. Porter1, Nico Kress1, Sam P. De Visser2, Nicholas J. Turner1 and Sabine L. Flitsch1 1School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom; 2School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester, United Kingdom. Hydroxylation of fatty acids to chiral hydroxy-acids yields a diverse array of high value compounds that have useful applications in the fragrances and flavouring industries. The synthesis of these compounds is challenging due to activation of unreactive C-H bonds, especially with regards to the appropriate stereo- and regioselective introduction of the new hydroxyl group. The chemical oxy-functionalisation typically requires harsh reaction conditions including transition metal catalysts, peracids and organic solvents. The regioselective targeting of a methylene group can be achieved under milder reaction conditions using cytochrome P450 biocatalysts. Whilst regioselective terminal fatty acid hydroxylation catalyzed by P450s has been reported, the oxygenation at highly subterminal positions remains challenging.

In this study we describe a chemo-enzymatic route to commercially valuable lactones via the regio- and stereoselective mid-chain hydroxylation of decanoic acid catalysed by a wild type class VII P450. A panel of class VII P450s was screened against saturated fatty acids, with chain lengths of 8, 10 and 12 carbon atoms. All the class VII P450s showed activity towards the fatty acid substrates, with a range of different regioselectivity from C5 to C11 hydroxylated products. Unexpectedly, P450-TT from Tepidiphilus thermophilus was observed to have high regioselectivity for the δ-hydroxylation of decanoic acid. Of particular note was the excellent stereoselectivity of this wild-type enzyme. This hydroxylated product afforded the precursor to the valuable δ-decalactone, with the reaction conducted on a preparative scale. The surprise was the observation that the wild-type enzymes were capable of yielding mid-chain hydroxylated products, with remarkable stereo- and regioselectivity and notably higher selectivity than P450-BM3 variants. Additional docking studies suggested a possible orientation of the decanoic acid, providing a plausible rationale for the mid-chain hydroxylation. We have demonstrated that wild-type class VII P450s are useful biocatalysts for ‘mid-chain’ C-H activation for the hydroxylation of fatty acids, with promising applications for the production of valuable fragrance compounds. References 1. Manning, J., Tavanti, M., Porter, J. L., Kress, N., De Visser, S. P., Turner, N. J. & Flitsch, S. L. Regio - and

enantio-selective chemo-enzymatic C-H-lactonization of decanoic acid to (S)-δ-decalactone, submitted. 2. Tavanti, M., Porter, J. L., Sabatini, S., Turner, N. J. & Flitsch, S. L. (2018). Panel of New Thermostable

CYP116B Self-Sufficient Cytochrome P450 Monooxygenases Catalyzing C-H Activation with a Diverse Substrate Scope. ChemCatChem, 10, 1042-1051.

3. Dietrich, M., Anh Do, T., Schmid, R. D., Pleiss, J. & Urlacher, V. B., (2009). Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM3 towards γ- and δ-positions. J. Biotechnol., 139, 115-117.


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Identification and characterization of new thermostable self-sufficient cytochrome P450 monooxygenases Nico Kress1, Michele Tavanti1, Joanne L. Porter1, Selina Sabatini1, Nicholas J. Turner1 and Sabine Flitsch1 1Manchester Institute of Biotechnology, University of Manchester, Manchester, United Kingdom. The fascinating oxyfunctionalization of unactivated C-H bonds catalysed by cytochrome P450 enzymes promises to revolutionize the way fine chemicals can be made. However, the usage of these enzymes, requiring the complex interplay of oxygenase and reductase protein partners, is hampered by poor protein stability, solubility as well as low enzyme expression and productivity. A desirable means to tackle these issues lies in the application of self-sufficient P450s in which both protein partners are fused in one entity, as for P450-RhF (class VII) or P450-BM3 (class VIII).

Intrigued by the expanding abundance of sequence data, this work aimed at the identification and characterization of novel thermostable self-sufficient P450s. Based on P450-BM3 and P450-RhF, sequence data was analysed to provide five novel members of class VII P450s. These enzymes were characterized phylogenetically, spectrally, with regard to substrate scope and nucleotide cofactor preference, as well as their thermostability. Moreover, further insights are derived from the crystal structure of P450-TT from Tepidiphilus thermophilus.

All candidates yielded high levels of soluble protein and a broad catalytic spectrum was found within the set of tested substrates. All enzymes showed a preference of the NADPH over the NADH cofactor. Notably, all members displayed high activity in the regioselective hydroxylation of the drug diclofenac. In a 100 mL approach, a productivity of 0.51 gL-1h-1 was found. The thermostability of the enzymes could be confirmed. In particular P450-AX and P450-TT displayed T50 values for residual activity of (59.3±0.2) and (62.9±0.3) °C, respectively. For the crystal structure of P450-TT, conservation of the P450 fold was found and active site residues could be classified. In comparison to the earlier known CYP116B class VII P450, the novel members outperform P450-RhF regarding protein expression, catalytic activity and thermal stability. In summary, this study showed how sequence data mining provides new stable catalysts with promising features that represent excellent starting points for enzyme evolution. This facilitates the practical usability of these highly promising enzyme catalysts. 1. Roberts, G. A., Grogan, G., Greter, A., Flitsch, S. L. & Turner, N. J. (2002). Identification of a New Class of

Cytochrome P450 from a Rhodococcus sp.. J. Bacteriol., 237, 1375-1376. 2. Tavanti, M., Porter, J. L., Sabatini, S., Turner, N. J. & Flitsch, S. L. (2018). Panel of New Thermostable

CYP116B Self-Sufficient Cytochrome P450 Monooxygenases that Catalyze C-H Activation with a Diverse Substrate Scope. ChemCatChem, 10, 1042 – 1051.

3. Tavanti, M., Porter, J. L., Levy, C. W., Castellanos, J. R. G., Flitsch, S. L. & Turner, N. J. (2018) The crystal structure of P450-TT heme-domain provides the first structural insights into the versatile class VII P450s. Biochem. Biophys. Res. Commun., 501, 846-850.


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Biocatalytic production of a potent inhibitor of adipocyte differentiation from apple-derived phloretin Ngoc Anh Nguyen, Jin Jang, Thien-Kim Le, Hyung-Sik Kang, Chul-Ho Yun

Department of Biological Sciences and Biotechnology, School of Biological Sciences and Technology, Chonnam National University, 77 Yongbongro, Gwangju 61186, Republic of Korea.

Introduction. Apples are the second fruit crop worldwide after bananas. Approximately 70 million tons of apples are produced worldwide per year. Valorization of apple pomace can reduce environmental impact and meet the requirement of sustainable development for the large-scale apple-processing industry. Phloretin is a natural polyphenol compound found mainly in apple trees it shows several biological activities. Aims. The aim of this research was to develop an enzymatic strategy for the efficient one-step production of a catechol compound with a high possibility of biological activity from phloretin and phloridzin, which are abundant in apple tree. Methods. A set of CYP102A1 variants was used to find highly active enzyme to catalyze phloretin hydroxylation. HPLC method was used to detect the products, which were catalyzed by CYP102A1, of phloretin into 3-OH phloretin with high conversion yields. Results. Almond β-glucosidase efficiently removed the glucose moiety of phlorizin to produce phloretin with high conversion yields. Bacterial CYP102A1 variants catalysed the highly regioselective C-hydroxylation of phloretin into 3-OH phloretin with high conversion yields. Furthermore, we found that differentiation of 3T3-L1 preadipocytes into adipocytes and lipid accumulation were dramatically inhibited by 3-OH phloretin, but promoted by phloretin. Consistent with these inhibitory effects of adipocyte differentiation, the expression of adipogenic regulator genes such as PPAR 2, C/EBP and CD36 was downregulated by 3-OH phloretin. Discussion. Here we present a highly efficient method to prepare 3-OH phloretin from phloretin which was developed using CYP102A1 variants. In addition, the hydroxylated metabolites can also be used as leads of bioactive compounds for inhibition of adipocyte lipogenesis to reduce the risk of obesity in humans. The eco-friendly and sustainable strategy in this study can be a platform for the valorization of bioactive compounds from food waste, such as apple pomace. Conclusion. We report an efficient enzymatic strategy for the production of a potentially valuable phloretin metabolite from phlorizin, a glucoside of phloretin which is abundant in apple pomace. Almond β-glucosidase can efficiently remove the glucose moiety of phlorizin to produce phloretin with high conversion yields. Phloretin itself is an efficient substrate for CYP102A1 variants to prepare 3-OH phloretin, a potent inhibitor for adipocyte differentiation. 1. Hyson DA (2011). A Comprehensive Review of Apples and Apple Components and Their Relationship to

Human Health. American Society for Nutrition., 2, 408-420.

http://advances.nutrition.org/search?author1=Dianne+A.+Hyson&sortspec=date&submit=Submit


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Regioselective hydroxylation of naringin dihydrochalcone, an artificial sweetener, to catechol product, neoeriocitrin dihydrochalcone, by Bacillus megaterium CYP102A1 Thi Huong Ha Nguyen, Chul-Ho Yun Department of Biological Sciences and Biotechnology, School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea. Introduction. Naringin dihydrochalcone (naringin DC) is well-known as an artificial sweetener with a strong antioxidant activity, that has potential applications in food and pharmaceutical fields. It is originally derived from the flavonoid naringin which occurs naturally in citrus fruits, especially in grapefruit. Naringin DC is a glycoside of phloretin which shows an inhibitory effect on active transport of glucose into cells by SGLT1 and SGLT2. It was suggested that naringin DC might be a potential therapeutic agent for the treatment of AD against multiple targets that include Aβ pathology, neuroinflammation and neurogenesis. Aims. In this study, we have tried to find an enzymatic strategy for the efficient synthesis of potentially valuable metabolites from naringin DC, which is a glycoside of phloretin. Methods. A set of Bacillus megaterium CYP102A1 variants was used to find efficient regioselective hydroxylases toward naringin DC. Activity analysis and purification of the metabolites were done by HPLC. Chemical identification of the metabolite was done by LC/MS and NMR. Results. We found three highly active CYP102A1 variants to hydroxylate naringin DC among wild type (CYP102A1) and its 60 variants. Highly active variants produced one major metabolite and its chemical structure was determined by LC/MS and NMR. The major metabolite is neoeriocitrin dihydrochalcone (neoeriocitrin DC), which has a catechol structure of naringin DC. Discussion. The synthesis of neoeriocitrin DC from naringin DC has been achieved by using biocatalytic strategy using CYP102A1 enzyme with highly efficient yields. At present, as neoeriocitrin DC is not commercially available, its biological functions have not been studied. This result suggests that neoeriocitrin DC can be used for further biological studies at the levels of cells and animals. Conclusion. Here, we reported an efficient synthesis of neoeriocitrin DC from naringin DC, a glycoside of phloretin, using CYP102A1. 1. Tang, N., Yan, W. (2016) Solubilities of naringin dihydrochalcone in pure solvents and mixed solvents at

different temperatures. J. Chem. Eng. Data, 61, 4085−4089. 2. Le, T. K., Jang, H. H., Nguyen, H. T., Doan, T. T., Lee, G. Y., Park, K. D., Ahn, T., Joung, Y. H., Kang H. S.,

Yun, C. H. (2017) Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3. Enzyme Microb. Technol. 97, 34-42.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Yun%20CH%5BAuthor%5D&cauthor=true&cauthor_uid=24474032

https://en.wikipedia.org/wiki/Active_transport

https://en.wikipedia.org/wiki/SGLT1

https://en.wikipedia.org/wiki/SGLT2


34A

The ability of P450s to be supported by oxygen surrogates is substrate-dependent Silja J Strohmaier1, James J De Voss1, Ulrik Jurva2, Shalini Andersson3, Elizabeth M J Gillam1

1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia; 2DMPK, Early Cardiovascular, Renal and Metabolism, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden; 3Discovery Sciences, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden. Introduction: Oxygen surrogates (OSs) have been used in cytochrome P450 research for diverse purposes, from mechanistic studies to biocatalytic applications. The opportunity to replace the complex electron transport chain, comprising NADPH and the one or more protein redox partners that are usually required for P450 catalysis, with cheap OSs could potentially allow more economically viable P450 catalysis and therefore has attracted considerable attention. Most studies have used either hydrogen peroxide or cumene hydroperoxide (CuOOH). However, peroxides have been shown to damage the haem prosthetic group of the P450. Moreover other OSs are available, namely hypervalent iodine compounds, yet little is known of the preference of individual P450s towards different OSs. Therefore, it was of interest to characterize the activity of human drug metabolizing P450s supported by a set of commonly available OSs. Aim: To explore the ability of the major human drug-metabolising P450s to use oxygen surrogates. Methods: The ability of five OSs to support product formation by the major drug metabolizing P450s, namely CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 was investigated. For activity measurements E. coli membranes were used, harbouring the recombinant human P450 reductase (hCPR) and the respective P450. The OS-supported activity was compared to hCPR/NADPH-supported reactions. Relative product formation was standardized to the hCPR/NADPH supported product formation and compared for each enzyme employing three different marker substrates. Results: The relative activity changed with the marker substrate used to varying extents. Furthermore, the OS resulting in highest relative activity of any individual P450 was dependent on the substrate used. In some cases, activity with an OS equalled or slightly exceeded that seen with hCPR and NADPH. Overall, CuOOH and t-butyl hydroperoxide (tBuOOH) were generally the most effective at supporting P450 activity, whereas little to no activity was seen with hydrogen peroxide. Discussion: The relative formation of product was found to be dependent on both the substrate and oxygen surrogate applied, suggesting that the binding of the substrate and OS to the P450 are not independent. For example, tBuOOH was more effective at supporting midazolam hydroxylation by CYP3A4 whereas testosterone hydroxylation was better with CuOOH. This may reflect alterations in the conformation at the active site due to binding of different OSs or substrates. Conclusion: A ‘one-size-fits-all’ approach is not useful for using OSs to support P450 activities. When OSs are used to support activity of P450s, the OS used should be tailored to both the P450 and the substrate under investigation. Directed evolution studies designed to engineer improved peroxygenase activity should employ screening methods that reflect the activity towards whatever substrate is relevant to the end application.


35B

NADPH - and redox partner-free catalysis by ancestral cytochrome P450 enzymes Silja J Strohmaier1, James J De Voss1, Ulrik Jurva2, Shalini Andersson3, Elizabeth M J Gillam1

1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia; 2DMPK, Early Cardiovascular, Renal and Metabolism, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden; 3Discovery Sciences, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden. Introduction: The stereo- and regioselective functionalisation of unactivated C–H bonds is a challenging reaction for the synthesis of valuable chemicals for which biocatalysts are often sought. Cytochrome P450 enzymes (P450s) are renowned for catalyzing such challenging reactions on a broad set of substrates, and are therefore potentially useful, candidate biocatalysts. However, naturally occurring P450s show low stability, poor expression, and typically modest catalytic efficiency. These factors, plus the requirement of most P450s for a redox partner and expensive cofactors, make them very cost intensive catalysts. Recent work has shown that the resurrected ancestors of extant, microsomal P450s exhibit high thermostability, while often retaining similar substrate specificity, properties that can be harnessed for biocatalysis. However, an equally robust redox support system is required. These P450s are routinely supported by the diflavin reductase, cytochrome P450 reductase (CPR), which is thermolabile and requires expensive cofactors to reduce the P450. A potential alternative solution is to use oxygen surrogates to support P450 activity, obviating the need for the redox partner, reducing cofactor or aeration of the reaction mixture. Aim:To explore the ability of ancestral P450s to be supported by oxygen surrogates. Methods: The ability of 10 oxygen surrogates (OSs) to support product formation by 24 ancestral was assessed compared to hCPR-supported reactions. Three peroxides, five λ3- and two λ5 – iodanes were tested using recombinant P450s expressed in E. coli membranes. Results: Of the 24 ancestral P450s tested, 14 were supported by at least one of the OSs examined, as well as, or better than, by the human CPR. Relative turnover compared to the CPR-supported reactions varied with enzyme and OS but 2-nitro-iodoso-compounds were generally the most effective, achieving improvements in product yield of up to 50-fold in one case. Discussion: Most ancestral P450s investigated here were effectively supported by at least one OS. Therefore, selected OSs may provide an economically viable alternative to hCPR-supported catalysis. The major advantages of using OSs over hCPR are: reduced cost, since no NADPH or coexpressed redox partner is required; the option to perform reactions at enhanced temperatures, as no thermostable redox partner is needed; and higher expression of P450s in the absence of a redox partner. Conclusion: The ability of thermostable P450s to use a low-cost and robust “chemical support system” may allow development of more economically viable processes based on P450-mediated catalysis.


36C

Profound alteration of P450 BM3 regioselectivity via a single point mutation Dominic R. Whittall, Hazel M. Girvan, Harshwardhan Poddar, Kirsty J. McLean, Andrew W. Munro1. 1Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom. Introduction. Cytochrome P450 BM3 (CYP102A1) is a highly efficient and soluble fatty acid hydroxylase, consisting of a natural fusion between a eukaryotic-like cytochrome P450 reductase and a bacterial fatty acid-binding P450 domain. Its amenability to engineering for novel functionality, ease of expression and rapid rate of turnover makes P450 BM3 an attractive target for industrial exploitation. Aims. A highly efficient fatty acid hydroxylase, wild-type BM3 produces predominantly ω-1, ω-2 and ω-3 hydroxylated products from medium to long chain fatty acids. A structurally disruptive point mutation (I263P) was introduced in the catalytically important I-helix region to examine its effect on substrate binding and catalysis. Methods. The BM3 I263P variant was subjected to a wide range of structural, catalytic and biophysical characterisation techniques. X-ray crystallography and GC-MS have been integral to the analysis of the structure and product profile of the BM3 I263P variant, respectively. Results. The BM3 I263P variant has profound effects on fatty acid substrate oxidation profiles. BM3 I263P was shown to produce a more diverse range of hydroxylated fatty acids in addition to the typical wild-type products. With dodecanoic (C12:0), tetradecanoic (C14:0) and hexadecanoic (C16:0) acids, BM3 I263P produces hydroxylated fatty acids in the range from ω-1 to ω-7, i.e. reaching the delta carbon in the case of C12:0. Discussion. By employing a rational approach to site-directed mutagenesis, additional substitutions to active site residues of BM3 I263P have yielded a variant capable of offering novel routes to valuable new hydroxylated compounds. Fatty acids hydroxylated at positions approaching the α-carbon are of particular interest to the food and cosmetic industries; serving as precursors to commercial flavour compounds. In addition, I263P-based BM3 variants have demonstrated altered regio- and stereoselective hydroxylation activity towards a variety of terpene compounds widely used within the fragrance industry. Conclusion. Additional studies of the BM3 variants featuring an I263P mutation may serve to further the understanding of how conformational properties of the I-helix regulate fatty acid binding modes and control regioselectivity of substrate oxidation by P450 BM3.


37A

P450-mediated oxidation cascade applied to vancomycin substrates: A serendipity

story

Julien Tailhades1,2, Yongwei Zhao3, Melanie Schoppet1,2, Anja Greule1,2, Max Cryle1,2

1The Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Australia; 2EMBL Australia, Monash University, Melbourne, Australia; 3School of Medicine, Nursing and Health Science, Monash University, Melbourne, Australia. Non-ribosomal peptide synthesis (NRPS) is central to the biosynthesis of a many compounds of medical importance, including glycopeptide antibiotics (GPAs). The potential of NRPS machineries to produce novel compounds has long been recognised as highly desirable. GPAs biosynthesis through the NRPS machinery includes an elongation (Step 1), a P450-mediated oxidation cascade (Step 2) and maturation including glycosylation, methylation and/or sulfonation (Step 3).1 The focus of this research is about the P450-mediated oxidation cascade (Step 2) in order to produce improved GPAs targeting resistant bacteria. In our complex enzymatic system, our limitation quickly became about the quantity of tricyclization and the scale of our protocol. For the latter, we partially addressed this issue by developing a chemical strategy to generate our crucial peptide-Co-enzyme A intermediate in a 5 to 10 mg scale.2 Consequently, the P450-mediated tricyclization cascade (OxyB, A and C) was tested on different substrates (~ 15 peptides) linked on the di-domain T-X following a structure activity relationship between substrates/P450s (Figure 1A). From this first study, we obtained a wide range of tricyclization production (0-40%, 12% in average). While trying to optimise our process, the addition of TCEP/cysteamine was identified to be promoting an usually high production of tricyclic compounds (Figure 1B). Consequently, the parameters around those chemicals were studied and further applied to the library of 15 peptides which highlighted +37% of tricyclic compound formation in average.

