Crystallographic Study To Determine the Substrate Specificity of anL-Serine-Acetylating Enzyme Found in the D-Cycloserine BiosyntheticPathway

Kosuke Oda, Yasuyuki Matoba, Takanori Kumagai, Masafumi Noda, Masanori Sugiyama

Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan

DcsE, one of the enzymes found in the D-cycloserine biosynthetic pathway, displays a high sequence homology to L-homoserineO-acetyltransferase (HAT), but it prefers L-serine over L-homoserine as the substrate. To clarify the substrate specificity, in thepresent study we determined the crystal structure of DcsE at a 1.81-Å resolution, showing that the overall structure of DcsE issimilar to that of HAT, whereas a turn region to form an oxyanion hole is obviously different between DcsE and HAT: in detail,the first and last residues in the turn of DcsE are Gly52 and Pro55, respectively, but those of HAT are Ala and Gly, respectively. Inaddition, more water molecules were laid on one side of the turn region of DcsE than on that of HAT, and a robust hydrogen-bonding network was formed only in DcsE. We created a HAT-like mutant of DcsE in which Gly52 and Pro55 were replaced byAla and Gly, respectively, showing that the mutant acetylates L-homoserine but scarcely acetylates L-serine. The crystal structureof the mutant DcsE shows that the active site, including the turn and its surrounding waters, is similar to that of HAT. Thesefindings suggest that a methyl group of the first residue in the turn of HAT plays a role in excluding the binding of L-serine to thesubstrate-binding pocket. In contrast, the side chain of the last residue in the turn of DcsE may need to form an extensive hydro-gen-bonding network on the turn, which interferes with the binding of L-homoserine.

D-Cycloserine (DCS), a cyclic structural analogue of D-alanine,is produced by Streptomyces garyphalus and S. lavendulae (1).

DCS prevents the actions of both alanine racemase (2, 3) andD-alanyl-D-alanine ligase (4, 5), which are necessary for the bio-synthesis of the bacterial cell wall. Thus, DCS functions as an in-hibitor of bacterial cell wall biosynthesis. This antibiotic is used asan antitubercular agent (6), but it is rarely prescribed and is usedonly in combined therapies because of its serious side effects (7).The side effects are caused by the binding of DCS to N-methyl-D-aspartate receptors as an agonist. However, applications of theseadverse effects to treatments for neural diseases have been dili-gently researched (8, 9).

We have recently reported the cloning and heterologous ex-pression of a biosynthetic gene cluster for DCS from DCS-pro-ducing S. lavendulae ATCC 11924 (10). The cluster has eight genes(dcsA to dcsH) that would be responsible for DCS biosynthesis andtwo genes (dcsI and dcsJ) as self-resistance determinants for theproducer organism (11, 12). Based on our studies (10) and thoseof another group (13–15), we have proposed a putative DCS bio-synthetic pathway in S. lavendulae ATCC 11924, as shown in Fig.1. Some of the enzymes catalyzing each reaction have already beenidentified by our group (10) and another (16). In the first biosyn-thetic step, the acetylation of L-serine by acetyl coenzyme A(acetyl-CoA)-dependent DcsE is necessary. The resulting O-acetyl-L-serine is used to generate O-ureido-L-serine, which maybe catalyzed by DcsD in the presence of hydroxyurea. Hydro-xyurea is obtained from the hydrolysis of N�-hydroxy-L-arginineby the arginase homologue DcsB. The stereochemistry of O-ureido-L-serine is then inverted by the putative racemase DcsC.Finally, cyclization and urea hydrolysis are suggested to be cata-lyzed by DcsG and/or DcsH. We have suggested that DcsA cata-lyzes the production of N�-hydroxy-L-arginine (17).

In general, O-acetyl-L-serine, which is generated from L-serinein the presence of acetyl-CoA by L-serine O-acetyltransferase

(SAT), is an intermediate for the biosynthesis of L-cysteine (18).Interestingly, DcsE, which displays no homology to well-knownSATs but high homology to L-homoserine O-acetyltransferases(HATs) in the L-methionine biosynthetic pathway (19, 20), acety-lates the hydroxyl group of L-serine (10). A previous report (21)showed that a putative HAT gene product found in Aspergillusnidulans, which shows 31% sequence identity with DcsE, is in-volved in L-cysteine biosynthesis; however, the enzymatic func-tion of the gene product has not yet been clarified. To understandthe substrate specificity of DcsE, we determined the X-ray crystalstructure of DcsE at a high resolution. The present study describesthe molecular mechanism whereby DcsE, which is homologous toHAT, preferentially acetylates the hydroxyl group of L-serine andnot that of L-homoserine.

