Introduction The rise of antibiotic resistance among microbial populations has surpassed the research and development of novel antibiotics (El Zoeiby et al., 2003). Successful antibiotics are often directed at a specific enzyme that is unique to microorganisms. Thus, the Mur family of enzymes (MurA-MurF) was selected for my research project, as these enzymes catalyze the biosynthesis of peptidoglycan in the bacterial cell wall (El Zoeiby et al., 2003 and Silver, 2006). Without peptidoglycan, the cell wall has less mechanical strength and is unable to withstand the forces of osmotic pressure, resulting in death of the bacteria. Human cells do not possess cell walls; thus, humans not affected by molecules that specifically inhibit the Mur family of enzymes. The mechanisms of action for these enzymes are well established and their X-ray crystal structures are available (Skarzynski, 1996). Understanding the structure and function of these enzymes enables researchers to develop specific inhibitors for peptidoglycan biosynthesis. Derivatives of phosphinic acid inhibit Mur C through Mur F; however, only one FDA-approved antibiotic (fosfomycin) targets what is arguably the most important enzyme in this family, MurA. MurA catalyzes the first and committed step of peptidoglycan synthesis; so, its inhibition would prevent peptidoglycan synthesis. While fosfomycin is an effective inhibitor, bacterial resistance to fosfomycin and other antibiotics has prompted the search for and development of novel antibiotics. Recent research has elucidated compounds which bind to and inhibit the Mur enzymes, but most of these compounds do not exhibit antibacterial activity (Baum et al., 2001 and

Silver, 2006). The most probable reason for MurA inhibition without antibacterial activity is the inability for the compounds to penetrate the bacterial cell wall. Because peptidoglycan synthesis occurs in the cytosol, compounds must overcome the cell wall to be effective. The goal of this project was to optimize a protocol for the generation and purification of the MurA enzyme, determine the activity of MurA, and characterize the biochemical properties of some potential inhibitors. First, the effect of fosfomycin on MurA activity was evaluated, followed by the effect of a putative inhibitor, BCB33b. I synthesized and purified BCB33b in the summer of 2011, and its formal name is 3-metheylcyclohex-4-ene-1,2-dicarboxylic acid (Figure 1). To produce MurA, various strains of Escherichia coli were transformed with specific plasmids. A plasmid is a circular

segment of DNA than can impart certain biological properties to a bacterium into which the plasmid is introduced. The plasmids we used contained genes for MurA, antibiotic resistance, and inducible expression. Isopropyl β-D-1-thiogalactopyranoside (IPTG), a non-cleavable lactose analog, acted as the inducer for the lac operator. The procedure for

 

BCB33b fosfomycin

Figure 1. Structure of BCB33b and Fosfomycin BCB33b is a putative inhibitor of the MurA enzyme, and fosfomycin is a known inhibitor.

MurA: production, purification, and characterization of antibiotic potential Ben Boone

transformation of the plasmid is well understood, and the plasmid itself has already been synthesized and characterized by prior research (Eschenburg et al., 2005). Successfully transfected bacteria are selected for in the presence of an antibiotic to which they are resistant. To ensure that the MurA gene is present within the supplied vectors, the polymerase chain reaction can be employed on the vectors using the forward and reverse MurA primers. A gene product of 1.3 kbp is expected for the MurA gene. A protocol for the overexpression of MurA, a process in which the production of MurA is sent into overdrive, is known for the bacterial strain we used; however, the generation of MurA by this protocol had been unsuccessful in our research group. Therefore, I worked to optimize a protocol for overexpression of MurA by reading a number of related research articles altering the process accordingly The pGEX-6P-1 plasmid codes for a glutathione S-transferase (GST) tag attached to the MurA enzyme, which enabled me to purify the enzyme using a glutathione agarose affinity column. L-glutathione is the natural substrate for the GST enzyme, so a solution of L-glutathione can be used to elute MurA-GST from the column. In addition, the MurA-GST has a restriction site for the PreScission™ Protease to cleave the GST tag. If PreScission™ Protease is added to a glutathione agarose column with MurA-GST bound, the tag will be cleaved and remain bound to the column while the MurA enzyme elutes from the column. After the MurA has been purified, the concentration of eluted protein can be quantified using the Bradford assay. The activity of MurA can be determined by simulating the reaction conditions in vitro by adding the substrates phosphoenolpyruvate (PEP) and uridine diphosphate N-

acetylglucosamine (UDP-NAG) to MurA (Figure 2).