Figure 2. Tricyclic production improvement

using TCEP and cysteamine.

1 Haslinger, K.; Peschke, M.; Brieke, C.;

Maximowitsch, E.; Cryle, M. J., X-domain of

peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis. Nature 2015, 521, 105.

2 Tailhades, J.; Schoppet, M.; Greule, A.; Peschke, M.; Brieke, C.; Cryle, M. J., A route to diastereomerically

pure phenylglycine thioester peptides: crucial intermediates for investigating glycopeptide antibiotic

biosynthesis. Chem. Commun. 2018, 54(17), 2146-2149.

HN

NH

HN

NH

HNO

O

HO

OH

O

OHOO

Cl O

O

NH

O

NH2

HN

O OH

R1R3

13

R2

> 15 compounds tested

Previous protocol

TCEPCysteamine

12%

49%

+37% production

A

B

T X

S

B

A

C


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P450 enzyme cascade to generate tetracyclic teicoplanin Yongwei Zhao1, Julien Tailhades2,3, Max J Cryle2,3 1School of Medicine, Nursing and Health Science, Monash University, Melbourne, Australia; 2EMBL Australia, Monash University, Melbourne, Australia; 3The Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Australia. Teicoplanin is an important member of the glycopeptide antibiotics (GPAs) and is in clinical use. The biosynthesis of teicoplanin includes heptapeptide backbone synthesis, mediated by non-ribosomal peptide synthases (NRPS), and four characteristic crosslinks of aromatic peptide side chains, mediated by cytochrome P450s called Oxy enzymes (OxyA, OxyB, OxyC and OxyE respectively). Previous studies have shown that the in vivo order of Oxy enzymes is B>E>A>C, and such a cascade also exists when generating tricyclic teicoplanin analogue in vitro. Our previous in vitro assays involving OxyE were achieved on non-natural teicoplanin substrates and without OxyC, which was unavailable at that time [1, 2]. Here, we report the first example of four-enzyme cascade (OxyB/OxyE/OxyA/OxyC) activity on teicoplanin sequence. Our method involves a peptide synthesis using previously reported Fmoc-based solid phase peptide synthesis (SPPS) [3], peptidyl-CoA formation and an Oxy enzyme mediated cyclisation reaction. When the peptide is converted into a CoA form, it is loaded onto a reconstituted Peptide Carrier Protein-X (PCP-X) di-domain using promiscuous phosphopantetheinyl transferase Sfp. Afterwards the turnover assay is performed with the presence of newly optimised conditions that include cysteamine and TCEP that we have shown to be able to increase P450 activities. The tetracyclisation of teicoplanin is obtained in vitro using our method, with ~40% yield of tetracyclic compound. Although tetracyclic compound could be formed by sequentially introducing OxyB, OxyA, OxyC and OxyE in any order of addition, the order B>E>A>C allowed the best yield of tetracyclisation and thus matches the previous in vivo results for this cyclisation cascade. 1. Hadatsch, B., Butz, D., Schmiederer, T., Steudle, J., Wohlleben, W., Süssmuth, R. and Stegmann, E.

(2007). The Biosynthesis of Teicoplanin-Type Glycopeptide Antibiotics: Assignment of P450 Mono-Oxygenases to Side Chain Cyclizations of Glycopeptide A47934. Chemistry & Biology, 14(9), pp.1078-1089.

2. Peschke, M., Brieke, C. and Cryle, M. (2016). F-O-G Ring Formation in Glycopeptide Antibiotic Biosynthesis is Catalysed by OxyE. Scientific Reports, 6(1).

3. Tailhades, J., Schoppet, M., Greule, A., Peschke, M., Brieke, C. and Cryle, M. (2018). A route to diastereomerically pure phenylglycine thioester peptides: crucial intermediates for investigating glycopeptide antibiotic biosynthesis. Chemical Communications, 54(17), pp.2146-2149.


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Defining the P450 repertoire across metamorphosis in spiny lobsters Courtney L Lewis1, Cameron C Hyde1, Abigail Elizur1, Quinn P Fitzgibbon2, Gregory G Smith2 and Tomer Ventura1 1GeneCology Research Centre, School of Science and Engineering, University of the Sunshine Coast, Sippy Downs, QLD, Australia; 2Institute for Marine and Antarctic Sciences, University of Tasmania, Hobart, TAS, Australia. Introduction. Nutrigenomics investigates the organism response to what it consumes, including molecular mediators and gene expression. Formulated feeds for species maintained in aquaculture is a large portion of the aquaculture budget, so it is necessary to use feeds that provide ideal, life-stage-specific nutrients to maximise farming efficiency. This is particularly significant for spiny lobsters whose life cycle comprises multiple, exceptionally varied phenotypes, that require different feed formulations (Fitzgibbon et al, 2013). Our research focuses on the Cytochromes P450 (P450s), a large, rapidly evolving superfamily of enzymes known to have roles in multiple pathways including lipid metabolism (Ventura et al, 2017). In crustaceans, P450s metabolise essential compounds responsible for the regulation of growth, development and reproduction. Research in the eastern spiny lobster identified a P450 which converts the ecdysone precursor to its bioactive form, 20-hydroxylated ecdysone (Ventura et al, 2017). Aims. Further research into the spiny lobster transcriptome at higher resolution to better understand the roles P450s play in lipid metabolism. Methods. A PFAM domain search across putative proteins derived from the ornate spiny lobster (Panulirus ornatus) transcriptome, retrieved the P450s. Digital expression patterns were used to identify the P450s that change across metamorphosis. A phylogenetic analysis annotated known P450s and highlighted unknown P450s suspected to be involved in lipid metabolism. Results. A total of 115 putative P450-encoding transcripts were recovered. Following size filtering to a minimum of 300 amino acids, 55 putative P450s were retrieved, of which 26 P450s had distinct differential expression correlated with 12 life-stages examined. Discussion. Clusters of genes were determined based on similar expression patterns, enabling characterization and function determination. Those associated with lipid metabolism could be ascertained using functional assays, to better our understanding of lipid metabolism throughout metamorphosis. Conclusion. The results of this research provide valuable insights that could be used by the industry to reduce lobster feed costs and increase product uniformity and quality. Further research is underway to define the function of P450 candidates that are suspected to be involved in lipid metabolism in the critical stage of phyllosoma to puerulus transition. 1. Fitzgibbon, Q.P. et al. (2013). The Achilles heel for spiny lobsters: the energetics of the non-feeding post-

larval stage. Fish and Fisheries, 15, 2, 312-326. 2. Ventura, T., Bose, U., Fitzgibbon, Q.P., Smith, G.G., Shaw, P.N., Cummins, S.F., Elizur, A. (2017). CYP450s

analysis across spiny lobster metamorphosis identifies a long sought missing link in crustacean development. J. Steroid Biochem. Mol. Biol., 171:262-269.


40A

Closing gaps in invertebrate development using crustacean P450 omics, 3D modelling and rapid in vitro assays Tomer Ventura1, Tuan Viet Nguyen1, Luke Ryan1, Quinn P Fitzgibbon2, Gregory G Smith2, Guiomar Rotllant3, Marc Le Groumellec4, Utpal Bose5 and Abigail Elizur1 1GeneCology Research Centre, School of Science and Engineering, University of the Sunshine Coast, Sippy Downs, QLD, Australia; 2Institute for Marine and Antarctic Sciences, University of Tasmania, Hobart, TAS, Australia 3Institut de Ciències del Mar (ICM-CSIC), Barcelona, Spain; 4Unima, Aqualma, Aquaculture de la Mahajamba, Mahajanga 401, Madagascar; 5CSIRO Agriculture and Food, St. Lucia, Australia. Introduction. The Cytochromes P450 (P450s) repertoire was identified in spiny lobsters transcriptomic libraries representing distinct life stages across metamorphosis, highlighting the differentially expressed genes. Using phylogenetic analysis, followed by three-dimensional modelling and finally an in vitro assay, a family of P450s that are responsible for converting the ecdysone precursor to its active molt hormone form, the 20-hydroxylated ecdysone, was discovered (Ventura et al, 2017). This pathway to discovery could be utilized to address broader gaps in the development of invertebrates like the absence of the aromatase gene which is converting testosterones to estrogens. Aims. Identify and characterize the P450s responsible for estrogens production in invertebrates. Methods. Use digital expression patterns of P450s that change between reproductive and non-reproductive phases of invertebrates, followed by phylogenetic analysis to annotate known P450s and highlight unknown P450s suspected to be involved in estrogen metabolism. These results were tested using a metabolome of the different reproductive stages. Results. Multiple P450s in decapod crustacean species were retrieved and analyzed. Candidate P450s that show differential expression across reproductive phases as well as 3D conservation with vertebrates’ Aromatase were identified. Discussion. A shortlist of candidate P450s which play a similar role to the vertebrate aromatase gene was defined. Functional assays are underway to define which gene can convert testosterones to estrogens in invertebrates. Conclusion. The results of this research provide a valuable pathway to discovery of key functions played by P450s in invertebrates. 1. Ventura, T., Bose, U., Fitzgibbon, Q.P., Smith, G.G., Shaw, P.N., Cummins, S.F., Elizur, A. (2017). CYP450s

analysis across spiny lobster metamorphosis identifies a long sought missing link in crustacean development. J. Steroid Biochem. Mol. Biol., 171:262-269.


41B

Potential steroid metabolising cytochrome P450s from Rhodococcus rhodochrous Ava Ho, Lauren J. Salisbury, and James J. De Voss School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia. Introduction: Rhodococcus rhodochrous was first isolated and studied for its ability to live on 1,8 cineole. A novel R. rhodochrous cytochrome P450, CYP176A1 (P450cin) initiates biodegradation of 1,8-cineole allowing the organism to use as its sole source of carbon and energy.1 More recently, genomic sequencing of R. rhodochrous identified a further 22 putative P450s, four of which were annotated as being involved in steroid metabolism. Two of these putative P450s are the focus of the current study – one with 77.9% amino acid identity to CYP51 (a lanosterol 14α demethylase) from Mycobacterium tuberculosis and a second that had 64.7% amino acid identity to CYP125 (a steroid C-26 monooxygenase) from Rhodococcus jostii (strain RHA1). Aim: The overall goal of this study is to clone both putative P450 genes, express them in Escherichia coli and purify them in order to determine the function of the CYP125 and CYP51 R. rhodochrous homologues. Methods: The putative CYP51 gene was codon optimised for E. coli expression before cloning into the pCW expression vector. Following successful expression in E. coli, CYP51 was then purified by ion-exchange and size exclusion chromatographic techniques. UV/Visible spectroscopy was used for initial characterization and included examination of the binding of potential substrates and inhibitors. In addition, reconstitution with heterologous redox partners was explored to generate enzymatic activity. This approach was also used for the cloning, expression, purification and characterisation of CYP125. Results and Discussion: CYP51 was expressed at approximately 350 nmol/L and purified to an R/Z of 0.92. UV/Visible characterisation demonstrated that it was a typical P450 and the coordination of CO to the reduced protein gave the expected difference spectrum (Figure 1). Preliminary binding and inhibition experiments have been performed for CYP51, but its native substrate is yet to be identified. Conclusion: The R. rhodochrous genome is a good source of P450s that can be expressed at high level in E. coli. The substrates for both enzymes expressed here are currently unknown and functional and structural characterisation is ongoing. 1. Hawkes, D.B., Adams, G.W., Burlingham, A.L., Ortiz

de Montellano, P.R. & De Voss, J.J. (2002). Cytochrome P450cin (CYP176A1), Isolation, Expression, and Characterization. J. Biol. Chem., 277, 27725-27732.

Figure 1 The absolute spectrum (solid line) and the carbon monoxide difference spectrum of CYP51 from R. rhodochrous.


42C

Exploring the impact of amino acid changes on fungal sterol 14α-demethylase structure and function using a yeast model. Brian C Monk1, Alia Sagatova1, Rajni K Wilson1, Joel D.A. Tyndall2, Michaela Lackner3, Yasmeen N. Ruma1, Parham Hosseini1, Mikhail V. Keniya1 1Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand; 2School of Pharmacy, University of Otago, Dunedin, New Zealand; 3Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria. Introduction. Azole drugs remain the primary class used to treat a wide range of fungal infections in humans, other mammals and crops. However, the incidence of azole resistance is increasing. This is driven by a variety of mechanisms including (a) acquisition of amino acid changes in the targeted sterol 14α-demethylases, (b) amino acid substitutions conferring intrinsic azole-resistance, (c) overexpression of the target enzyme, (d) bypassing of toxic metabolites using alternate pathways, and (e) drug efflux. Aims. Use phenotypic analysis and a library of crystal structures of full-length lanosterol 14α-demethylases from Saccharomyces cerevisiae and the major fungal pathogens Candida albicans and Candida glabrata in complex with azole drugs to better understand the effects of resistance-conferring substitutions/mutations and to direct the discovery of novel antifungals. Methods. Full-length recombinant C-terminal hexahistidine-tagged sterol 14α-demethylases were overexpressed constitutively from the PDR5 locus in a S. cerevisiae host deleted of 7 drug efflux pumps, in some cases in combination with a cognate NADPH-cytochrome P450 reductase overexpressed constitutively from a co-regulated PDR15 locus. Phenotypic, biochemical, and crystallographic analysis were used to determine structural and functional features of wild type and azole resistant versions of these enzymes in complex with various azole inhibitors. Results. Sterol 14α-demethylases from evolutionary divergent fungal species, including human and agricultural pathogens, were functionally overexpressed in S. cerevisiae. Co-expression with cognate reductases resulted in patterns of azole-susceptibility that closely matched the patterns of the native enzymes. Like the triazole fluconazole, the tetrazole VT-1161 showed resistance conferred by overexpression of the drug target, target mutations (tyrosine [Y] to phenylalanine [F] in the active site) and overexpression of MFS and ABC drug efflux pumps. VT-1161 and the medium-tailed triazole isavuconazole showed phenotypic behaviours intermediate between the short-tailed triazoles fluconazole and voriconazole that bind exclusively within the active site and the long-tailed triazoles itraconazole and posaconazole that have additional interactions with the substrate entry channel. Expression in S. cerevisiae of sterol 14α-demethylase F1 and F5 isoforms from the mucormycete Rhizopus arrhizus gave functional enzymes that showed the equivalent Y to F substitution confers intrinsic resistance. The effect of Y to F mutation or substitution, which alters a water-mediated hydrogen bond network in the active site, can be overcome by modifying the linker between the head and tail of the azole drug. Discussion. Phenotypic analysis and crystallographic studies of recombinant sterol 14α-demethylases expressed in yeast provide insights that facilitate structure-directed antifungal discovery. Conclusion. Amino acid changes within the active site of fungal sterol 14α-demethylases can be countered using structure-directed antifungal design.


43A

Development of Cryptococcus neoformans and Candida parapsilosis lanosterol 14α-demethylase as drug targets Yasmeen N Ruma1, Mikhail V. Keniya1, Joel D.A. Tyndall2, Brian C. Monk1

1Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand; 2School of Pharmacy, University of Otago, Dunedin, New Zealand. Introduction. Cryptococcus neoformans and Candida parapsilosis are opportunistic fungal pathogens that cause systemic infections in humans, especially in immunocompromised patients. Such infections are usually treated using the azole antifungals. These drugs inhibit lanosterol 14α-demethylase (CYP51), an enzyme that is essential for the synthesis of the fungal-specific sterol ergosterol. Azole prophylaxis and/or long-term use of azole drugs have led to the acquisition of azole resistance among these pathogens. NADPH-cytochrome P450 reductase (NCP1) is thought to be the redox partner that provides electrons for CYP51 activity. Aims. To assess the function and azole susceptibility of CYP51s from the fungal pathogens C. neoformans and C. parapsilosis and the impact of their cognate NCP1 in a Saccharomyces cerevisiae host system. Methods. Recombinant full-length, codon optimised and C-terminal hexahistidine-tagged CYP51 of C. neoformans (CnCYP51) and C. parapsilosis (CpCYP51) were constitutively overexpressed from the PDR5 locus of a Saccharomyces cerevisiae host strain deleted of 7 drug efflux pumps and with the galactose-regulated promoter (Gal1) replacing the promoter of the native CYP51. A cognate NCP1 was then constitutively co-expressed from the PDR15 locus. Agarose diffusion assays were used to evaluate the susceptibility of the S. cerevisiae host and recombinant strains to various azole antifungals during growth on complex synthetic medium containing galactose or glucose. The glucan synthase inhibitor micafungin was used as an independent control. Results. CpCYP51 and CnCYP51 expressed from the PDR5 locus supported growth in the presence of either galactose or glucose (native CYP51 suppressed) and showed functional activity that was inhibited by azole drugs in tests using agarose diffusion assays. The recombinant strains were susceptible to short- (fluconazole, voriconazole) and long-tailed (posaconazole, isavuconazole) triazoles and a medium-tailed tetrazole (VT-1161). Co-expression of CnCYP51 with its cognate NCP1 conferred reduced susceptibility to the short-tailed azoles but not the medium- or long-tailed azoles. The differential response to azole drugs is consistent with results obtained previously for CYP51 from the major fungal pathogen Candida albicans expressed in S. cerevisiae in the presence or absence of its cognate NCP1. Discussion. The heterologous overexpression in a hypersensitive S. cerevisiae host of the CYP51s of major fungal pathogens has revealed a differential response consistent with the binding efficacy of short-tailed azoles within the active site being significantly improved due to heme reduction in response to cognate NCP1 co-expression. For the longer-tailed azoles this effect appears to be countered due to interactions of those drugs with the CYP51 substrate entry channel. Conclusion. Structural and functional analysis of recombinant CYP51s from the fungal pathogens C. neoformans and C. parapsilosis is improving the understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery.