MATERIALS AND METHODSExpression and purification of DcsE. The pET-28a(�) plasmid carryingdcsE (10) was used to express DcsE with a His6 tag at the N terminus.Escherichia coli BL21(DE3)pLysS transformed with the plasmid was usedto obtain the recombinant DcsE protein. Expression and purification ofthe recombinant DcsE were conducted as described previously (10).

Mutagenesis. A QuikChange site-directed mutagenesis kit (Strat-agene) was used to generate DcsE mutants according to the supplier’sinstruction manual. The mutagenic primers containing the desired mu-tations (underlined) were as follows (sense only): 5=-GCTCGTGCTCACGGCCCTCTCACCGGAC-3= (G52A), 5=-CGGGCCTCTCAGGGGACGCGCACGCGG-3= (P55G), and 5=-CTCGTGCTCACGGCCCTCTCAG

Received 9 November 2012 Accepted 1 February 2013

Published ahead of print 8 February 2013

Address correspondence to Masanori Sugiyama, sugi@hiroshima-u.ac.jp.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.02085-12

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GGGAC-3= (G52A-P55G). To generate the G52A or P55G expressionvector, the pET-28a/dcsE plasmid was amplified using sense and antisenseprimers. To generate the G52A-P55G expression vector, the P55G expres-sion vector was amplified using the primers. Confirmation of the muta-tion was done by DNA sequencing analysis. Expression and purificationof the mutant proteins were conducted according to the same methodused for the wild type.

Kinetic analysis. The acetyltransferase activity of DcsE was measuredusing a continuous spectrometric assay (22) involving the formation of acolored product (ε � 13,600 M�1 cm�1 at 412 nm) from the reaction of5,5=-dithiobis(2-nitrobenzoic acid) with CoA, which is generated fromacetyl-CoA. Assays were performed in a 60 mM Tris-HCl buffer (pH 8.0)containing various concentrations of L-serine or L-homoserine (up to 400mM), a fixed concentration of acetyl-CoA (15 mM), 1.0 mM EDTA, and1 mM 5,5=-dithiobis(2-nitrobenzoic acid) in a final volume of 200 �l at30°C. Data for the steady-state kinetics of DcsE and its mutants were fittedto equation 1.

v �kcat · Et · X

Km � X(1)

In equation 1, v is the initial velocity, Et and X are the concentrations ofenzyme and substrate, respectively, and kcat and Km are the catalytic andMichaelis-Menten constants, respectively, which were evaluated by thenonlinear least-squares method. The kcat and Km values for L-serine of theDcsE mutants, which were designated the G52A and G52A-P55G mu-tants, could not be determined due to the fact that the enzyme activities ofthese mutants did not reach the plateau in the concentration range ofL-serine as a substrate. Since the enzymatic activity of these mutantsincreased linearly in relation to the increase in the concentration ofL-serine, data for the steady-state kinetics of these mutants were fittedto equation 2.

v �kcat · Et · X

Km(2)

Oligomeric analysis. The oligomeric state of DcsE in solution wasanalyzed by high-performance liquid chromatography (HPLC). A 100-�lportion of aliquots containing 5 �M DcsE was applied to HPLC using aSuperdex 75 10/300 GL column (GE Healthcare) equilibrated with a 0.1 MTris-HCl buffer (pH 8.0) containing 0.15 M NaCl, and DcsE was eluted atroom temperature at a flow rate of 1 ml/min. The detection of protein wascarried out at 280 nm with a diode array detector (MD-2010; Jasco). Themolecular mass of the protein was calibrated using a gel filtration calibra-tion kit (GE Healthcare).

Crystallography. Prior to crystallization, the DcsE protein solutionwas dialyzed against a 20 mM Tris-HCl buffer (pH 7.6) containing 0.2 MNaCl, 5 mM dithiothreitol, and 1 mM EDTA and then concentrated to 10mg/ml using an Amicon Ultra centrifugal filter unit (Millipore). The DcsEcrystals were grown using the sitting-drop vapor-diffusion method with a1:1 (vol/vol) ratio of protein solution to precipitant solution. Small andplate-like crystals were formed within 3 weeks by using a 0.1 M Tris-HClbuffer (pH 7.5) containing 25% (wt/vol) polyethylene glycol 4000(Sigma-Aldrich) and 0.2 M ammonium acetate as a precipitant solution.Crystals suitable for the diffraction analysis were obtained by using themicroseeding technique. The crystals were flash-frozen before data collec-tion with a cryoprotectant containing 35% (vol/vol) glycerol and 20%(wt/vol) polyethylene glycol 4000. The diffraction intensities of the crys-tals were collected using synchrotron radiation from BL41XU at SPring-8(Harima, Japan). The X-ray diffraction was measured with a charge-cou-pled-device camera equipped at the station, and the intensities were inte-grated and scaled using the HKL2000 program (23). The tertiary structureof DcsE was solved by the molecular replacement method using theatomic coordinates of the Haemophilus influenzae HAT (Protein DataBank accession number 2B61) (24) as a search model and the programMolrep in the CCP4 program suite (25). The model was refined by thesimulated annealing and conventional restrained refinement methods us-ing the CNS program (26). A subset of 5% of the reflections was used tomonitor the free R factor (Rfree) (27). Each refinement cycle includesrefinement of the positional parameters and individual isotropic thermalparameters of the temperature-dependent factor (B factors) as well asrevision of the model, which was visualized by the program Xfit in theXtalView software package (28). When the electron densities above 3� inthe Fo-Fc map (Fo and Fc mean observed and calculated structure factors,respectively) and 1� in the 2Fo-Fc map were found apart from the proteinregion, water molecules were assigned to the positions and included in thenext refinement. However, if the B factor of the added water was refined tobe larger than 55 Å2, it could be removed.