The amount of phosphate released, which is a measure of enzyme activity, can then be quantified using the malachite green assay (Feng et al., 2011). After establishing a baseline activity of MurA, the addition of known and putative inhibitors of MurA can be added to the reaction mixture to determine their effect on MurA activity. Methods Plasmids and Bacteria Used The pET41a and pGEX-6P-1 vectors with the MurA gene inserted were provided by Dr. Ernst Schonbrunn. Bacteria used for amplification of the plasmid DNA were One Shot® Chemically Competent E. coli (Invitrogen). Bacteria used for overexpression of the MurA protein were One Shot® BL21 Star™ (DE3) Chemically Competent E. coli (Invitrogen). PCR and Agarose Gel Electrophoresis Polymerase chain reaction (PCR) was carried out with either the amplified pGEX-6P-1 or pET41a vector as the template DNA. The PCR reaction mixture included 2 µL pDNA, 5 µL of 10 mM forward (5’-ATGGCGATTATGGATAAATTTC-3’) and reverse (5’-GGATTCTTCGCCTTTCACACGC-3’) MurA primers (Invitrogen), one bead of

Figure 2. Reaction Catalyzed by MurA MurA catalyzes the addition of phophoenolpyruvate (top-left) and N-acetylglucosamine (bottom-left) with the release of adduct and inorganic phosphate (http://chemwiki.ucdavis.edu/@api/deki/files/9989/=image070.png).

+  +  

illustra™ PuReTaq™ Ready-To-Go™ PCR beads (GE Healthcare), and 13 µL autoclaved ddH2O. PCR was run in an EdvoCycler with the following program: initial denaturation 93˚C for 180 s; denaturation 94˚C for 60 s, annealing 46˚C for 60 s, and extension 72˚C for 120 s (30 cycles); final extension 72˚C for 1800 s. PCR products and 1 Kb Plus DNA Ladder (Invitrogen) with PV92 Xylene Cyanole Loading Dye, 5x (BioRad) were run on a 1% agarose gel with ethidium bromide at 70V. E. Coli Transformation, Plasmid Amplification, and Protein Overexpression E. coli were incubated with 1 µL pDNA for 30 minutes on ice. After a 1 minute incubation at 42˚C, cells were placed back on ice for 2 minutes. Then 125 µL SOC Medium (cellgro®) was added. Cells were incubated for 1 hour at 37˚C and 225 rpm. Cells were then added to 5 mL Difco™ LB Broth, Lennox (Becton, Dickinson and Co.) with appropriate antibiotic, either kanamycin or ampicillin. This cell culture was incubated for 12 hours at 37˚C and 200 rpm. If cell cultures were for plasmid amplification, the plasmid was isolated using the QIAprep® Spin Miniprep Kit (Qiagen). If the cells were for protein overexpression, the 5 mL cell cultures were added to 200 mL LB broth with appropriate antibiotic and incubated for 3 hours in the at 37˚ and 200rpm. Then, 200 µL 1M IPTG was added for protein overexpression and cultures were incubated for 3 hours. The cell cultures were centrifuged for 20 minutes at 2,600g. Supernatant was discarded and cell pellets frozen at -20˚C. MurA-GST Purification Frozen pellets were resuspended in 5 mL GST Wash Buffer (20 mM Tris-HCl, pH 7.5, 0.25 M NaCl, 2 mM EDTA, 2 mM EGTA) and half a tablet of Pierce® Protease Inhibitor Tablets, EDTA-Free (Thermo Scientific). A few granules of Lysozyme Type VI (MP