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Two novel cytochrome P450s involved in the biosynthesis of benzastatins Hayama Tsutsumi1, Yohei Katsuyama1,2, Kazuo Shin-ya3, Yasuo Ohnishi1,2 1Grad. Sch. Agri. and Life Sci. The Univ. of Tokyo; 2CRIIM; 3AIST, Japan. Introduction. In the biosynthesis of natural products, various enzymes are involved in the generation of their unique and complex structures. Many cytochrome P450s play important roles in the formation of such structures. Thus, investigation of the functions of cytochrome P450s in the biosynthesis of natural products can lead to the discovery of novel biosynthetic reactions. Benzastatins (BSs), which are natural products isolated from several Streptomyces species, possess unique bicyclic structures (indoline and tetrahydroquinoline structures) derived from geranyl pyrophosphate (GPP) and p-aminobenzoic acid (PABA) [1]. Recently we revealed the whole pathway for the biosynthesis of BS derivatives in Streptomyces sp. RI18, in which two cytochrome P450s, BezC and BezE, catalyze unprecedented hydroxylation and cyclization reactions, respectively [2]. Results. We established a stable heterologous expression system of the BS biosynthesis gene cluster using Streptomyces lividans as a host and the function of each biosynthesis enzyme gene was analyzed by gene disruption. As a result, a putative methyltransferase (BezA) and a cytochrome P450 (BezC) were indicated to catalyze the sequential methylation and hydroxylation of geranylated PABA or GPP. Then in vitro enzyme assays using recombinant BezA and BezC proteins were carried out. First, BezA methylated GPP but did not geranylated PABA. Second, BezC did not hydroxylate methylgeranylated PABA. Finally, in the coupling reaction of BezA and BezC using GPP as a substrate, hydroxylation activity of BezC was observed. From these results, we concluded that BezA and BezC catalyze the sequential methylation and hydroxylation of GPP to form methylgeranyl pyrophosphate (MGPP) and hydroxymethyl geranyl pyrophosphate (HMGPP). We also showed that these compounds (GPP, MGPP, and HMGPP) can be transferred onto PABA by the prenyltransferase BezF to form geranylated PABAs. BezC showed type I substrate binding spectra in the presence of GPP. In contrast, BezC showed type II substrate binding spectra in the presence of methylgeranylated PABA. These results support that BezC catalyzes the hydroxylation of a GPP derivatives, and suggest that methylgeranylated PABA serves as an inhibitor of BezC. From the gene disruption experiment and in vitro enzyme analysis, the function of another cytochrome P450 (BezE) was also revealed. BezE plays a crucial role to form bicyclic structures using geranylated p-acetoxyaminobenzoic acid derivatives as substrates. We predicted that BezE catalyzes (i) elimination of acetic acid from the acetoxy group to form an iron nitrenoid intermediates, (ii) nitrene addition to a double bond to form a tricyclic structure containing an aziridine ring, and (iii) aziridine ring opening by nucleophilic addition of water or chloride to form indoline or tetrahydroquinoline structure, respectively. Conclusion. In this study, we discovered two novel cytochrome P450s, BezC and BezE. BezC catalyzes hydroxylation of MGPP. BezC is the first enzyme that was reported to catalyze the hydroxylation of prenyl pyrophosphates. BezE catalyzes an unprecedented cyclization reaction via nitrene formation and addition. BezE is the first natural cytochrome P450 that was reported to catalyze nitrene formation and addition. 1. Motohashi et al. (2011) J. Antibiot. 64, 281 2. Tsutsumi et al. (2018) J. Am. Chem. Soc. 140, 6631


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P450-catalysed citran-ring formation in bruceol biosynthesis Joel H.Z. Lee1, Aaron J. Day1, Hiu C. Lam1, Jonathan H. George1, Stephen G. Bell1 1Department of Chemistry, University of Adelaide, Adelaide, SA 5005, Australia. Introduction. The meroterpenoid bruceol (4) was isolated from Philotheca brucei in Western Australia in 1963.[1] The pentacylic structure of bruceol (4) contains a 5, 7-dioxy coumarin fused to a “citran” ring system. A separate meroterpenoid, isobruceol, has an opposite orientation of the dioxycoumarin to the “citran” ring system compared to bruceol. It is hypothesised that the final step of the biosynthesis of these two compounds commences via a stereoselective epoxidation of the homoprenyl substituent of protobruceol (1).[2] This epoxidation step is speculated to be mediated by a monooxygenase enzyme and triggers a cyclisation cascade through epoxide ring-opening to form an ortho-quinone methide (3) that subsequently undergoes an intra-molecular Diels-Alder reaction to form the final natural product. The synthesis of both bruceol (4) will be demonstrated using a P450-mediated biocatalytic epoxidation of protobruceol (1). Aims. To demonstrate the cyclisation cascade of (1) can be catalysed by a P450 enzyme to generate the meroterpenoid natural product, bruceol (4). Methods. Protobruceol (1) was synthesised and a small library of P450BM3 mutants was screened for in vitro oxidation activity. The presence of bruceol (4) and isobruceol were confirmed by chiral HPLC co-elution experiments with synthetic standards. Results. Chiral phase HPLC analysis of the turnover with P450BM3 variant KSK19/I263A/A328I revealed that bruceol (1) were formed as single enantiomers. No intermediates were formed that indicated the cyclisation cascade was triggered successfully by the P450 enzyme. Discussion. P450BM3 KSK19/I263A/A328I was able to catalyse the enantioselective epoxidation to form bruceol (4). Therefore, an as of yet unidentified P450 monooxygenase could potentially catalyse this biosynthetic step in Philotheca brucei. Conclusion. This study demonstrates the biosynthesis of bruceol (4) and isobruceol is possibly catalyzed by a P450 monooxygenase.

[1] A. M. Duffield, P. R. Jefferies, E. N. Maslen, A. I. M. Rae, Tetrahedron 1963, 19, 593-607. [2] A. J. Day, J. H. Z. Lee, Q. D. Phan, H. C. Lam, A. Ametovski, C. J. Sumby, S. G. Bell, J. H. George,

Angewandte Chemie International Edition 2019, 58, 1427-1431.


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The effect of the novel atypical antipsychotic drug asenapine on human cytochrome P450 1A (CYP1A) expression and activity Przemysław Danek , Jacek Wójcikowski, Władysława A. Daniel Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology Polish Academy of Sciences, Kraków, Poland. Introduction. Inhibition of cytochrome P450 enzymes is the most common cause of harmful drug–drug interactions. Asenapine is a novel atypical antipsychotic drug approved for the treatment of schizophrenia and bipolar disorders. It has a unique receptor-binding profile characterized by high affinity for serotonergic, dopaminergic, α-adrenergic and histaminergic receptor subtypes. Aims. The aim of the present study was to estimate the effect of asenapine on CYP1A2 in human liver. Methods. Experiments were carried out using two models. First model was performed using pooled human liver microsomes and cDNA-expressed human CYP1A2 enzyme in the absence and presence of asenapine. The inhibition constant (Ki) was obtained using a non-linear regression analysis (Program Sigma Plot 8.0; Enzyme Kinetics). Second model was performed using inducible-qualified human cryopreserved hepatocytes from three different donors. Asenapine was added to the culture medium at therapeutic concentrations of 0.01, 0.025 and 0.1 μM. The CYP1A2 activity was determined using CYP1A2-specific reaction caffeine 3-N-demethylation (HPLC). The level of CYP1A2 protein in hepatocytes was measured using human CYP isoform-specific ELISA kits. The expression of CY1A1 and CY1A2 gens (mRNA levels) was determined by qRT-PCR. Results. Asenapine potently inhibited CYP1A2 activity in liver microsomes and Supersomes (Ki= 4.0 and 4.5

M, respectively). At the tested concentrations asenapine significantly decreased the activity of CYP1A2 (to 67-78% of the control value) and the mRNA level of CYP1A2 (to 57-72% of the control value) in the cultures of human hepatocytes. The mRNA level of CYP1A1 was not changed. The neuroleptic did not exert any statistically significant effect on the protein level of CYP1A1 or CYP1A2. Discussion. The results obtained showed that asenapine exerts a direct inhibitory effect on the CYP1A2 enzyme protein. Asenapine may form reactive or intermediate metabolites, which inactivate CYP1A2 by binding directly to the enzyme protein. Direct inhibition of CYP1A2 by asenapine observed in vitro is expected to occur in vivo, since the calculated Ki values are close to the presumed concentration range of asenapine in the liver of patients. On the other hand, long-term exposure to the neuroleptic decreases CYP1A2 gene expression, which results in the lowered enzyme activity. The observed two inhibitory mechanisms (interaction with enzyme protein and gene expression) may lead to the inhibition of the metabolism of asenapine (which is a substrate of CYP1A2) and other co-administered drugs in the liver of psychiatric patients. Conclusion. By inhibiting the activity of CYP1A2 asenapine may diminish the metabolism of CYP1A2 substrates (e.g. caffeine, theophylline, phenacetin, tricyclic antidepressants, propranolol, fluvoxamine, clozapine). The results may also have physiological and toxicological significance, since CYP1A2 are implicated in the biotransformation of estrogens and environmental contaminants. (Supported by grant no. 2013/11/B/NZ7/01627 from the National Science Centre and by statutory funds from the Institute of Pharmacology, PAS, Kraków, Poland. Przemysław Danek acknowledges the support of InterDokMed project no. POWR.03.02.00-00-I013/16.)


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The inhibitory effects of Thai herb extracts on P450-mediated anticancer metabolism Porntipa Korprasertthaworn1,2,3, Chumaporn Rodseeda2,3, Paveena Yamanont1 and Darawan Pinthong1

1 Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand; 2 Toxicology Graduate Programme, Faculty of Science, Mahidol University, Bangkok, Thailand; 3 Center of Excellence on Environmental Health and Toxicology, Faculty of Science, Mahidol University, Bangkok, Thailand. Introduction. An increasing number of cancer patients are using complementary and alternative medicines (CAM) in combination with their conventional anticancer agents. CAM has been reported to cause pharmacokinetic interactions with chemotherapy, leading to a reduced efficacy or an unexpected toxicity. Tyrosine kinase inhibitors are widely used as targeted cancer therapy because they act with specific cell signalling pathways. Most of tyrosine kinase inhibitors undergo cytochrome P450 (P450)-dependent metabolism where anticancer-herb interactions have been identified. Aim. Inhibitory effects of Thai herb extracts on P450-mediated erlotinib and sorafenib were investigated. Methods. The inhibitory effects of Thai herb extracts including Andrographis paniculata (A. paniculata), Curcuma zedoaria (C. zedoaria), Ganoderma lucidum (G. lucidum), Murdannia loriformis (M. loriformis), Smilax corbularia (S. corbularia), Smilax glabra (S. glabra), Stemona collinsae (S. collinsae) and Ventilago denticulate (V. denticulate) were investigated. Pooled human liver microsomes were used as CYP450 enzyme sources. The depletion of erlotinib and sorafenib was examined using high-performance liquid chromatography (HPLC). Results. The inhibition of Thai herb extracts (20 µM) on P450-mediated erlotinib metabolism was in the order of C. zedoaria, A. paniculata, V. denticulate, S. glabra, M. loriformis, G. lucidum, S. corbularia and S. collinsae, respectively. In addition, the rank order of selected Thai herb extracts (20 µM) inhibition on sorafenib metabolism was C. zedoaria, V. denticulate, A. paniculata, S. collinsae, S. glabra, G. lucidum, M. loriformis and S. corbularia, respectively. The concentration required to achieve 50% of the maximum inhibition (IC50) of C. zedoaria on CYP450-mediated erlotinib and sorafenib were 4.59 ± 1.80 and 7.59 ± 3.54 µg/mL, respectively. Discussion. The results showed a potent inhibition of C. zedoaria on the metabolism of erlotinib and sorafenib by CYP450 enzymes. These interactions may cause the alteration in exposure of erlotinib and sorafenib in cancer patients and leading to a decrease in therapeutic response or an increase in drug toxicity. Conclusion. Potential interactions of C. zedoaria with erlotinib and sorafenib may become a safety concern in cancer chemotherapy.


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Allosteric interactions in CYP3A4 probed by site directed mutations Ilia G Denisov,1 Yelena V Grinkova,1 Stephen G Sligar1,2 1Department of Biochemistry and 2Chemistry, University of Illinois, Urbana, IL, 61801, USA Introduction. Human CYP3A4 is involved in metabolism of more than 30% of drugs administered in clinical practice, and concomitantly represents the main locus for drug-drug interactions. In addition to a spacious and malleable substrate binding pocket which can accommodate two substrate molecules, CYP3A4 has a peripheral allosteric binding site formed at the protein – membrane interface between F-F’ and G-G’ loops and lipid head groups. Binding of effector molecules at this site changes the shape and size of the substrate binding pocket, and also modulates the conformational dynamics of the protein. Results. Using CYP3A4 incorporated in Nanodiscs, we compared the functional properties of several single point mutations at positions 211-215 (F-F’ loop), which are known to be involved in cooperativity and allosteric interactions. Combination of model substrates progesterone (PGS) and carbamazepine (CBZ) has been used as a well-established example of asymmetric heterotropic interactions [1,2]. Activation of CBZ epoxidation in the presence of PGS, which is observed in wild-type CYP3A4, was inhibited in most mutants. In addition, spectral titration experiments revealed substantial changes in Type I binding of both substrates in several mutants, most pronounced in L211H and L211W with PGS as a substrate. Conclusion. Variations in substrate binding and metabolism, perturbations of cooperative properties and changes in the allosteric effect of PGS on the rate of CBZ epoxidation observed in CYP3A4 mutants indicate the presence of a highly sensitive regulatory mechanism in this important enzyme. Prediction of drug-drug interactions mediated by CYP3A4 requires better understanding of the specific details of allosteric interactions with various effectors at the protein-membrane interface. Supported by NIH R35 grant GM118145 to Stephen G. Sligar 1. Denisov, I.G., Grinkova, Y.V., Baylon, J.L. et al. (2015). Mechanism of drug-drug interactions mediated by human cytochrome P450 CYP3A4 monomer. Biochemistry, 54, 2227-2239. 2. Denisov, I.G., Grinkova, Y.V., Nandigrami, P. et al. (2019). Allosteric interactions in human cytochrome P450 CYP3A4: The role of phenylalanine 213. Biochemistry, 58, DOI: 10.1021/ acs. biochem.8b01268


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Time-dependent inhibition of CYP1A2 by stiripentol and its structurally related methylenedioxyphenyl compounds Yasuhiro Masubuchi1, Chieko Takahashi1 & Rina Gendo1 1Laboratory of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Choshi, Chiba, Japan. Introduction. Stiripentol (STP) is an antiepileptic agent, which is used for the treatment of Dravet syndrome by the combination therapies with clobazam and valproic acid. In addition to its direct anticonvulsant activity associated with diverse effects on GABAA receptor, STP inhibits several cytochrome P450 enzymes and increases plasma concentration of the concomitant drug, particularly clobazam, which also may play an important role in the combination therapies. But the inhibition mechanisms of P450 enzymes by STP have not been fully elucidated. Aims. STP has a methylenedioxyphenyl (MDP) group, which could be converted into carbene species and involved in inhibitory metabolic intermediate (MI) complex with P450 enzymes. Here, by using STP and its structurally related MDP compounds, we examined possible time-dependent inhibition of P450 enzymes via MI complex formation. In preliminary studies, we have found that STP inhibits P450 isoforms, nonspecifically, whereas time-dependent inhibition was most pronounced for CYP1A2, and thus, we focused on CYP1A2 in the present study. Methods. STP, methylenedioxybenzene (MDB), safrole (SF), isosafrole (ISF), dihydrosafrole (DSF) and piperonyl butoxide (PBO) were used as MDP compounds. Hepatic microsomes were prepared from β-naphthoflavone treated male Wistar rats. The reaction mixtures were subjected to difference spectra with and without MDP compounds in the presence of NADPH, which were monitored from 500 to 400 nm, to estimate MI complex formation. The same mixtures were preincubated with MDP compounds in the presence of NADPH, followed by the assay of phenacetin O-deethylation activity as a probe of CYP1A2. Results. Spectral analysis of hepatic microsomes incubated with STP and NADPH gave a Soret peak at approximately 455 nm. This absorption was largely disappeared by the addition of potassium ferricyanide. Similar spectra were also obtained for all other MDP compounds, whereas the increases in the absorbance at 455 nm varied among compounds, i.e., STP was most pronounced, followed by ISF, DSF and SF, and was minimum for MDB, which is the core structure of MDP compounds but has no side chain. The peak formation by PBO, a classical P450 inhibitor, was smaller than MDP compounds except MDB. STP and other MDP compounds inhibited CY1A2 activity in a concentration-dependent manner. The preincubation of microsomes with these compounds and NADPH potentiated the inhibitory potency, revealing leftward shifts on the inhibition curves and lowering of IC50 values. The extents of the time-dependent CYP1A2 inhibition, which are estimated by the lowering of the IC50 values obtained after preincubation, were largest in STP, followed by ISF and DSF. These results were in good agreement with those of the spectral analysis. Discussion. We found that STP like other MDP compounds caused a time-dependent CYP1A2 inhibition, which should be associated with MI complex formation. STP could be converted into corresponding carbene species, which generate the complex with CYP1A2. It is suggested that small alkyl groups are favourable to generate the postulated inhibitable carbenes. Conclusion. STP could inhibit P450 enzymes via the MI complex formation. This may lead to continuous inhibition of the metabolism of concomitant antiepileptic agents, which is beneficial for keeping their blood concentrations.


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Increased plasma tacrolimus concentration after a single intravenous administration of voriconazole: a case of drug-drug interaction Hideo Shiohira1, Satoshi Yamada1, Hitoshi Uehara1, Jose Carlos S. Tayag1, Nobuo Hokama1, Shinichiro Ueda2, Katsunori Nakamura1 1Department of Pharmacy, University of the Ryukyus Hospital. Okinawa, Japan; 2Department of Clinical Pharmacology & Therapeutics, Faculty of Medicine, University of the Ryukyus. Okinawa, Japan. Introduction: Tacrolimus is known to have many drug-drug interactions. As a cytochrome P450 (CYP) 3A4/5 substrate, the metabolism of tacrolimus is inhibited by many drugs, such as azole antifungal agents which affect CYP3A4/5. Thus, implementation of therapeutic drug monitoring is recommended during treatment with tacrolimus to maintain optimal plasma concentration levels and to avoid toxicity. Case: We report the case of a 58-year-old Japanese female with lupus nephritis whose plasma concentration of orally administered tacrolimus increased after an initial intravenous administration of 300 mg voriconazole for suspected Aspergillus infection. Results: Therapeutic drug monitoring was initiated before the second dose of voriconazole. Although the tacrolimus dosage did not vary, the plasma tacrolimus concentration was markedly increased with a concentration dose ratio 2.1-fold greater than prior to voriconazole treatment. Discussion: We infer from this case that even a single intravenous dose of voriconazole may seriously affect the metabolism of tacrolimus due to drug-drug interaction involving CYP3A4/5. Conclusion: A single intravenous dose of voriconazole, which is assumed to have a minimal effect on the gut metabolism of tacrolimus, may result in a marked increase in the tacrolimus plasma concentration. Optimization of tacrolimus dosage or performance of TDM immediately upon administration of voriconazole is warranted to minimize the risk of tacrolimus toxicity.