The crystals of the G52A-P55G mutant were obtained by using almostthe same condition used for the wild type, except for the use of a slightlylower concentration of polyethylene glycol 4000. The X-ray diffractionintensities of the crystal were collected using synchrotron radiation atSPring-8, and the structure was refined by CNS. Details of the data collec-tion and refinement statistics are shown in Table 1.

Model structure. A tetrahedral intermediate structure, which shouldbe formed after the nucleophilic attack of the hydroxyl group of substrate(L-serine or L-homoserine) to the carbonyl carbon in the acetyl moietyattached to active site Ser149, was manually created. In detail, the tetra-hedral intermediate structure, which is formed between the acetylated Serresidue and L-serine or L-homoserine as the substrate, was constructed byusing the ChemBioDraw Ultra (version 12.0) suite (CambridgeSoft Cor-poration), and the energetically minimized structure was theoreticallycalculated. Then, the intermediate structure was manually fit into theactive site of DcsE by using a molecular visualization system, PyMOL (29).In this step, only the torsion angles were changed.

Protein structure accession numbers. The atomic coordinates andstructure factors of the wild-type and G52A-P55G mutant of DcsE havebeen deposited in the Protein Data Bank with accession codes 3VVL and3VVM, respectively.

FIG 1 Predicted biosynthetic pathway of DCS in S. lavendulae ATCC 11924.HS-CoA, reduced CoA.

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RESULTSKinetic analysis of DcsE. In a previous study, we demonstratedthat DcsE acetylates the hydroxyl group of L-serine in the presenceof acetyl-CoA (10). Since the protein sequence is homologous tothe HAT sequence, we expect that DcsE may also acetylate L-ho-moserine, confirming experimentally that DcsE catalyzes the O-acetylation of L-homoserine in the presence of acetyl-CoA. For theenzyme kinetic study, we measured the acetylating activity of DcsE

under the given concentrations of L-serine or L-homoserine in thepresence of a sufficient (15 mM) amount of acetyl-CoA (Table 2).The kinetic response of DcsE showed that the Km value for L-serine(4.9 mM) was lower than that for L-homoserine (98 mM). How-ever, the kcat value for L-serine (95 min�1) was almost the same asthat for L-homoserine (88 min�1), indicating that the DcsE pro-tein homologous to HAT preferentially acetylates L-serine ratherthan L-homoserine.

Overview of structure of DcsE. The structures of HATs fromH. influenzae (24) and Leptospira interrogans (30), which show33% and 35% sequence identities with DcsE, respectively, are al-ready known. The size-exclusion profile of DcsE on HPLC indi-cates that DcsE behaves as a dimer in solution, like two structurallydefined HATs. An asymmetric unit of the DcsE crystal containstwo subunits that form a dimer. The structure of the DcsE mono-mer reveals a two-domain organization in which one domain, the�/�-hydrolase domain, consists of residues 1 to 176 and 289 to374, and the other domain, the helical domain, is composed ofresidues 179 to 286 (Fig. 2). The �/�-hydrolase domain contains astrongly twisted eight-stranded � sheet (�1 to �8). Four � helices(�1-�2 and �8-�9) are flanked on one side of the sheet, and two �helices (�10-�11) are on the opposite face. The helical domain,which is composed of five � helices (�3 to �7), is inserted betweenthe �6 strand and the �8 helix. In the dimer structure, the �3 and�4 helices from each subunit form an antiparallel four-helix bun-dle surrounded by three helices (�5 to �7) from each subunit. Thisindicates that the helical domain is involved in the formation ofthe dimer structure. The average B factor of the atoms in thehelical domain is higher than that in the �/�-hydrolase domain(Table 1). In particular, the electron density of residues 244 to 249in one monomer and residues 244 to 248 in the other monomer isinvisible. These residues are part of a long loop between �4 and�5. Furthermore, the Glu208 residue in one monomer and theAsp205 residue in the other monomer adopt energetically unfavor-able torsion angles in the Ramachandran plot. The Asp205 andGlu208 residues are located in the turn region between �3 and �4,and the electron densities are weak. These observations indicatethat the increased value of the average B factor in the helical do-main is the result of a higher number of flexible loops. A deeptunnel is formed by the juxtaposition of two domains in eachsubunit. The residues lining the wall of the tunnel are predomi-nantly polar and include residues Ser54, Asp56, Asp65, Thr67,Ser149, Arg218, Tyr225, His352, and Asp353.