Biomedicals, LLC) were added to the resuspended pellet to lyse the cells. The mixture was incubated on ice for 20 minutes with frequent vortexing. To assist in cell lysis, the cell mixture was sonicated with the Cell Disrupter 16-850 (VirSonic) at 30% power for 10 cycles of 10 s on with 10 s off. The resulting lysed cell mixture was centrifuged for 20 minutes at 9,100g. The supernatant was filtered using a 12 mL Monoject™ Syringe (Kendall) and 0.2 µm PES Syringe Filter (Whatman™) to yield filtered cell lysate. A volume of 1mL Pierce® Glutathione Agarose was washed with GST Wash Buffer with ten bed volumes and then combined with the cell lysate. The resin and lysate were incubated on an end-over-end rotator for 1 hour at 15°C. A series of 4 10 mL washes with GST Wash Buffer was carried out, with light centrifugation between washes, to remove unbound proteins. The loaded resin was added to a 1x10 cm Flex Column (Kimble Chase). A volume of 3 mL Elution Buffer (Wash Buffer + 20 mM L-Glutathione reduced, pH 8.0) was added to the column and 0.5 mL elutions were collected. SDS-PAGE 26 µL of protein sample were combined with 4 µL NuPage® Sample Reducing Agent (10X) (Invitrogen) and 10 µL NuPage® LDS Sample Buffer (4X) (Invitrogen). This mix was boiled for 5 minutes and then 30 µL loaded into Mini-PROTEAN® Precast Gels (BioRad). 4 µL of Precision Plus Protein™ Dual Color Standards (BioRad) were loaded, and the gel was run to completion at 70V. Gels were stained with colloidal blue staining solution. Cleavage of GST-tag from MurA-GST Taking 1 mL glutathione agarose loaded MurA-GST as described above, the resin was washed with 10 bed volumes PreScission Cleavage Buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, pH

7.5). The resin was then incubated with 1 mL PreScission™ Protease Mix (920 µL PreScission™ Cleavage Buffer and 80 µL PreScission™ Protease) for 4 hours at 4°C with an end-over-end rotator. The resin was then added to a Flex Column and the cleavage elution was eluted. Then 3 separate additions of 1 mL PreScission™ Cleavage Buffer were eluted. Bradford Assay Coomassie Plus™ Protein Assay Reagent (Thermo Scientific) was used to quantify protein concentration of samples. A volume of 50 µL protein sample was added to 1 mL of the Coomassie reagent. Protein standards were prepared with Albumin Standard (Thermo Scientific). The A595 was measured and protein concentration determined by sample comparison to standards. Malachite Green Assay Malachite green color solution was prepared by adding 22.5 mg malachite green to 50 mL ddH2O, taking 25 mL of this solution and adding 7.33 mL 4.2% ammonium molybdate in 2M HCl. Final substrate concentrations were 1mM for both phosphoenolpyruvate (PEP) and uridine diphosphate N-acetylglucosamine (UDP-NAG). Reactions were carried out in 100 µL reaction mixtures (1 mM substrates, pH 7.8 25 mM Tris-HCl, and 7.5 µM MurA) for 20 minutes before addition to 800 µL malachite green color solution. After 1 minute exposure to the color solution, reactions were quenched with 100 µL 34% sodium citrate. For inhibition studies, MurA samples were incubated with inhibitors for 10 minutes prior to addition of substrates. Imaging and Spectrophotometry Visualization of agarose and SDS-PAGE was performed with Molecular Imager® Gel Doc™ XR Imaging System (BioRad) and The Discovery Series™ Quantity One® 1-D

Analysis Software. Spectrophotometry for Bradford and malachite green assays was performed using the Cary® 50 Bio UV-Visible Spectrophotometer (Varian, Inc.) and Cary ® WinUV Software (Varian, Inc.). Results PCR and Agarose Gel Electrophoresis PCR products of the amplified pGEX-6P-1 and pET41a MurA vectors using forward and reverse MurA primers revealed the presence of the 1.3 kbp MurA gene in both vectors (Figure 3).

E. coli transformation and protein overexpression BL21 Star cells transformed with the pGEX-6P-1 MurA-GST or pET41a MurA vectors yielded MurA-GST or MurA, respectively (Figure 4). Unsurprisingly, a number of other proteins were present in the lysates at levels less than the MurA proteins.