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Dopamine formation from p-tyramine mediated by human CYP2D6 in the brain Toshiro Niwa1, Mayumi Yanai1, Shizuya Sugimoto1 1 School of Pharmacy, Shujitsu University, Okayama, Japan. Introduction. Dopamine is formed from p- and m-tyramine in the brain by human CYP2D6 and rat CYP2D4. CYP2D6 is polymorphic; CYP2D6*2 and CYP2D6*10 alleles are present in Caucasian and Japanese populations as frequencies of 27-32% and 31-38%, respectively [1]. Aims. Dopamine formation from p-tyramine mediated by CYP2D6 variants, CYP2D6.2 (Arg296Cys, Ser486Thr) and CYP2D6.10 (Pro34Ser, Ser486Thr) was compared, and the effect of genetic polymorphism on the inhibitory effects of various compounds including antidepressants and endogenous steroid hormones were investigated. Methods. CYP2D6.1, CYP2D6.2, and CYP2D6.10 expressed in recombinant Escherichia coli (Bactosomes, Cypex Ltd) were used. Dopamine formation from p-tyramine in the absence or presence of four antidepressants including imipramine, desipramine, fluvoxamine, and fluoxetine and five steroid hormones including cortisol, cortisone, corticosterone, dehydroepiandrosterone, and pregnenolone was determined by HPLC as described previously [2]. Results. CYP2D6.10 had higher Michaelis constant (Km) of dopamine formation than CYP2D6.1 and CYP2D6.2. Inhibitory constant (Ki) of imipramine and desipramine against CYP2D6.10 were higher than that against CYP2D6.1. The maximal velocity (Vmax) values for dopamine formation by all CYP2D6 variants gradually increased with increasing the concentrations of fluvoxamine and fluoxetine, indicating that these SSRIs stimulated dopamine formation. Progesterone inhibited dopamine formation and Ki values against CYP2D6.10 was approximately twice that for CYP2D6.1 and CYP2D6.2, whereas marked inhibition was not observed for other steroid hormones. Discussion. CYP2D6.10 had lower affinity of dopamine formation and weaker inhibitory effect by imipramine and desipramine than CYP2D6.1 and CYP2D6.2. Interestingly, fluvoxamine and fluoxetine stimulated dopamine formation. We previously reported that fluoxetine stimulated progesterone 21-hydroxylation [3]. These SSIRs might activate some kinds of CYP2D6-mediate reactions. Conclusion. CYP2D6 polymorphism would affect dopamine formation and the inhibition by various compounds in the brain. Fluvoxamine and fluoxetine stimulated CYP2D6-mediated dopamine formation. 1. Niwa T. et al. (2011). Comparison of cytochrome P450 2D6 and variants in terms of drug oxidation rates

and substrate inhibition. Curr. Drug Metab., 12, 412-435. 2. Niwa T. et al. (2017). Effect of genetic polymorphism on the inhibition of dopamine formation from p-

tyramine catalyzed by brain cytochrome P450 2D6. Arch. Biochem. Biophys., 620, 23-27. 3. Niwa T. et al. (2008). Effect of psychotropic drugs on the 21-hydroxylation of neurosteroids,

progesterone and allopregnanolone, catalyzed by rat CYP2D4 and human CYP2D6 in the brain. Biol. Pharm. Bull., 31, 348-351.


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Bacterial P450 engineering for production of valuable drug metabolites Sian Thistlethwaite1, Laura N Jeffreys1, Hazel M Girvan1, Kirsty J McLean1, Richard B Tunnicliffe1, Matthew Cliff1, Andrew W Munro1 1Manchester Institute of Biotechnology, University of Manchester, Manchester, United Kingdom. Introduction. The US Food and Drug Administration (FDA) requires human drugs and their metabolites to be submitted for safety testing. Cytochromes P450 account for approximately two thirds of human drug metabolism. However, membrane-bound human P450s are often unstable and catalytically slow proteins that do not yield sufficient amounts of metabolic products for thorough analysis. Chemical synthesis routes also prove challenging due to poor yields, cost and the potential for production of unwanted side products. To combat this issue we have used a double mutant (DM) variant of the P450 BM3 P450-P450 reductase fusion enzyme from Bacillus megaterium. This natural fusion protein has been used to produce human-derived and novel drug metabolites. Aims. To study the interactions of the BM3 DM with a diverse (978 compound) FDA library, and (for identified substrates) to extract, quantify and compare the metabolites formed to those of human-derived metabolites. Methods. Analysis of the interactions of the BM3 DM with the FDA compound library and elucidation of metabolites will be achieved by using several biophysical techniques. Recent work has focused on developing a high throughput screen of the FDA library in order to identify compounds that are rapidly metabolised by the BM3 DM. Furthermore, development of an NMR analysis method to elucidate metabolite structures is ongoing. Results. A novel high throughput binding screen has shown that the BM3 DM elicits a significant spin shift indicative of binding for 59% of drugs in the FDA library. Drugs in the antidiabetic and fibrate classes have been shown via LCMS and NMR to produce human-derived metabolites. Work that is ongoing includes development of a method that successfully separates out metabolites for thorough analysis by LCMS and NMR. The LCMS method has given successful separation of metabolites for a compound with a complex metabolic profile. Discussion. The promiscuity of the BM3 DM has enabled production of specific metabolites and has indicated that this enzyme can be an important new biotechnological tool for use in a range of applications, including pre-clinical testing and the production of novel compounds that are difficult to synthesise via traditional organic chemistry routes. Conclusion. To date, significant progress has been made in identifying and quantifying BM3 DM-derived metabolites. Further work includes identifying human drug metabolites produced by the enzyme.


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Metabolic activity testing can underestimate acute drug cytotoxicity as revealed by HepG2 cell clones overexpressing cytochrome P450 2C19 and 3A4 Susanne Steinbrecht1, Rosalie König1, Kai-Uwe Schmidtke1, Natalie Herzog1, Katrin Scheibner1, Anne Krüger-Genge2, Friedrich Jung1, Sarah Kammerer1 & Jan-Heiner Küpper1

1Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany; 2Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany. Introduction. Preclinical drug safety assessment includes in vitro studies with physiologically relevant cell cultures. As an in vitro system for hepatic toxicology testing, we have been generating cell clones of human hepatoblastoma-derived cell line HepG2 by lentiviral transduction of phase I cytochrome P450 (CYP) enzymes. Aims. We aimed to generate a HepG2 clone overexpressing CYP2C19 in addition to our previously established HepG2-3A4 clone. Further, we aimed to use the cell clones for analyzing acute cytotoxicity and genotoxicity of the model drug cyclophosphamide (CPA) since CPA is metabolized by those CYP2C19 and CYP3A4 enzymes. Methods. The effects of 2.5 – 10 mM CPA on the CYP-overexpressing HepG2 cell clones were detected using various metabolic activity tests (CellTiter-Glo® Luminescent Cell Viability Assay, Cell Proliferation Kit II (XTT)) and investigation of cell death as well as DNA damage formation. Results. Here, we present a stable CYP2C19-overexpressing HepG2 cell clone (HepG2-2C19 C1) showing an enzyme activity of approximately 82 pmol x min-1 x mg-1 total cellular protein. Upon 10 mM CPA exposure, we were able to detect the metabolites 4-hydroxy-CPA and acrolein in CYP3A4- and CYP2C19-expressing cell clones, but not in parental HepG2 cells. XTT and ATP assays showed a modest reduction of cell viability of not more than 50 % with high dose (10 mM) CPA treatment. By contrast, dramatic acute cytotoxic and genotoxic effects of CPA were evident by the formation of nuclear yH2AX foci and by increased cell death events. These effects were paralleled by substantial decreases of cell membrane integrity as measured by the trypan blue exclusion test. Discussion. The phenotypic stability over many passages of the presented HepG2-2C19 C1 and HepG2-3A4 renders those cell clones a suitable reference for the study of CYP2C19 and CYP3A4 enzyme expression and activity. Furthermore, the investigation of CPA effects on the generated CYP-overexpressing HepG2 cell clones showed a clear difference to the parental HepG2 cells, especially through the detection of DNA damage, meaning that those cell clones also present interesting tools to study CYP2C19- or CYP3A4-mediated drug metabolism in vitro. Conclusion. We successfully established HepG2 clones stably overexpressing CYP2C19 and CYP3A4 with CYP enzyme activity levels of physiological relevance. Our data on CYP enzyme overexpressing HepG2 cell clones clearly showed that cytotoxicity of CPA is dramatically underestimated by standard metabolic activity tests. Thus, additional tests to quantitate DNA damage formation and cell death induction might be required to realistically assess cytotoxicity of such compounds.


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Effects of arsenic exposure on CYP expression and drug metabolism Qing-Yu Zhang, Xiaoyu Fan, Pengfei Liu, Weiguo Han, Xiangmeng Wu, Liang Ding, Donna Zhang, Xinxin Ding Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721, USA Introduction. Environmental exposure to arsenic is associated with increased incidences of multiple adverse health consequences in humans, posing a significant health risk to people living near contaminated sites, for example, populations residing near mining sites who may be exposed to mine tailings through the inhalation of dust particles or ingestion of contaminated water or food. Previous studies in rodents reported the ability of short-term exposures to arsenic at high doses to increase or decrease the expression of several xenobiotic metabolizing CYPs in liver, lung, heart, or kidney, raising the possibility that exposed human individuals may have altered ability to metabolize drugs and other xenobiotic compounds. Aims. This study is to examine effects of chronic arsenic oral exposure at a relative low level on expression of various CYPs in the liver and intestine. Methods. In the first experiment, mice were fed control or sodium arsenite-containing drinking water (25 ppm) for 20 weeks. Hepatic gene expression was analyzed using RNA-seq. In a follow-up study, both hepatic and intestinal transcript levels were determined by real-time PCR after 4 weeks of exposure to arsenic. CYP2A5 and CYP3A11 protein levels were determined by immunoblot analysis and their in vitro activities in microsomal preparations were also determined with model substrates. Results. RNA-seq analysis revealed significant increases in transcript levels for several CYPs, including CYP2A5 (by 10-fold), an enzyme known for its activities toward nicotine, various environmental toxicants, as well as endogenous compounds. Further studies with mice after 4 weeks of exposure to arsenic demonstrated that hepatic CYP2A5 transcript level was also increased (by 2.5-fold). Like CYP2A5, hepatic expression of CYP3A11, a major drug-metabolizing CYP enzyme, was also induced by arsenic exposure for 4 or 20 weeks. Induction of CYP2A and CYP3A protein was confirmed by immunoblot analysis of both sets of samples. CYP expression in the intestine was also examined in the 4-week exposed groups. CYP3A11, but not CYP2A5, is expressed in the intestine. Induction of CYP3A11 expression was observed in the proximal, but not distal, intestine, and it was less than 2-fold. The increased CYP expression was accompanied by increased CYP-mediated nifedipine and coumarin metabolism in vitro in liver microsomes. Discussion. The mechanisms for the observed induction of hepatic CYP2A5 and CYP3A11 expression by arsenic are unclear. It also remains to be determined whether the induction plays a role in arsenic’s toxic effects, including diabetes. The impact of this model of arsenic exposure on xenobiotic metabolism in vivo is currently under study. Conclusion. Our data reveal significant induction of hepatic CYP2A5 and CYP3A11 expression by chronic exposure to a relatively low dose of arsenic in drinking water. (Supported in part by NIH grants ES020867 and GM082978.)


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Characterization of epalrestat as a highly selective CYP4A11 inhibitor Satoshi Yamaori1,2, Noriyuki Araki3, Mio Shionoiri3, Kurumi Ikehata3, Shinobu Kamijo2, Shigeru Ohmori1,2 & Kazuhito Watanabe3,4 1Department of Pharmacy, Shinshu University Hospital, Matsumoto, Japan, 2Department of Biochemical Pharmacology and Toxicology, Graduate School of Medicine, Shinshu University, Matsumoto, Japan, 3Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan, 4Center for Supporting Pharmaceutical Education, Daiichi University of Pharmacy, Fukuoka, Japan. Introduction. Cytochrome P450 4A11 (CYP4A11) is a physiologically important enzyme that catalyzes ω-hydroxylation of lauric acid and arachidonic acid in the human liver and kidneys. CYP4A11 is also involved in the metabolism of some marketed drugs such as febuxostat, tofogliflozin, and epalrestat. To elucidate the involvement of a certain CYP isoform in drug metabolism, selective chemical inhibitors and antibodies are used for inhibition studies. Although several CYP4 inhibitors, such as 17-octadecynoic acid (17-ODYA) and HET0016, have been developed1, no chemical inhibitors entirely selective for CYP4A11 have yet been found. Thus, a selective inhibitor of CYP4A11 is essential to clarify its roles in drug metabolism as well as physiological function. Aims. The selectivity and mode of CYP4A11 inhibition by epalrestat were examined with recombinant CYP (rCYP) enzymes and human liver microsomes (HLMs). Methods. Individual catalytic activities in the presence or absence of epalrestat were measured using high performance liquid chromatograph, fluorescence microplate reader, or luminometer. Results. Epalrestat exhibited the strongest inhibition against rCYP4A11 (IC50 = 1.82 μM) among the 17 recombinant enzymes tested. The inhibitory effect of epalrestat on rCYP4A11 was at least 10 times more potent than those on rCYP4F2, rCYP4F3B, and rCYP4F12. In contrast, HET0016 reduced rCYP4A11 and rCYP4F2 activities (IC50 = 0.0137 – 0.0182 μM); 17-ODYA inhibited activities of rCYP4A11, rCYP4F2, rCYP4F3B, and rCYP4F12 to a similar extent (IC50 = 5.70 – 17.7 μM). Epalrestat strongly inhibited the CYP4A11 activity of HLMs (IC50 = 0.913 μM); the drug moderately inhibited a marker activity of CYP2C9 (IC50 = 23.7 μM) and exhibited no or weak inhibition against marker activities of CYP1A2, CYP2C19, CYP2D6, and CYP3A4/5 (IC50 > 50 μM). Furthermore, epalrestat showed a mixed-type inhibition against the activity of rCYP4A11 and HLMs. Discussion. Since epalrestat selectively inhibited CYP4A11 activity of HLMs as well as rCYP4A11, this compound is suggested to be useful to identify the involvement of CYP4A11 in oxidation of fatty acids and drugs. Conclusion. We demonstrated that epalrestat is the most selective CYP4A11 inhibitor among the chemical inhibitors reported so far. 1. Edson, K.Z. & Rettie, A.E. (2013). CYP4 enzymes as potential drug targets: focus on enzyme multiplicity,

inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ω-hydroxylase activities. Curr. Top. Med. Chem., 13, 1429-1440.

Epalrestat


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Functional characterization of 11 allelic variants of CYP2C9 identified in 3554 Japanese individuals Masaki Kumondai1, Akio Ito1, Takahiro Saito1, Sakae Saito2, Jun Yasuda2, Masao Nagasaki2, Kengo Kinoshita2, Masayuki Yamamoto2, Akifumi Oda3, Noriyasu Hirasawa1, 4, 5, Masahiro Hiratsuka1, 2, 4, 5 1Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan, 2Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan, 3Faculty of Pharmacy, Meijo University, Nagoya, Japan, 4Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan, 5Advanced Research Center for Innovations in Next-Generation Medicine, Tohoku University, Sendai, Japan. Introduction. Variations in genes implicated in drug pharmacokinetics can influence inter-individual viability of drug efficacy and adverse drug reactions. Particularly so for warfarin, a widely prescribed anticoagulant used as a long-term treatment and prevention of thromboembolic events. However, some patients could experience heavy bleeding or thromboembolic events due to this drugs’ narrow therapeutic window. Notably, (S)-warfarin, which is predominantly metabolized by CYP2C9, has a three- to five-fold anticoagulant effect when compared to that of (R)-warfarin. By analysing the whole-genome sequences for 3554 healthy Japanese individuals, several novel CYP2C9 allelic variants have been recently identified. Thus, revealing their enzymatic activities which provide useful pharmacokinetic information for the improvement warfarin dosing guidelines. Aims. In order to advance the development of personalized medicine regarding warfarin therapy, we performed the functional characterization of the novel CYP2C9 allelic variants identified in Japanese individuals. Methods. Wild-type and 11 CYP2C9 variant proteins were heterologously expressed in 293FT cells. The CYP2C9 content in the microsomal fraction extracted from 293FT cells was determined by western blotting. The enzymatic activities of CYP2C9 variants were assessed by (S)-warfarin 7-hydroxylation. Reduced CO-difference spectra of microsomal fractions were determined by measuring the increase in the maximum absorption wavelength at 450 nm following CO treatment. Additionally, we performed a 3D modelling analysis to determine the effect of amino acid substitutions on CYP2C9 conformation. Results. Kinetic parameters of (S)-warfarin 7-hydroxylation were determined for 8 CYP2C9 variants. The kinetic parameters for the remaining 4 variants could not be determined due to insufficient metabolite amounts. As for reduced CO-difference spectra, we detected an increase in the maximum absorption wavelength at 450 nm after CO treatment for all variants excluding C13R, G332D, and E400X. In the case of G332D, conformational changes caused by the formation of a hydrogen bond between D332 and Q454 affect the stability of J-, J’-, and L-helix regions. Discussion. The results show that several variants were either catalytically inactive for the substrates used or had low enzymatic activity due in part to structural changes. Interestingly, the variants which showed an increase in the maximum absorption wavelength at 450 nm after CO treatment seem to retain their enzymatic activities suggesting that the formation of the holo-protein plays an important role in drug metabolism. Additionally, we consider 3D modelling as a helpful tool for the advance prediction of the effect of amino acid substitutions in newly identified variants. Conclusion. These findings can be used to predict pharmacokinetics based on CYP2C9 genotyping and would contribute to the development of personalized medicine and optimized warfarin dosing algorithms.


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Variability in drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase (POR) Maria Natalia Rojas Velazquez1,2, Shaheena Parween1,2, and Amit V Pandey1,2 1Pediatric Endocrinology, University Children’s Hospital, Bern, Switzerland; 2Department of Biomedical Research, University of Bern, Bern, Switzerland. Introduction. A broad spectrum of human diseases, including abnormalities in steroidogenesis, are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR) (1-2). Cytochrome P450 proteins perform several reactions, including metabolism of steroids, drugs and other xenobiotics. Therefore, genetic variations in POR can impact many different metabolic pathways by changing the activities of cytochromes P450 (1). In 2004 the first human patients with defects in POR were reported, and over 200 variations in POR are known (2). Information about effects of POR variants on drug metabolizing P450s is limited and has not received much attention. Aims. Analysis of cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities due to variations in POR. Methods. By analyzing the POR sequences from sequencing projects, we identified potentially disease-causing variations and characterized these by functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded and assayed using fluorogenic substrates on a microplate spectrofluorometer. Results. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284T, P284L and A287P and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20-50 activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. Similarly, the common polymorphism in POR, A503V showed several fold higher activities with all drug metabolizing P450s studied. Discussion. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities have important consequences for monitoring and treatment of patients. Similarly, higher drug metabolism activities from A503V variant of POR suggests monitoring the patients with this variant carefully. The A503V is the common polymorphism in POR present in about 25% of all alleles and is often ignored in diagnostic reports. These results indicate that detailed knowledge of POR effects is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism due to variations in POR needs to be addressed. Conclusion. Changes in drug metabolism due to genetic variations in POR should be considered in addition to variations in P450 genes. Differences in drug metabolism can be addressed using personalized metabolic profiling and supplementation. 1. Parween S et al. P450 Oxidoreductase Deficiency: Loss of Activity Caused by Protein Instability From a

Novel L374H Mutation. J Clin Endocrinol Metab. 101:4789-4798 (2017). 2. Burkhard FZ et al. P450 Oxidoreductase deficiency: Analysis of mutations and polymorphisms. J Steroid

Biochem Mol Biol. 165:38-50 (2017).


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Pharmacogenomics of cytochromes P450 in breast cancer patients Pavel Soucek1,2, Viktor Hlavac1,2, Maria Kovacova3, Veronika Brynychova1,2, Karel Raus4, Katerina Kopeckova5, Jiri Gatek6, Radka Vaclavikova1,2

1Laboratory of Pharmacogenomics, Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic; 2Toxicogenomics Unit, National Institute of Public Health, Prague, Czech Republic; 3Third Faculty of Medicine, Charles University, Prague, Czech Republic; 4Department of Breast Services, Institute for the Care for Mother and Child, Prague, Czech Republic; 5Department of Oncology, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic; 6Department of Surgery, EUC Hospital and University of Tomas Bata in Zlin, Zlin, Czech Republic. Introduction. Breast cancer still belongs to the most frequent cancers in women. Efficacy of breast cancer therapy is influenced by a number of cellular processes that in some cases lead to the tumour resistance. Most currently used cytotoxic drugs are metabolized by biotransformation enzymes in liver and extrahepatic tissues. Consequently, germline genetic variability in biotransformation enzymes is considered as important factor determining individual patient sensitivity to an administered drug. Although cytochromes P450 (CYPs) constitute a major group of drug (in)activation enzymes, comprehensive germline genetic variability screen of these enzymes in breast cancer patient cohorts is virtually missing. Aims. The aim of our study was to explore germline genetic variability of panel consisting of all known human CYP genes and evaluate their predictive and prognostic value in breast cancer patients. Methods. All exons with short overlaps into intronic sequences in both directions of 57 CYP genes were sequenced using massive parallel sequencing in blood DNA from 105 breast cancer patients in the testing phase. Variants with minor allele frequency (MAF) over 0.05 were further prioritized for validation phase based on newly developed decision tree using emerging in silico tools and pharmacogenomic databases for functional predictions and associations with response to cytotoxic therapy or disease-free survival of patients. Putative variants identified in the testing phase were used for validation in 805 patients with clinical follow up using KASPTM technology. Results. A total of 1622 variants were identified of which 120 were novel variants (without rs number in dbSNP build 151) in the testing phase. Variants in CYP2D6 (rs1065852 and rs28371725) and CYP4F12 (rs12460651) significantly associated with the response of tumours to preoperative cytotoxic therapy while those in CYP2C9 (rs1934969) and CYP2E1 (rs2515641 and rs2070677) associated with disease-free survival after adjuvant therapy. These variants were genotyped by different technology in a large group of patients in the validation phase, but failed to replicate. Discussion. The modest size of the testing set may be seen as a limitation of the study. Due to this fact, the importance of very rare (MAF<0.001) and rare (<0.01) variants could not be addressed. On the other hand, ethnical homogeneity and completeness of clinical follow up is considered beneficiary. Moreover, study may be extended by addition of more patients or compiling with similarly designed sets of patients with whole exome or genome data. Conclusion. Germline variability within a panel consisting of all CYP genes was assessed through massive parallel sequencing in breast cancer patients of Czech origin for the first time. Although several variants significantly associated with prognosis or therapeutic response on the small scale, none of these relationships was replicated in large cohort of patients. The study was funded by the Czech Medical Council project no. 17-28470A, Charles University project no. GAUK 1776218, National Sustainability Program I project no. LO1503 and The National Centre for Medical Genomics project no. CZ.02.1.01/0.0/0.0/16_013/0001634.


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The association of CYP2D6(C100T) allele and blood lipid levels with plasma endoxifen level in breast cancer patients in Taiwan Ta-Chung Chao1, Wen-Chi Pan3, Yi-Fang Tsai1, Yu-Rong Liu4, Yueh-Ching Chou2, 9, Sheng-Fan Wang2, Yi-Kai Wang2, Ying-Jen Chen7,8, Yune-Fang Ueng 4,5,6,10* 1Departments of Oncology and 2Pharmacy, Taipei Veterans General Hospital, Taipei, Taiwan; 3Institute of Environmental and Occupational Health Sciences, School of Medicine, National Yang-Ming University, Taipei, Taiwan; 4National Research Institute of Chinese Medicine, Taipei, Taiwan; 5Department and Institute of Pharmacology, School of Medicine, and 6Institute of Biopharmaceutical Sciences, School of Pharmacy, National Yang-Ming University, Taipei, Taiwan; 7Division of General Internal Medicine and Geriatrics, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan City, Taiwan; 8Chang Gung University, College of Medicine, Taoyuan City, Taiwan; 9Department of Pharmacy and 10Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan. Introduction. Poorer therapeutic outcome was reported in breast cancer patients with high cholesterol biosynthesis signature. Cytochrome P450 (CYP) 2D6 is crucial in the metabolic activation of tamoxifen to generate 4-hydroxytamoxifen and endoxifen. CYP2D6 haplotypes with C100T substitution encode null or poor functional proteins. Dosage adjustment has been recommended in patients with plasma endoxifen level below 5.97 ng/mL. Aims. This study aims to examine the association of genotypes and serum cholesterol with plasma drug levels in patients. Methods. Breast cancer patients (women) taking tamoxifen for more than one month were recruited in this study. C100T genotypes of CYP2D6 were analyzed by DNA sequencing. Blood lipid levels and drug concentrations were determined. Results and Discussion. Taiwanese patients with high body mass index (BMI) (> 40) and high serum cholesterol (> 200 mg/dL) had an increased risk of an ineffective endoxifen level. Plasma tamoxifen concentration was positively associated with serum triglyceride concentration, but not BMI or cholesterol level. Compared to the low-cholesterol group, the high-cholesterol group had a lower 4-hydroxytamoxifen or endoxifen level in C100T carriers. Compared to the low-cholesterol group, T/T carriers with high cholesterol levels had an increased risk of an ineffective endoxifen level. Patients with hot flash/flushing did not have higher levels of tamoxifen or its metabolites. It is suggested that increased doses of tamoxifen might not increase the hot flash risk. Further studies of a large hypercholesterolemic population should be conducted to examine the outcome of increased doses of tamoxifen. Conclusion. These results reveal that high serum cholesterol could be an additive risk factor for ineffective endoxifen levels in overweight and C100Tallele-carrying patients.


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The expression of cytochrome P450 in the liver is regulated by the GluN2B subunit NMDA receptor antagonist CP-101,606 Ewa Bromek , Anna Haduch, Marta Rysz, Renata Pukło, Władysława A. Daniel Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland. Introduction. The most important physiological regulators of the cytochrome P450 expression in the liver comprise such hormones as growth hormone, thyroid hormones (triiodothyronine T3 and thyroxine T4), glucocorticoids, and sex hormones, which, via activation of the membrane, cytoplasmic or nuclear receptors, regulate the transcription of CYP. The hypothalamus, a centre of neuroendocrine regulation, is also heavily innervated by glutamatergic system, where signals are transmitted through different glutamate receptors. A detailed engagement of individual glutamate receptors in hormonal regulation has not been fully recognized so far. Aims. The aim of the present study was to test the effect of selective GluN2B subunit NMDA receptor antagonist (a potential antidepressant drug) on the cytochrome P450 expression and activity in the liver. Methods. The studies included five-day administration of the selective GluN2B subunit NMDA receptor antagonist: CP-101,606 (20 mg/kg ip.), followed by determination of cytochrome P450 activity in liver microsomes using CYP enzyme specific reactions (HPLC). The pituitary GHRH level and hormone levels were measured by ELISA. The protein level was assessed by Western Blot. Results. The repeated, administration of CP-101,606 significantly decreased the activity of CYP2A, 2B, 2C11 and CYP3A measured as a rate of testosterone hydroxylation in specific positions. The activity of the CYP1A (caffeine 3-N-demethylation) and CYP2D (1’-bufuralol hydroxylation) was also significantly lower compared to the control group. In case of CYP2C11, CYP3A and CYP2B, the protein level showed similar tendency as the changes of the activity. However, considering CYP2A and CYP2D there were discrepancies between activity and protein amount. Pituitary GHRH and serum corticosterone hormone levels were significantly diminished. Discussion. The selective GluN2B subunit NMDA receptor antagonist CP-101,606 produced significant decreases in the activities of hormone-dependent cytochrome P450 enzymes and of CYP2D in the liver. The observed changes in the cytochrome P450 function are probably due to neuroendocrine regulation of enzyme by the investigated compound, since the level of GHRH and corticosterone were significantly altered. Conclusion. The tested NMDA receptor agonist affect liver cytochrome P450 expression and activity which may have an impact on the metabolism of drugs. Therefore further clinical studies are necessary to examine the effect of the glutamatergic system on cytochrome P450 activity in the liver of patients (metabolic tests) during therapy with future drugs acting as selective antagonists of glutamate receptors. (This study was financially supported by the OPUS 12 grant no 2016/23/B/NZ7/02283 from the National Science Centre, Kraków, Poland and by statutory funds from the Institute of Pharmacology, PAS, Kraków, Poland).


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The positive allosteric modulator of mGlu5 receptor (VU 0360172), but not the mGlu2/3 receptor agonist (LY354740) affects liver cytochrome P450 Wladyslawa A. Daniel, Ewa Bromek, Anna Haduch, Renata Pukło, Marta Kot Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland. Introduction. Cytochrome P450 is physiologically regulated by the hypothalamic-pituitary-adrenal axis, the hypothalamic-pituitary-thyroidal axis, and the so-called hypothalamic-pituitary-liver axis. These axes are under the control of the brain nervous system. Our earlier studies showed an important role of monoaminergic systems in the neuroendocrine regulation of liver cytochrome P450 expression. However, a role of glutamate has not been studied in this respect, though the hypothalamus is heavily innervated by glutamatergic system and glutamate receptors are present also in the liver. Aims. The aim of our study was to investigate the effect of the specific positive allosteric modulator of mGlu5 receptor VU 0360172 and the mGlu2/3 receptor agonist LY354740 (potential antipsychotic drugs) on the regulation of cytochrome P450 in the liver. Methods. Male Wistar Han rats were administered intraperitoneally VU 0360172 or LY354740 (30 or 10 mg/kg, respectively) for 5 days. The following analyses were performed: (1) activities of cytochrome P450 enzymes in liver microsomes, based on velocity of specific metabolic reactions (HPLC); (2) cytochrome P450 enzyme protein levels in liver microsomes (Western blot); (3) mRNA levels of CYP genes in the liver (qRT-PCR); (4) serum hormones and interleukins (growth hormone, corticosterone, thyroid hormones T3 and T4, testosterone; IL-2, IL-6) using ELISA method. Results. The specific mGlu5 receptor positive allosteric modulator VU 0360172 increased the activity of CYP enzymes: CYP2B, CYP2C11 and CYP3A (testosterone hydroxylation at positions 16β; 2α and 16α; 2β and 6β, respectively), but did not affect that of CYP1A (caffeine 8-hydroxylation and 3-N-demethylation), CYP2C6 (warfarin 7-hydroxylation) or CYP2D (bufuralol 1’-hydroxylation). The CYP2C11 protein level was decreased and adequate mRNA level tended to do so. The protein levels of CYP2B1/2, CYP3A1 and CYP3A2, as well as serum hormone and interleukin concentrations were not significantly changed. The activities of the investigated CYP enzymes were not affected by the mGlu2/3 receptor agonist LY354740. Discussion. The observed increases in the activities of CYP2B, CYP2C11 and CYP3A (main rat CYP pool) by the specific mGlu5 receptor positive allosteric modulator VU 0360172, which did not correlate with protein and mRNA levels or serum hormone and interleukin concentrations, suggest some posttranslational modifications by the parent compound or its metabolites. Conclusion. The results obtained indicate increased activities of liver CYP2B, CYP2C11 and CYP3A by the specific mGlu5 receptor positive allosteric modulator VU 0360172, which may have an impact on the metabolism of steroids and drugs. On the other hand, the mGlu2/3 receptor agonist LY354740 does not change the activity of the investigated CYP enzymes in the rat. (This study was financially supported by the OPUS 12 grant no 2016/23/B/NZ7/02283 from the National Science Centre, Kraków, Poland and by statutory funds from the Institute of Pharmacology, PAS, Kraków, Poland.)


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Nutritional modulation of intestinal cytochrome P450 enzymes in broiler chicken Gábor Mátis1, Anna Kulcsár1, Andor Molnár2, Máté Mackei1, Károly Dublecz2, Zsuzsanna Neogrády1 1Division of Biochemistry, Department of Physiology and Biochemistry, University of Veterinary Medicine, Budapest, Hungary; 2Department of Animal Sciences, Georgikon Faculty, University of Pannonia, Keszthely, Hungary.

Introduction. The short chain fatty acid butyrate is produced in the hindgut and is widely used as a natural growth promoting feed additive in monogastric animal farming, particularly in poultry nutrition. Based on its epigenetic effects, butyrate can modify the expression of certain genes, such as those of drug-metabolizing cytochrome P450 (CYP) enzymes. It was described in our previous studies that orally applied butyrate modulated the expression of CYPs in the liver of broiler chickens. Beside hepatic CYP-mediated drug metabolism, intestinal CYP enzymes are also involved in the biotransformation of orally ingested xenobiotics by forming a primary metabolic barrier, thus their dietary modulation can be of high importance.

Aims. The main goal of this study was to investigate the effects of the dietary cereal type (wheat vs. maize) and oral butyrate supplementation on the activity of intestinal CYP enzymes in chicken. Wheat contains high amount of soluble non-starch polysaccharides (NSP), providing substrates for the enhanced caecal microbial butyrate production, in contrast to maize as a crop with lower NSP content.

Methods. Ross 308 broilers (n=22/group) were fed for 6 weeks with maize-based or wheat-based diet, referring to lower or higher dietary NSP levels. Diets were supplemented with non-protected or protected forms of butyrate. In order to study the effects of butyrate on intestinal drug metabolism, at first the kinetics of butyrate was assessed from the ingesta of various gut sections and from portal blood by gas chromatography. The activity of CYP1A2, CYP2H2 and CYP3A37 was measured from the duodenal mucosa with luminescent assays. Data were analyzed by two-way ANOVA and pairwise comparison using the R 2.14.0 software.

Results. Butyrate concentration in the ileum was significantly increased by protected butyrate, while NSP-rich wheat-based diet elevated caecal butyrate levels. According to differing absorption sites, butyrate of different origin significantly increased the butyrate concentration in various vessels of the hepatic portal system. Duodenal CYP1A2 and CYP2H2 activities were significantly increased by non-protected butyrate supplementation and by wheat-based diet, while CYP3A37 was affected only by the dietary cereal type.

Discussion. Based on our results it can be assumed that the protected form of butyrate could reach the ileum due to the prolonged butyrate release. Wheat-based diet successfully stimulated the microbial butyrate production in the caecum, reflected by the elevated caecal butyrate concentration. Based on the diet-driven microbial butyrate production, wheat-based diet remarkably stimulated all the investigated intestinal drug-metabolizing CYP enzymes; CYP1A2 and CYP2H2 activities were also increased by the orally applied non-protected butyrate. This butyrate-triggered enhanced intestinal xenobiotic biotransformation may have important practical implications by altering the kinetics of orally administered drugs and further xenobiotics.

Conclusion. The observed stimulatory action of butyrate of different origin on intestinal CYPs can be of high importance from food safety and pharmacological point of view by possibly modifying the metabolism of simultaneously applied drugs or toxicants derived from the feed.

The study was supported by the CEPO (Centre of Excellence for Poultry) project, funded by the European Regional Development Fund, Cross-border Cooperation Programme Austria-Hungary and by the 17896-4/2018/FEKUTSTRAT grant of the Hungarian Ministry of Human Capacities.


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Investigations on hepatic and intestinal cytochrome P450 enzymes in wild boar and domestic pig Kata Orbán1, Ádám Kurucz1, Máté Mackei1, Hedvig Fébel2, Zsuzsanna Neogrády1, Gábor Mátis1 1Division of Biochemistry, Department of Physiology and Biochemistry, University of Veterinary Medicine, Budapest, Hungary; 2Research Institute for Animal Breeding, Nutrition and Meat Science, National Agricultural Research Center, Herceghalom, Hungary. Introduction. Drug-metabolizing cytochrome P450 (CYP) enzymes are of special importance in wild animals, directly exposed to environmental pollutants as potential CYP inducers or inhibitors. Concerning hepatic CYPs of hunted game species in Central Europe, only limited data are available regarding wild ruminants, while absolutely no information can be found about wild boar and with regard on the intestinal drug metabolism in any wild species. Aims. In the present research, we aimed to monitor the activity of certain CYP enzymes playing key role in xenobiotic biotransformation in wild boar and, as a comparative study, in domestic pig. Further, various factors (such as age, sex, sexual maturation, season) were considered as possibly modulating CYP function in wild boar. Methods. Liver and intestinal mucosa (from duodenum, jejunum, ileum and caecum) samples were freshly collected from 49 hunted wild boars and 15 wild boar fetuses in Western Hungary, while domestic pig samples (n=40) were gained from a slaughter house. The microsome-enriched post-mitochondrial supernatant containing CYP enzymes was isolated after homogenization of tissue samples by a multi-step differential centrifugation. Specific activity of CYP1A2, CYP2C9 and CYP3A4 enyzmes was assessed by luminometric P450-Glo assays. Results. The activity of hepatic CYP1A2 enzyme was significantly (P=0.008), approx. 4-fold higher in wild boars than in domestic pigs. Similarly, the activity of CYP3A4 was found to be significantly (P<0.001), approx. 8-fold increased in the liver of wild boars when compared to those of domestic pigs. In contrast, hepatic CYP2C9 had a significantly (P<0.001), 50% lower activity in wild boars than in domestic pigs. The activity of all intestinal CYPs was under detection level in both species in all intestinal sections. The modulatory role of certain investigated factors (sex, sexual maturation and season) on hepatic CYP activity was also confirmed in wild boars. The activity of hepatic CYP2C9 and CYP3A4 was detectable in wild boar fetuses, but their activities were remarkably lower than those of adult animals. Discussion. According to our results, great differences were found in the activity of hepatic drug-metabolizing CYP enzymes between wild boars and their domestic counterparts. The described species-related alterations might be in connection with the different exposure of wild and domesticated animals to specific CYP modulators, taken up from the environment or with the diet. For instance, it can be hypothesized that pesticides, such as organophosphates may have induced CYP1A2 and CYP3A4 activity, while flavonoids as inhibitors could have decreased the CYP2C9 activity in wild boars. However, further studies are required to assess xenobiotic exposures related to CYP-mediated drug metabolism in wild animals. Conclusion. As the activity of CYPs in wild boars can be highly affected by environmental pollutants, CYP enzymes may also serve as ecotoxicological markers in wild game species, reflecting the presence of agricultural or industrial toxicants. Investigating CYP-related drug metabolism in wildlife species may clarify some possible toxicokinetic interactions, thus having huge importance in the production of safe game meat, being free of toxic residues. The study was supported by the grants EFOP-3.6.3-VEKOP-16-2017-00005 and 17896-4/2018/FEKUTSTRAT of the Hungarian Ministry of Human Capacities.


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Stilbene compound trans-3,4,5,4´-tetramethoxystilbene regulates constitutive androstane receptor (Car) target genes, but does not possess proliferative activity in mouse liver Jan Dusek1, Josef Skoda1, Alzbeta Horvatova1, Albert Braeuning3, Ondrej Holas2, Petr Pavek1 1Department of Pharmacology and Toxicology, 2 Department of Pharmaceutical Technology, Faculty of Pharmacy, Charles University, Ak. Heyrovského 1203, Hradec Kralove, 500 05, Czech Republic, EU. 3Department of Food Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589, Berlin, Germany, and Department of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074, Tübingen, Germany. Introduction. The constitutive androstane receptor (CAR) is a primary regulator of drug detoxification. CAR activation is connected with mitogenic effects leading to liver hyperplasia and tumorigenesis in rodents. Therefore currently known mouse CAR activators, including phenobarbital and the potent agonist TCPOBOP (1,4-bis[2–(3,5-dichloropyridyloxy)]benzene), are considered rodent non-genotoxic carcinogens. Recently, the stilbenoids resveratrol and trans-3,4,5,4´-tetramethoxystilbene (TMS) have been shown to alleviate N-nitrosodiethylamine/phenobarbital-induced liver carcinogenesis. Aims. Thus in the present work, we examined if TMS may be an inverse agonist of mouse CAR. Methods. We used luciferase reporter assays, in silico docking, primary human hepatocytes or we performed animal experiments with C57BL/6 mice. Results. Unexpectedly, we identified TMS as a novel moderate murine CAR agonist. This was consistently observed in in silico docking experiments, in in vitro analyses comprising reporter gene experiments, studies in mouse hepatocytes and AML12 hepatic cells, as well as in C57BL/6 mice in vivo. TMS significantly up-regulated Cyp2b10, Cyp2c29 and Cyp2c55 mRNAs, but down-regulated expression of genes involved in gluconeogenesis and lipogenesis such as Pck1, G6pc, Scd1, Acaca and Fasn to a similar degree as TCPOBOP. Importantly, TMS did not promote EdU incorporation in AML12 cells, did not increase liver weigh and had no statistically significant effect on Ki67 and Pcna labeling indices in mouse liver in vivo. In line with that, TMS did not induce genes involved in liver proliferation or apoptosis such as Mki67, Foxm1, Myc, Mcl1, Pcna, Bcl2, Bax or Mdm2 in mice, but slightly up-regulated Gadd45β mRNA. Discussion. We conclude that TMS is a mouse CAR ligand with limited effects on hepatocyte proliferation, while at the same time controlling CAR target genes involved in xenobiotic and endobiotic metabolism. Since TMS is a potential anticancer drug, further studies should clarify the Car-dependent and Car-independent effects of TMS on hepatocyte proliferation. All these data suggest that TMS is a novel Car ligand without proliferative activity in C57BL/6 mice. Funded by GAČR 19-14497S to P.P.