 ––  1.3  kbp    

         1          2          3          4                  

Figure 3. Agarose Gel of pGEX-6P-1 and pET41a MurA PCR Products with MurA Forward and Reverse Primers Electrophoresis of PCR products from both pGEX-6P-1 and pET41 MurA vectors with MurA primers reveals the presence of the MurA gene in both vectors at the expected size of 1.3 kbp.

         1                2        3                4                  5

Figure  4.  SDS-­‐PAGE  of  Unpurified  MurA-­‐GST  and  MurA  Unpurified  cell  lysates  were  analyzed  by  SDS-­‐PAGE  and  staining  with  colloidal  blue.  MurA-­‐GST  (1&2)  migrated  to  70.7  kDa  (left  arrow)  as  expected.  MurA  (4&5)  migrated  to  44.7  kDa  (right  arrow)  as  expected.  Contaminating  protein  is  observed  in  both  lysates.  Protein  standard  is  shown  (3).  

0

0.5

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E1 E2 E3 E4

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Figure  8.  Bradford  Assay  of  GST-­‐tag  Cleavage  of  MurA-­‐GST  The  GST  tag  of  MurA-­‐GST  was  cleaved  with  PreScission  Protease,  and  successful  tag  cleavage  led  to  the  elution  of  1.31  mg/mL  of  tagless  MurA  in  elution  1  (E1).  

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200

400

600

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M M+U M+P M+U+P

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Figure  10.  Malachite  Green  Assay  of  Tagless  MurA  The  reaction  mixture  with  MurA  (M),  UDP-­‐NAG  (U),  and  PEP  (P)  was  the  only  sample  that  yielded  high  levels  of  phosphate,  which  was  detected  by  malachite  green.  

 

MurA-GST Purification Purification of MurA-GST resulted in 3.6 mg/mL of protein in Elution 2 (Figure 5).

The purity of elution 2 was confirmed by SDS-PAGE, revealing only MurA-GST at the expected 70.7 kDa (Figure 6).

MurA-GST Activity A malachite green assay using the MurA-GST revealed enzyme activity (Figure 7).

Cleavage of GST Tag from MurA-GST Cleavage of the GST tag from the MurA-GST protein using PreScission™ Protease was successful (Figure 8).

The successful cleavage of the GST-tag from MurA-GST was confirmed by SDS-PAGE of elution 1, revealing only MurA at the expected 44.7 kDa (Figure 9).

MurA Activity A malachite green assay with the cleaved MurA was performed to determine if enzymatic activity was affected by the GST-tag, and it appeared that whether or not the tag was part of MurA had no effect on activity (Figure 10).

The Effect of Fosfomycin and BCB33b on MurA-His6 Activity For inhibition analysis of MurA, an alternatively tagged version of MurA that was produced previously was used. This MurA

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4  

E1 E2 E3 E4 E5

[Protein] (mg/mL)

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Figure  5.  Bradford  Assay  of  MurA-­‐GST  Purification  Elutions  MurA-­‐GST  was  purified  and  a  Bradford  assay  of  the  elutions  revealed  a  peak  protein  concentration  in  elution  2  (E2),  with  diminishing  protein  concentration  in  successive  elutions.  

Figure  6.  SDS-­‐PAGE  of  Purified  MurA-­‐GST  SDS-­‐PAGE  of  purified  MurA-­‐GST  revealed  a  single  band  at  70.7  kDa.  

 

0 100 200 300 400 500 600 700

[Phosphate]  (µM)    

[Phosphate] (µM)

MG MG+U MG+P MG+U+P

Figure  7.  Malachite  Green  Assay  of  Purified  MurA-­‐GST  The  reaction  mixture  with  MurA-­‐GST  (MG),  UDP-­‐NAG  (U),  and  PEP  (P)  was  the  only  sample  that  yielded  high  levels  of  phosphate,  which  was  detected  by  malachite  green.  

Figure  9.  SDS-­‐PAGE  of  MurA  SDS-­‐PAGE  of  tagless  MurA  revealed  a  single  band  at  44.7  kDa.  

 

had a hexahistidine tag and was titled MurA-His6. A malachite green assay with MurA-His6, both substrates, and fosfomycin revealed 50% inhibition with 1 mM fosfomycin (Figure 11).