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Human CYP1B1 protects cancer cell apoptosis through TRAIL-FOXO3-Skp2 pathway Yeo-Jung Kwona, Dong-Jin Yea, Hyoung-Seok Baeka, Tae-Uk Kwona, Eunah Choa, Donghak Kimb and Young-Jin Chuna aCollege of Pharmacy and Center for Metareceptome Research, Chung-Ang University, Seoul, Korea; bDepartment of Biological Sciences, Konkuk University, Seoul, Korea. Human cytochrome P450 1B1 (CYP1B1) is an important enzyme that catalyzes metabolism of 17β-estradiol (E2) to catechol estrogens, including carcinogenic metabolite 4-OHE2. CYP1B1 overexpression occurs frequently in many cancer cells and CYP1B1 may play a crucial role in estrogen-mediated carcinogenesis. However, the detailed mechanism of CYP1B1-dependent carcinogenesis remains unclear. Here, we investigated the association between TRAIL, FOXO3, Skp2, and CYP1B1 expression in human tumor cells, including MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 can inhibit expression of TRAIL and the death receptor DR4/5 to prevent apoptosis. Furthermore, knockdown of CYP1B1 by shRNA promotes TRAIL-induced apoptosis and inhibits MDM2. Interestingly, we found that CYP1B1 knockdown can induce FOXO3A expression and nuclear translocation. Phosphorylation of FOXO3 at Ser7, which may promote nuclear translocation was also significantly increased by CYP1B1 deficiency. In addition, the expression of Skp2, an F-box protein of the SCF E3 ligase complex, was significantly inhibited by CYP1B1 knockdown. Treatment with TRAIL in CYP1B1-overexpressed cells markedly interrupted the effect of CYP1B1 on FOXO3, Skp2, and p27. Furthermore, we also identified that FOXO3 directly modulates Skp2 expression through binding to the promoter region and subsequently suppressing the transcription level of Skp2. Taken together, these data suggest that CYP1B1 protects cancer cells from apoptosis through induction of Skp2 and suppression of TRAIL and FOXO3. Funding Source: This work was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (Grant No. NRF2017R1E1A1A01074032 and NRF2015R1A5A1008958). Disclosure Statement: None of the authors have any conflicts of financial interest to declare.


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The Study of cytochromes P450 from environmentally sensitive species Emily Fox1, Hazel M Girvan1, Kirsty J Mclean1, Sarah J Matthews1, Richard Tunnicliffe1, Harshwardhan Poddar1, Marina Golovanova1 and Andrew W Munro1 1Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN, UK. Introduction. Cytochromes P450 (CYPs) in many species are responsible for catalyzing the oxidation of numerous toxic substrates. The Environmental Protection Agency (EPA) estimated that there are more than 85,000 chemicals which are classified under the Toxic Substances Control Act (TCSA)1. Current regulations require chemicals and their metabolites to be assessed for their potential harm within the environment through direct/non-direct exposure. Determining the functional similarities and differences of species-specific membrane-bound cytochromes P450 and their response to environmental toxins will help demonstrate the versatility of these enzymes. This will highlight the hazards presented by environmental toxins and how they interact with these species-specific CYPs. Obtaining this information will produce a predictive model of toxicity, eliminating the requirement of traditional models to determine the harm and risk of a product. This will be of benefit to several industries. Aims.

• Express and characterize CYP enzymes form environmentally sensitive species.

• Develop toxicity profiles to inform the environmental risks of toxins. Methods. Generally, membrane bound cytochromes P450 are difficult to express in bacterial expression systems and the previous data are very limited for the expression of fish CYP enzymes. However, the equivalent proteins in Homo sapiens have been successfully expressed using this system. Through an iterative process, the truncated CYPs have been successfully expressed using E. coli and soluble protein has been obtained by using a range of additives in the buffers. The activity and functional properties of the proteins have been compared to their equivalent membrane-bound proteins using previous data in the literature. Results and Discussion. Here we show successful overexpression and purification of recombinant trout CYP1A in E. coli. CYP1A shows a typical UV-visible absorbance spectrum with Soret maximum at 424 nm. It is capable of binding CO in the ferrous state again giving a typical shift to 450 nm with a large P420 feature at 420 nm. A range of substrates and inhibitors including caffeine, ciprofloxacin, melatonin and naproxen have been tested for binding to CYP1A by UV-visible absorption and EPR. Stability has been investigated by calorimetric methods and data will be presented. Various biophysical techniques have been utilized to analyze the structure of the proteins which will also be presented. Conclusion. Cytochromes P450 can theoretically be used as biochemical markers, since defining their function and mechanism of action (including their metabolites) for known toxins will provide an in vitro determination of such hazards. The development and use of in vitro and predictive models to determine the risk of chemicals has increased recently, moving away from traditional animal model systems as society becomes more aware of their environmental impact and ethical issues. The fish species selected are widely employed as bioindicator species due to their sensitivity to changes in their habitat. Therefore, studying their CYP enzymes will provide data to determine the harm and risks of these changes. These data can be used to produce a predictive model which will allow industries to determine the risk and harm of a product or by-products using these enzymes. Isolation of microsomal CYPs orthologous to known human CYPs and analyzing their function will be of considerable interest to several industries. 1. Erikson, B. E., How many chemicals are in use today? EPA struggles to keep its chemical inventory up to

date. Chem. Eng. News, February 2017, 95 (9), 23-24.


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Localization and quantification of cytochrome P450 27C1 in human skin Sarah M. Glass1, Ambra Pozzi2, F. Peter Guengerich1 1Deparment of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA; 2Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA and Veterans Affairs Medical Center, Nashville, TN, USA. Introduction. Cytochrome P450 27C1 (CYP27C1) is preferentially expressed in the skin and catalyzes the desaturation of all-trans retinol to 3,4-dehydroretinol in vitro. The catalytic activity of CYP27C1 is dependent on the additional of the mitochondrial electron transfer partners adrenodoxin and adrenodoxin reductase. Many aspects of the CYP27C1 catalytic mechanism are well understood, but questions remain regarding its localization and biological function in humans. Aims. (1) Assess localization of CYP27C1 within the skin and (2) determine cellular concentrations of CYP27C1 and required catalytic partners. Methods. Localization of CYP27C1 within the skin is assessed by tissue immunofluorescence and by immunoblotting separated skin components, including cultured primary keratinocytes. CYP27C1 and P450 catalytic partners (adrenodoxin, adrenodoxin reductase, cytochrome P450 reductase, and cytochrome b5) are quantified in the skin using a LC-MS/MS quantification concatemer (QconCAT) strategy. Results. Within the skin, CYP27C1 is localized to the basal keratinocytes of the epidermis. Primary keratinocytes continue to express CYP27C1 through at least passage 7. With successful expression of an isotopically-labelled QconCAT, quantification of CYP27C1 and P450 catalytic partners can be assessed simultaneously within individual skin samples. Discussion. The localization of CYP27C1 is consistent with studies that show that the biosynthesis of 3,4-dehydroretinol from all-trans retinol occurs in keratinocytes within the skin. Utilizing results from the LC-MS/MS quantification, the abundance of CYP27C1 can be compared with previously reported values in the skin from quantitative immunoblotting and physiological ratios of P450:catalytic partners within the skin can be determined. This is the first developed QconCAT to include peptides for quantification of all P450 catalytic partners and is the first time this strategy has been used to quantify a P450 outside of the liver. Conclusion. With the consistent expression of CYP27C1, primary human keratinocytes could serve as a potential system for probing the biological function of CYP27C1. CYP27C1 and P450 catalytic partners within the skin can be quantified using a QconCAT-based LC-MS/MS strategy.


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Differentiation of human iPS cells into enterocyte-like cells using cAMP-activating compounds and these functions Tamihide Matsunaga, Tomoki Kabeya, Shimeng Qiu, Takahiro Iwao Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan. Introduction. In drug disposition, the small intestine is involved in the absorption and metabolism of orally administered drugs. Therefore, the small intestine together with the liver is a key organ related to the first-pass effect of drugs. The Caco-2 cell line, a human intestinal epithelial cell model derived from a colon carcinoma, is commonly used to predict drug membrane permeability in the small intestine. However, Caco-2 cells differ from normal human small intestinal cells in the expression patterns of drug metabolizing enzymes and transporters. Therefore, there is no pharmacokinetic system that can simultaneously predict the intestinal metabolism and drug absorption. Aims. Human induced pluripotent stem (iPS) cells have the potential to form almost any type of cell. Recently, we found that cyclic adenosine monophosphate (cAMP) signaling promotes the differentiation of human iPS cells into enterocyte-like cells [1]. In this study, we aimed to clarify the effects of cAMP-activating compounds in the intestinal differentiation of human iPS cells and the pharmacokinetic functions of the differentiated enterocyte-like cells. Methods. Human iPS cells were differentiated into enterocytes according to the protocol established in our laboratory previously [1]. Human iPS cell-derived intestinal stem cells were treated with small molecular compounds that activate cAMP signaling during the intestinal differentiation. After terminal differentiation, we analyzed the expression levels of intestinal specific markers and the pharmacokinetic functions in the differentiated enterocyte-like cells. Results and Discussion. The expression levels of intestinal markers and pharmacokinetics-related genes in the differentiated cells were significantly increased by using cAMP-activating compounds. In particular, the mRNA and protein expression levels of CDX2, a transcription factor involved in the differentiation into the intestine, were markedly increased. In the enterocyte-like cells differentiated with the compounds, the metabolic activities of CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, and UGT were significantly increased. The activity of PEPT1, a transporter involved in uptake transport of peptides from the lumen, was also markedly increased. Furthermore, in the enterocyte-like cells, CYP3A4 was significantly induced by 1α,25-dihydroxyvitamin D3 and rifampicin. CYP3A4/5 activity of the enterocyte-like cells was much higher than Caco-2 cells. These results suggested that the activation of cAMP signaling is useful for the promotion of intestinal differentiation. Conclusion. These results indicate that the activation of cAMP signaling contributes to the intestinal differentiation and maturation of human iPS cells. 1. Kabeya T. et al., Cyclic AMP signaling promotes the differentiation of human induced pluripotent stem

cells into intestinal epithelial cells. Drug Metab. Dispos., 46:1411-1419, 2018.


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Comparative study of 25-hydroxyvitamin D3 treatment between Cyp27b1 knockout and Vdr (R270L) rats to evaluate clinical effects of 25(OH)D3 Miyu Nishikawa, Keisuke Abe, Kaori Yasuda, Hiroki Mano, Shinichi Ikushiro, Toshiyuki Sakaki Fac. of Engineer, Toyama Pref. Univ., Imizu, Toyama, Japan. Introduction. Vitamin D3 plays important roles in osteogenesis and calcium (Ca) homeostasis. The active form of vitamin D3, 1 , 25-dihydroxyvitamin D3 (1,25D3), is generated from 25-hydroxyvitamin D3 (25D3) by CYP27B1 in the kidney. In the previous study, we showed that 25D3 acts as VDR ligand in vitro [1]. To evaluate the direct action of 25D3 in vivo, we administrated 25D3 to Cyp27b1 knockout (KO) mice, in which plasma 1,25D3 was under-detectable. Surprisingly, Cyp27b1 independent generation of 1,25D3 was caused by the high dose of 25D3 in Cyp27b1-KO mice [2]. The results suggested the 25D3 might be useful as a prodrug to generate 1,25D3 in CYP27B1 deficient patient. However, we could not confirm the direct action of 25D3 in vivo. Aims. To evaluate the direct action of 25D3 via VDR, we generated genetically modified rats in Cyp27b1 or Vdr. Rat mutant Vdr (R270L), which corresponds to human VDR (R274L) from the rickets patient, has significantly decreased affinity toward 1,25D3. There might be no high-affinity ligands in the Vdr (R270L) rats. Thus, Vdr (R270L) rats might be useful to evaluate VDR ligands without 1 -hydroxyl group such as 25D3. Methods. Cyp27b1-KO and Vdr (R270L) knocked-in rats were generated by CRISPR/Cas9 genome editing systems. These rats were raised with normal diet or 25D3 containing diet after weaning. Femur was analysed by computed tomography to determine bone mineral density (BMD). Plasma concentrations of bone metabolism parameters were also analysed. Plasma 25D3 and its Cyp24a1-dependent metabolites were determined by LC/MS/MS. Plasma 1,25D3 was determined by ELISA. Results. Cyp27b1-KO rats showed significantly decreased 1,25D3 with rickets symptoms including decreased BMD in the femur and hypocalcemia. Treatment of 25D3 fully normalized BMD and plasma Ca without hypercalcemia, which is a typical adverse effect of 1,25D3 treatment. Normal level of 1,25D3 was detected in the plasma of Cyp27b1-KO rats. Liver mitochondrial fraction prepared from Cyp27b1-KO rats converted 25D3 to 1,25D3. Vdr (R270L) rats also showed the rickets symptoms such as deceased BMD whereas plasma 1,25D3 was remarkably elevated, suggesting that the 1,25D3 could not act as high-affinity Vdr ligand. 25D3 treatment to the Vdr (R270L) rats dramatically improved BMD and plasma Ca. It was important notice that these ameliorations in Vdr (R270L) rats were observed in high plasma level of 25D3 and quite low level of 1,25D3. Discussion. Cyp27b1-KO rats fed 25D3 diet generated 1,25D3. 25D3 was converted to 1,25D3 in the liver mitochondrial fraction prepared from Cyp27b1-KO rats. We previously showed CYP27A1 had weak 1 -hydroxylation activity toward 25D3 [3] Taken together, the most probable candidate is Cyp27a1. 25D3 diet also ameliorated the rickets symptoms in Vdr (R270L) rats with decreased 1,25D3 and increased 25D3 plasma levels, suggesting that 25D3 acts as a Vdr ligand in Vdr (R270L) rats. Conclusion. These findings showed the clinical importance of 25D3 as following two points: (1) 25D3 is useful as a prodrug to generate active vitamin D form in the CYP27B1 deficient patients. (2) 25D3 may be act as an active VDR ligand in VDR (R274L) rickets patients. It was also revealed that Vdr (R270L) was useful as evaluation model of natural vitamin D and its derivatives. Thus, we now evaluate the activity of vitamin D analogues in Vdr (R270L) rats. [1] Munetsuna, E. et al. (2014). Mol Cell Endocrinol., 382, 960-970. [2] Nishikawa, M. et al. (2019). J Steroid Biochem Mol Biol., 185, 71-79. [3] Sawada, N. et al. (2000). Biochem Biophys Res Commun., 273, 977-984.


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Cytochrome P450 aromatase (CYP19A1) deficiency caused by a novel mutation in the NADPH cytochrome P450 oxidoreductase Amit V Pandey1,2, Shaheena Parween1,2, Sara Benito-Sanz3, Juan-Pedro López-Siguero4, Núria Camats5, Mónica Fernández-Cancio5, Sameer S Udhane1, Norio Kagawa6, Christa E Flück1,2, Laura Audí5 1Biomedical Research, University of Bern, Bern, Switzerland, 2Pediatric Endocrinology, University Children's Hospital Bern, Bern, Switzerland, 3Instituto de Genética Médica y Molecular, Hospital Universitario La Paz, Madrid, Spain, 4Pediatric Endocrinology Unit, Universidad de Málaga, Málaga, Spain, 5Growth and Development Research Unit, Autonomous University of Barcelona, Barcelona, Spain, 6School of Medicine, Nagoya University, Nagoya, Japan. Introduction. Cytochrome P450 oxidoreductase (POR) deficiency (PORD) is a form of congenital adrenal hyperplasia (CAH) and results in loss of steroid and drug metabolizing cytochrome P450 activities. Mutations in POR cause mild to severe forms of CAH with/without bone malformation. We report a novel POR Arg550Trp mutation identified in a 46, XX patient with signs of CYP19A1 deficiency. Child and mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. Sequencing of the CYP19A1 gene did not reveal any defects, and further analysis revealed compound heterozygous mutations c.70_71delTC/p.Leu25PhefsTer93 and c.1648C>T/p.Arg550Trp in POR. Aims. Analysis of molecular and biochemical basis of CYp19A1 deficiency due a novel POR variant. Methods. DNA was analyzed with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform. We analyzed the ability of POR wild-type (WT) and Arg550Trp to reduce ferricyanide, MTT, cytochrome c and activity towards the drug and steroid metabolizing cytochromes P450. The POR WT and Arg550Trp variants were expressed and produced as recombinant proteins in bacteria and combined with recombinant P450 proteins and small molecule substrates for enzyme assays. Results. We found severe effects of Arg550Trp mutation on activities with different substrates. Arg550Trp showed 41% of the WT activity in cytochrome c and only 7.7% activity towards the reduction of MTT. A 2.75 fold increase in Michaelis constant (Km) was observed in ferricyanide reduction assays compared to WT POR. Severe effect on NADPH binding by R550W mutation was observed using both cytochrome c and MTT as substrates with variable NADPH concentration. Assays of CYP19A1 activity showed loss of >90% activities using androstenedione as substrate. Discussion. The mutation Arg550Trp is located in the NADPH binding region of POR. Computational analysis predicted instability in the NADPH binding region of POR by R550W mutation, Computationally predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH were confirmed by experiments using recombinant proteins. Conclusion. These results suggest a pathological effect of POR R550W and a diagnosis of PORD in the patient with p.Arg550Trp/p.Leu25PhefsTer93 in POR.


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Exploring cytochromes P450-mediated diversification of the strigolactone structure via ancestral sequence reconstruction of the CYP711A family Marcos Hamborg Vinde1,2, Elizabeth M.J. Gillam2, Claudia E. Vickers1 1Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia QLD 4072, Australia. 2School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia QLD 4072, Australia.

Developing new methods to increase efficiency of agricultural crops has become a key objective in order to meet the agricultural demands of a growing world population without constantly disrupting existing ecosystems by expanding farmland. Strigolactones belong to a structurally diverse group of terpene phytohormones which, in addition to their involvement in regulation of secondary growth and plant stress signalling, also function as germination signals to certain parasitic weeds and as recruitment signals for symbiotic arbuscular mycorrhiza interactions1. Thus, potential agricultural uses of strigolactones include beneficial modifications to plant architecture, improvement of crop resilience, suicidal seed germination of parasitic weeds, and improvement of interactions with soil probiotics. To date over 25 different naturally-occurring strigolactones have been identified and more are still being discovered. While little is known about the enzymes responsible for diversification of the strigolactone structure, the cytochromes P450 from the CYP711 family play a key role. Known catalytic activities of CYP711As in the strigolactone pathway include: 1, the sequential oxidation of the common precursor carlactone C19 to form carlactonic acid; 2, the hydroxylation of carlactone C18 initiating the B-C ring closure to form the canonical strigolactone ABCD-ring structure of 4-deoxyorobanchol (4DO); and 3, the hydroxylation of 4DO to yield orobanchol2 (See Figure). While P450s are expected to be responsible for much of the diversity among the canonical strigolactones, the hydroxylation of 4DO by CYP711A is still the only known occurrence of P450s in strigolactone diversification. The aim of this study was to further elucidate the mechanisms behind the diversification of the canonical strigolactone structure by exploring the evolution of the CYP711A family. Ancestral versions of CYP711A were inferred and characterized to provide a deeper understanding of how this enzyme family evolved, and its impact on diversity of strigolactones. A related objective of this project is to incorporate ancestral CYP711As with interesting activities into a microbial production platform for strigolactones, solving the challenge of otherwise limited access to these compounds. 1. Waters, M. T. et al. (2017). Strigolactone Signaling and Evolution. Annu. Rev. Plant Biol., 68, 291-322. 2. Yoneyama, K. et al. (2018). Conversion of carlactone to carlactonoic acid is a conserved function of MAX1

homologs in strigolactone biosynthesis. New Phytol., 218, 1522-1533.


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Ancestral reconstruction of cytochrome P450 family 1: improved thermostability for biocatalysis and insights into CYP1 evolution Kurt L Harris1, Yosephine Gumulya1, Gabriel Foley1, Parnayan Syed1, Tomasz Janosik2, Ann-Sofie Sandinge3, Ulrik Jurva 3, Shalini Andersson4, Mikael Bodén1, Elizabeth M J Gillam1 1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia, 2RISE Surface, Process and Formulation, Forskargatan 18, Södertälje, Sweden, 3DMPK, Early Cardiovascular, Renal and Metabolism, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden, 4Discovery Sciences, R&D BioPharmaceuticals, Astrazeneca, Gothenburg, Sweden. Introduction: While cytochrome P450 family 1 (CYP1) enzymes play an important role in the detoxification of xenobiotic and environmental compounds, their historical function and subsequent diversification remains a mystery. Ancestral sequence reconstruction (ASR) is a bioinformatic approach to resurrect ancestral proteins, many of which have been found to demonstrate higher thermostability than extant forms. Aims: To investigate the physical and functional properties of ancestors belonging to the vertebrate CYP1ABCD clade in order to explore their evolution and potential biocatalytic applications. Methods: ASR was carried out using a maximum-likelihood, joint reconstruction approach. Eleven ancestors were reconstructed, synthesized and expressed as recombinant proteins in E. coli. Bacterial membranes were used for catalytic and thermostability analyses. Results: Most ancestors demonstrated significant increases in thermostability of up to 30 °C over the corresponding extant forms. Thermostability correlated with the evolutionary age of the enzymes. However, ancient forms generally showed less activity than younger forms towards typical CYP1 substrates. Caffeine was primarily metabolised by human (h) CYP1A1/2 and corresponding mammalian ancestors of CYP1A forms. CYP1A and CYP1B ancestors metabolised estradiol to 2- and 4-hydroxy metabolites. However, CYP1B1 ancestors showed different regioselectivity towards estradiol to hCYP1B1. The aryl hydrocarbon receptor (AhR) ligand and potential endogenous substrate of CYP1 forms, 6-formylindolo-3,2-b-carbazole (FICZ), was metabolised by all forms to numerous products, including several metabolites not seen previously. Interestingly, the mammalian CYP1A2 ancestor showed markedly broader substrate specificity and generally higher turnover towards most substrates tested than all other enzymes investigated. Discussion: The oldest ancestor is estimated to date from ~500 million years ago, a time when global temperatures did not differ much to the present day, so we propose that the increased stability of the ancestral CYP1 forms is a carry-over from more ancient enzymes present in thermophilic organisms. Since caffeine was not metabolised by older ancestors, caffeine metabolism by CYP1A forms may be a recent evolutionary event, which is consistent with the recent evolution of caffeine synthesis in plants. The fact that CYP1B1 ancestors produced both 2- and 4-hydroxyestradiol suggests that the pronounced regiospecificity of mammalian forms also emerged recently. The diversity of FICZ metabolism shown by CYP1 ancestors suggests that CYP1s play an important role in FICZ metabolism across all species. Conclusion: The varied activities of the ancestors provide insights into the functional evolution of the CYP1 family whereas the high thermostability of these proteins makes them ideal scaffolds to engineer for biocatalytic applications.


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Ancestral sequence reconstruction of the CYP2U subfamily Connie Ross1, Gabriel Foley1, Mikael Boden1 and Elizabeth Gillam1 1School of Chemistry and Molecular Biology, University of Queensland, Brisbane, Australia. Introduction. CYP2U1 is an orphan human cytochrome P450 and is proposed to have an important, but presently unknown, endogenous function in humans. It is highly conserved in the vertebrate lineage and rare mutations in CYP2U1 have been shown to cause a hereditary form of spastic paraplegia in humans (Dhers et al., 2017). We sought to use ancestral sequence reconstruction (ASR) to investigate the evolutionary history of this cytochrome P450. Results from our lab suggest that ancestors from the CYP1, 3 families and selected CYP2 subfamilies are more heat-tolerant than their extant counterparts. This has not yet been explored for the CYP2U and CYP2R subfamilies. Since the CYP2U subfamily is generally considered the most basal of the CYP2 subfamilies (Kirischian et al., 2010), it is a good system for testing the validity of this hypothesis. Aims. Infer, resurrect, express and characterise the expression and thermostability of the ultimate and intermediate ancestors of CYP2U1 to determine what changes in protein structure and stability CYP2U1 has undergone throughout its evolutionary history. Methods. The vertebrate, fish, tetrapod and mammalian ancestors of CYP2U1 were inferred by ASR using multiple sequence alignments generated in MAAFT (L-INS-i), a phylogenetic tree generated in RaxML and the ASR program GRASP. Human CYP2U1 and ancestors were expressed in E. coli and thermostability was measured as described in (Gumulya et al., 2018). Results. The CYP2U ancestors were all successfully expressed in E. coli, producing a measurable P450 peak in whole cells. Although CYP2U ancestors show similar thermostability to the extant counterparts the ancestors lacking lineage-specific insertions showed increased thermostability compared to the ancestors with the insertions. Discussion. Unlike many of the ancestors of the CYP3 family and other CYP2 subfamilies that have been reconstructed, CYP2U1 does not appear to show increased thermostability with evolutionary age. However, CYP2U lineage-specific insertions do appear to decrease the overall thermostability of the ancestor. When these insertions are removed, the ancestors show a large increase in thermostability. It is unclear whether removing these insertions will affect the catalytic activity of the enzyme. Conclusions. The thermostability of CYP2U ancestors is similar to the extant human CYP2U1 protein. However, when lineage-specific insertions are removed, these ancestors are much more thermostable. Dhers, L., Ducassou, L., Boucher, J. L., et al., (2017). Cytochrome P450 2U1, a very peculiar member of the human P450s family. Cell Mol Life Sci, 74, 1859-1869. Gumulya, Y., Huang, W., D'cunha, S. A., et al., (2018). Engineering thermostable CYP2D enzymes for biocatalysis using combinatorial libraries of ancestors for directed evolution (CLADE). Chem Cat Chem. Kirischian, N., McArthur, A. G., Jesuthasan, C., et al., (2010). Phylogenetic and Functional Analysis of the Vertebrate Cytochrome P450 2 Family. Journal of Molecular Evolution, 72, 56-71.


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Investigation of structural residues required for metabolism of loganic acid by Camptotheca acuminata secologanic acid synthases (CYP72As) Justin C Miller1, Mary A Schuler2,3. 1Department of Chemistry, 2Department of Biochemistry, 3Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, U.S.A. Introduction. Strictosidine is a monoterpenoid indole alkaloid produced by the seco-iridoid pathway conserved in many medicinal plants. The identification of this pathway’s genes in vinca alkaloid-producing Catharanthus roseus (Cra) has shown that a number of CYPs are involved in generating secologanin from geraniol to form strictosidine—the last common intermediate in the pathway. Metabolic analyses of another medicinal plant, Camptotheca acuminata (Caa), have recently revealed divergence in the later steps of its pathway that alternately produces strictosidinic acid from secologanic acid. Based on Cra and Caa metabolomics, Caa appears to lack the methyl transferase required to produce loganin from loganic acid and, instead, utilizes an as-yet-unidentified CYP72A to transform loganic acid into secologanic acid. Aims. We initially aimed to identify the number of Caa secologanic acid synthases (SLAS) that transform loganic acid into secologanic acid. We next compared these with Cra secologanin synthase (SLS) using molecular modeling and ancestral protein reconstruction to identify key structural changes enabling the utilization of loganic acid instead of loganin. Methods. Caa and Cra cDNAs for relevant CYP72As were cloned into expression vectors and co-expressed with Arabidopsis thaliana P450 reductases in yeast or purified from E. coli and reconstituted with purified Caa P450 reductases. In vitro reaction products were assessed by HPLC, compared with authentic standards, and confirmed by LC-MS.

Results. Based on transcriptome and genome data, Caa has two closely related CYP72A candidates (CYP72A564, 72A565) that potentially metabolize loganic acid, and Cra has two known CYP72A variants (CYP72A1v1,v2) that process loganin but not loganic acid. Although multiple sequence alignments reveal multiple changes in the SRSs, it is the F131D132 of Cra SLSs positioned adjacent to loganin and the H131H132 of Caa SLASs that most likely account for these catalytic differences. When compared with other annotated CYP72As, the Caa SLASs and a number of putative SLSs form a single, monophyletic group. Ancestral sequence reconstruction of this data revealed that H131H132 is likely the ancestral state for all CYP72As in the strictosidine/strictosidinic acid-producing pathway. Discussion. That the Caa SLASs studied herein are distantly related to Cra SLSs is unsurprising given the differences in their substrates as well as the phylogenetic relationships of these plant species. What is surprising is that the H131H132 motif is predicted as the ancestral state for the SLS/SLAS lineage, although Caa SLASs are the only ones reported to cleave the C-C bond in unmodified loganic acid. Moreover, this suggests that the ancestral CYP72A for the SLS/SLAS and the 7-deoxyloganic acid hydroxylase (7DLH) lineages was probably able to hydroxylate 7-deoxyloganic acid to form loganic acid (7DLH-like) and subsequently transform loganic acid into secologanic acid(SLAS-like). Conclusion. We have identified two CYP72As from Caa that function as SLASs and are currently testing the importance of the H131H132

motif for utilizing loganic acid using site-directed mutagenesis and in vitro analyses.


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Heterologous expression of chalcone 3-hydroxylase from Dahlia variabilis in E. coli as a soluble enzyme Christian Molitor1, Julia Weissensteiner1, Johanna Hausjell1, Rosaria Gandini2, Christina Divne2, Oliver Spadiut1, Heidi Halbwirth1 1Institute of Chemical and Biological Engineering, Vienna University of Technology, Vienna, Austria; 2Department of Industrial Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden. Introduction. Chalcone 3-hydroxylase from Dahlia variabilis (DvCH3H) clusters in the CYP75 family containing the subfamilies of flavonoid 3’-hydroxylases (CYP75B) and flavonoid 3’,5’-hydroxylases (CYP75A). Chalcone 3-hydroxylases catalyze the ortho-hydroxylation of 6’-deoxychalcones in Asteraceae species [1]. Chalcones are precursors of aurones and both belong to the group of anthochlor pigments [2]. Even though the production of soluble CYPs lacking the membrane anchor is reported frequently [3], the production of a soluble form of a CYP75, has not been reported so far. Aims. The aim of this study is to produce and purify high amounts of a functionally active and soluble form of CH3H, lacking the membrane anchor. This will enable structural studies, which in turn will provide insights into the reaction mechanism and substrate specificity of this specialized CYP75. Methods. A truncated form of DvCH3H, lacking the N-terminal membrane anchor, was cloned into the pNIC-CTHO expression vector and was heterologously expressed in E. coli (BL21 (DE3)). The enzyme was purified by affinity chromatography, assayed for functional activity and characterized biochemically. Results. DvCH3H was expressed as a soluble protein and the purified enzyme showed functional activity. The enzyme’s photometric characterization revealed however, that only a portion of DvCH3H contained the prosthetic heme group. Discussion. The results demonstrate, that E. coli is a suitable host for the production of soluble DvCH3H, albeit the only partial occupancy of the heme group. The heme reconstitution of DvCH3H is currently in progress. Conclusion. The recombinant production of soluble DvCH3H in E. coli will enable its biochemical characterization and will also allow structural studies of this specialized enzyme. 1. Schlangen, K. et al. (2009). Chalcone 3-hydroxylation is not a general property of flavonoid 3′-

hydroxylase. Plant Sci., 177, 97-102. 2. Miosic, S. et al. (2013). 4-Deoxyaurone formation in Bidens ferulifolia (Jacq.) DC. PLoS ONE 8. 3. Bart, A. G. & Scott, E. E. (2018). Structures of human cytochrome P450 1A1 with bergamottin and

erlotinib reveal active-site modifications for binding of diverse ligands. J. Biol. Chem. 293, 19201-19210 Financial support: European Union Horizon 2020 (Marie Skłodowska-Curie: 675657), Austrian Science Fund (FWF): P 29552-B29.


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Dioscorea transversa CYP90B57 catalyses the C22R-hydroxylation of cholesterol, an essential step in steroidal saponin biosynthesis Lauren J. Salisbury, Stephen J Fletcher, Jeanette E. Stok, James R. Beckett, Margaret C. Noble, Joanne T. Blanchfield, James J. De Voss School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia. Steroidal saponins are a class of natural products that are prevalent in many plants and have been reported to possess a wide range of physiological effects. However, they are difficult to obtain pure through extraction/purification or synthesis and so their bioactivities have been difficult to characterise. Elucidating the biosynthetic pathway may facilitate eventual in vivo synthesis to produce these compounds for future studies and/or clinical application. It is hypothesised that steroidal saponins are biosynthesised from cholesterol. Hydroxylations at C-22, C-16 and C-26 (Fig. 1) are essential to generate the saponin core, and these reactions are thought to be performed by cytochromes P450. A previously assembled transcriptome of Dioscorea transversa, an Australian yam that produces a high concentration of steroidal saponins, revealed multiple candidate P450s for these hydroxylations (1, 2). One, a CYP90B homologue (CYP90B57) was hypothesised to perform C-22 hydroxylation of cholesterol in the plant.1 f Aim: This study will characterise and elucidate the function of CYP90B57, especially with respect to its role in saponin biosynthesis. Methods: The N-terminus of CYP90B57 was modified and the gene was codon optimised for expression in E. coli. Heterologous expression of CYP90B57 and required redox partners in E. coli were undertaken. After reconstitution of the activity of CYP90B57 in the presence of NADPH and its redox partner with a number of potential substrates, potential products were analysed by GCMS. Results: CYP90B57 was expressed to 81 nmol L-1, shown to bind cholesterol and oxidise it to (22R)-22-hydroxycholesterol (Fig. 2). Conclusion: Clarification of the substrate specificity and catalytic activity of CYP90B57 from D. transversa led to the conclusion that this P450 can stereospecifically hydroxylate C-22 of cholesterol, which is an essential step in steroidal saponin biosynthesis (Fig. 2). 1. Challinor, V. & Smith, D.M. & De Voss, J.J. (2011). Steroidal saponins isolated from an Australian Yam (Dioscorea sp.) Aust. J. Chem. 64:5, 545-549.

Fig. 1 1. 1β-hydroxyneogracillin and 2. Protoneogracillin: steroidal saponins isolated from Dioscorea transversa1.

Fig. 2 The role of D. transversa CYP90B57 in steroidal saponin biosynthesis.


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Characterization of a full-size CYP75A in Euphorbia pulcherrima Julia Weissensteiner1, Daria Nitarska1, Christian Molitor1, Lisa Führer1, Johanna Hausjell1, Oliver Spadiut1, Heidi Halbwirth1 1Institute of Chemical, Environmental and Biosciences Engineering, Vienna University of Technology, Vienna, Austria. Introduction. Flavonoid 3',5'-hydroxylase (F3'5'H, CYP75A) [1] catalyzes the hydroxylation of various flavonoids in position 3' and 5' [2] yielding delphinidin based anthocyanins. Most Poinsettias (Christmas stars) show intense red bract color. Blue coloration is currently unavailable for poinsettia bracts and artificial staining is common, particularly in North America, underpinning the potential of a niche market for the biotechnologically breeding of blue poinsettias. Aims. The aim of this study is to identify the molecular reason of the functional inactivity of EpF3'5'H in order to explain why of blue colored Poinsettias are not available. Methods. Based on Euphorbia pulcherrima transcriptome studies, EpF3'H and EpF3'5'H transcript levels were determined by qPCR. The putative EpF3'5'H enzyme was heterologously expressed in yeast (INVSc1). Based on sequence alignments and homology models, site-directed mutagenesis was performed. Microsome preparations of the heterologous gene expressions were assayed for EpF3'5'H activity [3]. Results. Transcriptomic and phylogenetic analysis as well as gene expression studies revealed the presence of a putative EpF3'5'H clustering in the CYP75A subfamily transcript in poinsettia bracts. Even though the EpF3'5'H was successfully expressed, no enzymatic activity could be detected. Discussion. Various differences in the sequences and thereby structural differences were identified between EpF3'5'H and functionally active flavonoid 3'5'-hydroxylase from other sources. Alternatively, the gene encodes an enzyme with unknown functionality. Conclusion. We demonstrated that a functionally inactive F3'5'H is present in Euphorbia pulcherrima. The identification of the molecular reason of the inactivity will explain the absence of blue Christmas stars. [1] Werck-Reichhart, D. & Feyereisen, R. (2000). Cytochromes P450: a success story, Genome Biol., 1: 3003.3001-3003.3009. [2] Menting J. G. T., et al., (1994). Characterization of Flavonoid 3′,5′-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers, Plant Physiol., vol. 106, no. 2, pp. 633–642. [3] Schlangen et al., (2010). Cloning, functional expression, and characterization of a chalcone 3-hydroxylase from Cosmos sulphureus, J.Exp.Bot. 61, 3451-3459. Financial support: European Union Horizon 2020 (Marie Skłodowska-Curie: 675657), Austrian Science Fund (FWF): P 29552-B29, Christiana HÖRBIGER Award.


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Terpene-metabolising cytochromes P450 from Rhodococcus rhodochrous

Peter D. Giang, Jeanette E. Stok and James J. De Voss

aSchool of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia.

Introduction: Abundant throughout nature, terpenes are essential for life.1 Volatiles monoterpenes that are present in plants are the main components of essential oils and are used for insect attraction and predator repulsion. In addition to these roles in the plant, bacteria have been shown to utilise these reduced hydrocarbons as a sole source of energy and carbon, often initiated by oxidative processes catalysed by cytochromes P450. The initial monoterpene hydroxylation is the archetypal chemical transformation catalysed by bacterial P450s. Rhodococcus rhodochrous has been shown to grow on the monoterpenes p-cymene, R-limonene, and 1,8-cineole present in Eucalyptus globulus.2

Aim: Characterise two P450s, P450TolR and P450TerpR found in the genome of R. rhodochrous and determine if they play a role in monoterpene biodegradation.

Methods: The P450 genes were codon optimised for expression in E. coli and cloned into pCW. Both P450s were expressed at high level (240-550 nmol/L) and purified via traditional methods. The proteins were characterised by UV/Visible spectroscopy and their binding to a variety of monoterpenes investigated. Catalytic activity was reconstituted in vitro by using the known bacterial P450 redox partners, cindoxin and putidaredoxin and the products of catalysis determined by GCMS and NMR.

Results and Discussion: Both P450s were successfully expressed, purified and characterised by UV/Vis spectroscopy. P450TolR was shown to oxidise p-cymene to 4-isopropylbenzyl alcohol, and R-limonene to perillyl alcohol through both in vivo and in vitro catalytic turnovers. In vitro catalytic turnovers were successful with putidaredoxin and putidaredoxin reductase as redox partners.