A malachite green assay with MurA-His6, both substrates, and varying concentrations of BCB33b revealed 68% inhibition with 1mM BCB33b (Figure 12), compared to only 54% inhibition with the same concentration of fosfomycin (Figure 11).

Discussion The MurA gene was present in both the pGEX-6P-1 and pET41a vectors, with only the pGEX-6P-1 vector containing the gene for the fusion protein MurA-GST. The GST-tag attached to the MurA enzyme allowed for the

purification of MurA-GST. Both MurA-GST and MurA without the GST tag possessed enzymatic activity. MurA-His6 was active and, with the substrate concentration and reaction conditions described, was inhibited by 1 mM fosfomycin to 46% normal activity. Under the same reaction conditions, the putative inhibitor BCB33b (3-metheylcyclohex-4-ene-1,2-dicarboxylic acid) inhibited MurA-His6 to 32% normal activity. These results appear to implicate BCB33b as a more potent inhibitor of the MurA enzyme. In order to clarify whether or not these results are correct in asserting the inhibitory quality of BCB33b, three future directions must be pursued: (i) test if the acidic pH induced by BCB33b is responsible for inhibiting MurA, (ii) perform inhibition studies with the MurA protein with no tag, and (iii) replicate the substrate concentrations and reaction conditions of Baum et al. (2001) for the malachite green assay. The third (iii) direction will help to show the inhibitory properties of fosfomycin and/or BCB33b at much lower concentrations. Acknowledgments This work was supported by the John C. Young Scholarship Fund and Centre College Faculty Development Committee. I would like to thank Dr. Ernst Schönbrunn of the H. Lee Moffitt Cancer Center and Research Institute for sending me two vectors to carry out protein expression. I would also like to thank Andrea Frost (Centre College ’15) for her contribution of the MurA-His6 and fosfomycin inhibition data. References Silver, L.L. (2006). Does the cell wall of bacteria remain a viable source of targets for novel antibiotics? Biochemical Pharmacology, 71(7), 996-1005.

Figure 12. Malachite Green Assay with MurA-His6 and Varying Concentrations of BCB33b Enzyme activity is expressed as a percent of the enzyme activity with MurA-His6, PEP, and UDP-NAG. Increasing concentrations of BCB33b inhibited MurA-His6 at increasing amounts. 1 mM BCB33b inhibited MurA-His6 to 32% normal activity.

0 20 40 60 80

100 120

% Activity

of No

Inhibitor

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100 120

1 mM fosfomycin

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Inhibitor

Figure 11. Malachite Green Assay with MurA-His6 and 1 mM Fosfomycin Enzyme activity is expressed as a percent of the enzyme activity with MurA-His6, PEP, and UDP-NAG. 1 mM fosfomycin inhibited MurA-His6 to 46% normal activity.

Skarzynski, T., Mistry, A., Wonacott, A., Hutchinson, S.E., Kelly, V.A., Duncan, K. (1996). Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin. Structure, 4(12), 1465-74. Baum, E.Z., Montenegro, D.A., Licata, L., Turchi, I., Webb, G.C., Foleno, B.D., Bush, K. (2001). Identification and characterization of new inhibitors of the Escherichia coli MurA enzyme. Antimicrobial Agents and Chemotherapy, 45(11), 3182-8. El Zoeiby, A., Sanschagrin, F., Levesque, R.C. (2003). Structure and function of the Mur enzymes: development of novel inhibitors. Molecular Microbiology, 47(1), 1-12. Eschenburg, S., Priestman, M.A., Abdul-Latif, FA, Delachaume, C., Fassy, F., Schönbrunn, E. (2005). A novel inhibitor that suspends the induced fit mechanism of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA). Joural of Biological Chemistry, 280(14), 14070-5. Feng, J., Chen, Y., Pu, J., Yang, X., Zhang, C., Zhu, S., Zhao, Y., Yuan, Y., Yuan, H., Liao, F. (2011). An improved malachite green assay of phosphate: Mechanism and application. Analytical Biochemistry, 409(1), 144-149.