Conclusion: P450TolR appears to play a role in initiating the biodegradation of p-cymene and/or (R)-limonene but the current function of P450TerpR is still under investigation.

Figure 1. Hydroxylation of p-cymene and R-limonene catalyzed by P450TolR from R. rhodochrous. 1Davis, E. M. and Croteau, R. (2000). Cyclization enzymes in the biosynthesis of monoterpenes, sesquiterpenes, and diterpenes Top. Curr. Chem. 209 53–95. 2Hawkes, D., Adams, G., Burlingham, A, Ortiz de Montellano, P. & De Voss, J.J. (2002). Cytochrome P450cin (CYP176A1), Isolation, Expression, Characterization. J. Biol. Chem., 277, 27725-32.


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Synthesis of hydroxycholesterols and derivatives for exploration of cytochromes P450 in steroidal saponins biosynthesis and M. tuberculosis inhibition James R. Beckett, Lauren J. Salisbury, Jeanette E. Stok, James J. De Voss School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia. This work describes the synthesis of key hydroxycholesterols and their derivatives which will be used for two purposes: the exploration of steroidal saponin biosynthesis in the Australian yam Dioscorea transversa, and inhibition of CYP125 of M. tuberculosis as a method of inhibiting tuberculosis growth. Steroidal saponins are a class of terpenoid natural product that are often reported to have medicinal properties, but whose biosynthetic pathway remains largely unexplored in plants. D. transversa provides an ideal model plant to study saponin biosynthesis as its phytochemical profile contains only two steroidal saponins, both of which are formed by sequential oxidation at the 16, 22 and 26 positions of a cholesterol precursor (see Figure 1). These oxidations are likely performed by Cytochromes P450 but their identity and the order in which the oxidations occur is currently unknown. Synthesis of each possible molecule from combined oxidation at these three positions generates a suite of substrates to use for enzyme turnover and standards for product comparison. Using this information, it will be possible to determine the natural substrate of this enzyme and shed light on the biosynthetic pathway of steroidal saponins. Fortunately, the hydroxycholesterols synthesised for this project can also be applied to the study of M. tuberculosis inhibition both directly and as scaffolds to synthesise derivatives. Previous studies have shown that a degradation resistant analogue of cholesterol could be used in wild type tuberculosis as a potential new anti-tubercular agent.1 Surprisingly, it was found that installation of hydroxyl groups at C16 on the cholesterol core was a key modification that caused the compound to become resistant to degradation, and hence act as a potent growth inhibitor. The commercially available steroids diosgenin and stigmasterol were chosen as starting materials for these syntheses because they could be efficiently converted to the desired products through a variety of degradative reactions. To date, 6 of the 7 possible biosynthetic intermediates were synthesised. These hydroxycholesterols were then further derivatised at the 16 and 26 positions to provide substrates for testing against M. tuberculosis. 1. Frank, D., Zhao, Y., Wong, S., Basudhar, D., De Voss, J., & Ortiz de Montellano, P. (2016). Cholesterol

Analogs with Degradation-resistant Alkyl Side Chains Are Effective Mycobacterium tuberculosis Growth Inhibitors. J. Biol. Chem., 291(14), 7325-7333.

Figure 1: Biosynthesis of steroidal saponins in D. transversa involves cholesterol hydroxylation.


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Arginine-124 is involved in electron transfer in a protozoan cytochrome P450 CYP51 enzyme Stella A Child1, Tatiana Y Hargrove1, Galina I Lepesheva1,2, F Peter Guengerich1 1 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee U.S.A. 2 Center for Structural Biology, Vanderbilt University, Nashville, Tennessee U.S.A. Introduction. Electron transfer to the heme of cytochrome P450s is proposed to occur by several different pathways. In bacterial P450-BM3 and mitochondrial P450scc the heme-coordinating cysteine is involved.1,2 In P450cam, the porphyrin prosthetic group participates, as well as an Arg residue in Helix C.3 Aims. To probe the electron transfer pathway in CYP51 from the protozoan Trypanosoma cruzi using site-directed mutagenesis of an amino acid, Arg-124, located near the proximal surface of the enzyme. Methods. The R124A mutant of T. cruzi CYP51 was produced, and X-ray crystallography of substrate-bound and ligand-free CYP51 enzyme was performed. The spectral response of the R124A CYP51 to substrate, substrate conversion, and the rate of enzymatic reduction were measured (under anaerobic conditions). Results. Comparison of the substrate-bound and substrate-free CYP51 structures showed the guanidine of Arg124 moving away from the heme propionate OH and protruding from the proximal surface of the enzyme in the presence of substrate. The R124A mutant was expressed in a functional form, though the enzymatic

activity was reduced by more than 100-fold. The rate of reduction of the R124A mutant (0.65 0.02 min-1)

was less than half that of the WT (1.56 0.24 min-1) in the presence of the substrate eburicol. The reduction is the apparent rate limiting step for the WT (Kcat = 2.4 min-1) but not for the R124A mutant (Kcat = 0.02 min-

1). Without substrate, the rate of reduction of the WT enzyme was over 7-fold slower (0.2 0.02 min-1). Discussion. The shift in position of the Arg-124 group upon substrate binding suggests a role in facilitating electron transfer from CPR to the heme of CYP51. The reduction of the R124A mutant was slower than for the WT enzyme, in support of this conclusion. This mechanism of electron transfer is more similar to bacterial P450cam than mitochondrial P450 enzymes. Conclusion. The heme-binding Arg124 residue of T. cruzi CYP51 is involved in the electron transfer mechanism, which occurs in a P450cam-like manner. 1. Sevrioukova, I. F., Li, H., Zhang, H., Peterson, J. A., & Poulos, T. L. (1999). Structure of a cytochrome P450–

redox partner electron-transfer complex. Proc. Natl. Acad. Sci. U.S.A. 96, 1863–1868. 2. Strushkevich, N., MacKenzie, F., Cherkesova, T., Grabovec, I., Usanov, S., & Park, H.-W. (2011) Structural

basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system. Proc. Natl. Acad. Sci. U.S.A. 108, 10139–10143.

3. Tripathi, S., Li, H., & Poulos, T. L. (2013) Structural basis for effector control and redox partner recognition in cytochrome P450. Science 340, 1227-1230.

4. Hargrove, T.Y., Wawrzak, Z., Fisher, P.M., Child, S.A., Guengerich, F.P., Waterman, M.R., & Lepesheva, G.I. (2018) Binding of a physiological substrate causes large scale conformational reorganization in cytochrome P450 51. J. Biol. Chem. 293, 19344-19353.


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Synthesis of intermediates in steroidal saponin biosynthesis Luke R. Churchman, Lauren J. Salisbury and James J. De Voss School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia. The saponin class of natural products present in higher plants consists of many large, structurally diverse glycosylated terpenoids. Steroidal saponins in traditional herbal medicines are believed to confer the observed therapeutic effects of such remedies. Developing semi-synthetic methods to produce pure steroidal saponins would greatly aid in activity studies as well as providing standards for quality control of commercial herbal medicines. P450s are a ubiquitous family of haem-dependent enzymes that catalyse many different reactions including hydroxylation, epoxidation and demethylation. The sole

CYP51 (C-14 demethylase) in Dioscorea transversa may provide crucial information about the synthesis of observed cholesterol metabolites in this organism. To elucidate the role of this enzyme, a suite of potential substrates for this enzyme must be synthesised. The aim of this work was to synthesise a range of potential substrates for D. transversa CYP51. Lanosterol was chosen as a starting material for all synthesis due to its similarity to the desired compounds and commercial availability. Synthetic routes were designed using lanosterol that allowed the production of 24,25-dihydrolanosterol and eburicol, as well as allowing isolation of pure lanosterol from the impure commercial sample. C-4 demethylation via a ring opening/closing methodology adapted from Cohen gave access to obtusifoliol, 4-demethyl-24,25-dihydrolanosterol, and 4-demethyllanosterol.1 Four potential substrates of the CYP51 from D. transversa were synthesised from commercial lanosterol: 24,25-dihydrolanosterol (94%); 4-demethyl-24,25-dihydrolanosterol (11%); eburicol (6%); and pure lanosterol (20%). 1H and 13C NMR spectra of the compounds produced were in agreement with literature values obtained for the same compounds isolated from natural sources. Structures of all synthesised compounds including key intermediates were confirmed by above techniques as well as 2D NMR, GCMS, IR, melting point, and crystal structures when obtainable. Synthetic routes are currently being optimised to allow for comprehensive testing of the compounds with the putative CYP51 from D. transversa, however preliminary substrate binding experiments are already being explored. 1. Cohen. K. & Pinhey, J. (1973). A general method for removal of a 4-methyl group from triterpenoids. J.

Chem. Soc. 2076-2082. 2. Challinor, V. & Smith, D.M. & De Voss, J.J. (2011). Steroidal saponins isolated from an Australian Yam

(Dioscorea sp.) Aust. J. Chem. 64:5, 545-549.

Figure 1. The hypothesised potential substrates of the CYP51 from D. transversa, the synthetic targets of this work.


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Some reactions require commitment: processivity of human P450 11B2 reactions Michael J. Reddish1, F. Peter Guengerich1 1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, United States of America. Introduction. Aldosterone synthase is a human cytochrome P450 enzyme responsible for the formation of aldosterone, the major human mineralocorticoid steroid. Aldosterone is important for regulation of electrolyte homeostasis, but aldosterone synthase mutations and overexpression can lead to deleterious effects such as hypertension, congestive heart failure, and diabetic nephropathy. The enzyme is therefore a target for drug development to treat various conditions. Aldosterone synthase catalyzes the formation of aldosterone from 11-deoxycorticosterone through three distinct oxidation steps. It is currently unknown if these reactions happen in sequence without the intermediate products dissociating from the enzyme (i.e., processively) or if these reactions happen distributively where the intermediate products must first dissociate and then rebind to the enzyme prior to subsequent oxidation. Aims. 1 – Quantitatively determine the degree of processivity of human P450 11B2 steroid reactions. 2 – Establish the molecular mechanism by which a distributive or processive mechanism is favoured. Methods. Steady state and pre-steady state measurements using purified, recombinant enzyme indicate the reactivity of P450 11B2 with each reactant. Equilibrium binding and stopped-flow binding studies establish reactant binding parameters. Pulse-chase studies directly probe the processivity of each reaction step. We then combine the results of all of these studies together using a computational modelling approach to quantitatively assess processivity. Results. Our results show that P450 11B2 reactivity, in terms of both kcat and kcat/KM, is 11-deoxycorticosterone > corticosterone >> 18-OH corticosterone. Our results measure, for the first time, the steady state reaction of P450 11B2 with 18-OH corticosterone. We establish the equilibrium binding parameters of the enzyme with each of its reactants and products, including the importance of lipids vs. detergents and redox partner protein on binding parameters. Discussion. The production of aldosterone by human P450 11B2 must be proceed predominantly through a processive mechanism because the enzyme produces aldosterone significantly faster when starting with 11-deoxycorticosterone or corticosterone than 18-OH corticosterone. The enzyme can bind 18-OH corticosterone to be able to support faster turnover; however, it appears that this binding does not lead to a productive enzyme-substrate complex. We believe this is because free 18-OH corticosterone exists in a hemiketal, lactol state that makes the 18-carbon inaccessible to further oxidation. However, if the 18-OH corticosterone is produced by the enzyme it is stabilized in an open-ring, ketone form that can still be oxidized. Conclusion. P450 11B2 must act processively to form aldosterone to avoid the inactive hemiketal form of 18-OH corticosterone that exists free in solution. This is not a concern for the substrates 11-deoxycorticosterone or corticosterone because they do not form a lactol; therefore, these reactions can proceed distributively or processively.


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Inhibitory effects of Curcuma longa extracts on the steroid metabolizing cytochrome P450 enzymes Patricia Rodríguez Castaño1,2, Amit V Pandey1,2

1Pediatric Endocrinology, Diabetology and Metabolism, University Children’s Hospital Bern, 3010 Bern, Switzerland; 2Department of BioMedical Research, University of Bern, Bern, Switzerland. Introduction. Turmeric is a popular ingredient in the cuisine of many Asian countries. It is also known for its use in Chinese and Ayurvedic medicine. It comes from the root of the Curcuma longa. Turmeric is rich in curcuminoids, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Curcumin has potent anti-inflammatory and anti-carcinogenic activities. Since many anti-cancer drugs target enzymes from the steroidogenic pathway, we tested the bioactivity of curcuminoids on cytochrome P450 CYP17A1, CYP21A2, and CYP19A1 enzyme activities. Aims. To test the effects Curcuma longa extracts on steroid metabolizing cytochrome P450 CYP17A1, CYP19A1 and CYP21A2. Methods. Curcuminoids were extracted from turmeric with organic solvents. We conducted a cell-based assay for CYP17A1 and CYP21A2 activities using human adrenal cell line NCI-H295R. 3H-pregnenolone was used for CYP17A1 assays, and 3H-17α-hydroxyprogesterone was used as a substrate for CYP21A2. Curcuminoids were added at different concentrations and incubated for 24h. Steroids were separated by thin layer chromatography and analyzed by phosphorimager analysis. For CYP19A1 activity, an in vitro assay using endoplasmic reticulum from JEG3 were used with 3H-androstenedione as the substrate. Curcuminoids were incubated for 1h, and the formation of 3H-water from the androstenedione breakdown was measured by scintillation counting. Results. The CYP17A1 hydroxylase activity, when using 10 µg/ml of curcuminoids, was reduced to ~15%, whereas CYP17A1 lyase activity was reduced to ~30% of control. On the other hand, CYP21A2 activity was only reduced to a ~50%. Furthermore, CYP19A1 activity was reduced to 80~20% when using 1-100 µg/ml of curcuminoids in a dose-dependent manner. No effect on the activity of 5alpha reductase for the conversion of testosterone to dihydrotestosterone was observed. Discussion. These studies show that curcuminoids may potentially cause some inhibition of steroid metabolism, especially at higher dosages. The activities of CYP17A1 and CYP19A1 were inhibited by curcuminoids which indicate potential anti-carcinogenic effects in case of prostate cancer as well as breast cancer which can be targeted by inhibition of steroidogenesis. Also, the recent popularity of turmeric powder/curcumin as a dilatory supplement needs further evaluation of the effect of curcuminoids on steroid metabolism. Conclusion. Curcuminoids present in curcumin may affect activities of steroid metabolizing cytochrome P450 enzymes.


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Evaluating the resistance potential of Drosophila melanogaster cytochrome P450 genes by transgenic overexpression Alex Giang1, Roberto Fusetto, Trent, Perry1, Philip Batterham1 1School of Biosciences, Bio21 Institute, University of Melbourne, Melbourne, Australia. Introduction.Strong selection pressure imposed by insecticide usage has allowed resistance to evolve and spread in insect populations. One mechanism underlying resistance, increased insecticide metabolism, is often linked to elevated levels of transcription of cytochrome P450 genes. This has led to the hypothesis that insect populations may contain an array of metabolic enzymes that can potentially provide resistance to insecticides if their level of expression in the appropriate tissues is increased. Aims. To explore this hypothesis, this project examined whether the upregulation of P450 genes most similar to a well characterized P450 gene, Cyp6g1, would confer insecticide resistance. Methods. Candidate genes were overexpressed using the GAL4/UAS system and their resistance profiles for the insecticides nicotine, imidacloprid and nitenpyram were determined using a standard toxicological bioassay and an assay of larval movement, the Wiggle Index1. Following this, LC/MS analysis was used to examine the metabolites produced in these overexpression lines after exposure to nicotine and imidacloprid.

Results. The overexpression Cyp6g1 and Cyp6g2 confer resistance towards all 3 insecticides tested. Cyp6v1 overexpression also results in slight resistance towards nicotine. An increase in the metabolites of nicotine and imidacloprid were also observed in these resistant overexpression lines. Discussion. P450s that confer resistance towards a specific insecticide are likely to show cross-resistance to other chemically similar insecticides. A key developmental gene, Cyp6g22, was shown to confer resistance when expressed in metabolic tissues. Transcript data on field-derived lines suggests that Cyp6g2 may indeed contribute to imidacloprid resistance in natural populations3. Conclusion. The in vivo expression approach taken is useful in determining the capacity of the P450 arsenal of insects, pest and beneficial, in surviving insecticide exposure. 1. Christesen, D. et al. (2017). Transcriptome Analysis of Drosophila melanogaster Third Instar Larval Ring

Glands Points to Novel Functions and Uncovers a Cytochrome P450 Required for Development. Genes Genomes Genetics, 7(2), pp.467–479.

2. Denecke, S. et al. (2017). Multiple P450s and Variation in Neuronal Genes Underpins the Response to the Insecticide Imidacloprid in a Population of Drosophila melanogaster. Scientific Reports, 7(1), p.11338.

3. Denecke, S. et al. (2015). The wiggle index: An open source bioassay to assess sub-lethal insecticide response in Drosophila melanogaster. PLoS ONE, 10(12), pp.1–19.


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CYP27A1 but not CYP2R1 can metabolise vitamin D3-3-sulfate Robert C. Tuckey1, Chloe Y. S. Cheng1 & Yuhan Jiang1 1School of Molecular Sciences, The University of Western Australia, Perth, Australia. Introduction. 25-Hydroxyvitamin D3-3-sulfate (25(OH)D3-3-sulfate) is a major form of vitamin D3 in human serum, particularly in the fetal circulation where its concentration is higher than that of 25(OH)D3 (1). Its specific function is unknown but it likely serves to buffer serum 25(OH)D3 levels. 25(OH)D3-3-sulfate may be formed from 25(OH)D3 by the action of sulfotransferases, such as SULT2A1 (1). Since vitamin D3-3-sulfate is also present in human serum, another possible pathway for the formation of 25(OH)D3-3-sulfate is by the action of CYP2R1 or CYP27A1, the two major vitamin D 25-hydroxylases in the human liver. Aims. To test the abilities of CYP27A1 and CYP2R1 to metabolise vitamin D3-3-sulfate. Methods. CYP2R1 and CYP27A1 were expressed in E. coli, purified, reconstituted with their redox partners and activity assayed with substrates dissolved in 2-hydroxypropyl-β-cyclodextrin. Products were measured by reverse-phase HPLC. Results. Purified CYP2R1 reconstituted with cytochrome P450 oxidoreductase was unable to metabolise vitamin D3-3-sulfate whereas the unsulfated substrate was hydroxylated at C25, as expected. In contrast, purified CYP27A1 reconstituted with adrenodoxin and adrenodoxin reductase, converted vitamin D3-3-sulfate to 1 major and 3 minor products. The major product was identified as 25(OH)D3-3-sulfate based on its identical HPLC retention time to authentic standard. The kcat values for the 25-hydroxylation of vitamin D3-3-sulfate (1.1 min-1) and vitamin D3 by CYP27A1 were similar but the Km for vitamin D3-3-sulfate (44 µM) was 4-fold higher than for vitamin D3. Discussion. The higher Km for vitamin D3-3-sulfate compared to vitamin D3 displayed by CYP27A1 suggests that it is more difficult to bring the charged sulfate group into the hydrophobic active site of this enzyme than the free 3-hydroxyl group. For CYP2R1, the presence of the 3-sulfate group on the vitamin D3 presumably prevents entry of the substrate into the active site, at least in an orientation that permits hydroxylation. Conclusion. This study shows that 25-hydroxylation of vitamin D3-3-sulfate by CYP27A1, but not by CYP2R1, can provide an alternative pathway for the production of 25(OH)D3-3-sulfate to the sulfation of 25(OH)D3 by SULT2A1 in the liver. 1. Tuckey, R. C., Cheng, C. Y. S. & Slominski, A. T. (2019). The serum vitamin D metabolome: What we know and what is still to discover. J. Steroid Biochem. Mol. Biol. 186, 4-21.

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