Interestingly, although DcsE prefers L-serine over L-homoser-ine as a substrate, the protein possesses a three-dimensional struc-ture similar to that of HAT but distinct from the well-known SATstructure. A comparison among the monomer structures of DcsE

TABLE 1 Data collection and refinement statistics

Parameter

Value(s) fora:

Wild type G52A-P55G mutant

Data collection statisticsSpace group P212121 P212121

Unit cell dimensionsa (Å) 46.7 46.6b (Å) 102.6 102.3c (Å) 146.8 147.3

Wavelength (Å) 1.00000 1.00000Resolution (Å) 100–1.81 100–1.70Unique reflection 65,230 78,450Redundancy 7.0 (6.8) 7.2 (6.7)Completeness (%) 99.6 (96.9) 99.9 (99.9)Rmerge (%)b 6.5 (24.9) 5.7 (22.7)I/� 33.9 (5.5) 38.1 (5.4)

Refinement statisticsResolution (Å) 30–1.81 30–1.70Used reflections 64,077 77,226No. of atoms

Protein 5,578 5,574Solvent 398 414

R (%) 19.6 19.2Rfree (%) 22.4 21.4RMSDc

Bond length (Å) 0.005 0.004Bond angle (°) 1.3 1.3

Mean B factor (Å2)Protein 22.9 20.9

�/�-Hydrolase domain 21.2 19.4Helical domain 27.0 26.0

Solvent 27.9 26.1Ramachandran plot

Favored (%) 88.6 88.3Allowed (%) 11.3 11.4Disfavored (%) 0.2 0.3

a Values in parentheses are for the highest-resolution bin.b Rmerge � |I � I� |/I, where I is the observed intensity and I� is the mean value of I.c RMSD was calculated by CNS (26).

TABLE 2 Kinetic parameters of DcsE and its mutants

Enzyme

L-SerineaL-Homoserine

SpecificitybKm (mM)kcat

(min�1)kcat/Km

(min�1 mM�1) Km (mM)kcat

(min�1)kcat/Km

(min�1 mM�1)

Wild type 4.9 � 1.3 95 � 8.7 19 � 5.4 98 � 29 88 � 10 0.90 � 0.29 21 � 9.1G52A ND ND 0.0022 � 0.00038 25 � 6.6 13 � 0.91 0.52 � 0.26 0.042 � 0.0022P55G 0.50 � 0.083 37 � 1.1 74 � 10 1.0 � 0.11 38 � 1.1 38 � 4.5 1.9 � 0.35G52A-P55G ND ND 0.0029 � 0.0012 2.0 � 0.20 47 � 1.3 24 � 2.8 0.00012 � 0.000052a ND, not determined by the nonlinear least-squares method.b Values were defined as the ratio of kcat/Km for L-serine to that for L-homoserine. As the enzyme preferentially acts on L-serine rather than L-homoserine, the values are higher.

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and two structurally defined HATs indicates that DcsE is moresimilar to HAT from H. influenzae (Fig. 3A), which can be super-imposed with a root mean square deviation (RMSD) of 0.95 Å forthe 266 C-� atoms, than to that from L. interrogans (Fig. 3B),which can be superimposed with an RMSD of 1.39 Å for the 257C-� atoms. The �/�-hydrolase domains in the three enzymes have

similar structures. The �/�-hydrolase domain of DcsE can be su-perimposed on that of the H. influenzae HAT with an RMSD of0.98 Å for the 176 C-� atoms and on that of the L. interrogans HATwith an RMSD of 0.99 Å for the 182 C-� atoms. However, al-though the structures of the helical domains are similar betweenDcsE and the H. influenzae HAT, the structure of the L. interrogansHAT is different from the structures of DcsE and the H. influenzaeHAT. In detail, �3, �4, and �7 in the helical domain are superim-posable among DcsE and two structurally elucidated HATs. How-ever, the positions and orientations of �5 and �6 in the helicaldomain of the L. interrogans HAT are largely different from thoseof DcsE and the H. influenzae HAT. Furthermore, the positionalrelationship of two subunits in the DcsE dimer is similar to that inthe H. influenzae HAT but considerably different from that in theL. interrogans HAT (Fig. 3C and D). The dimer organization maybe altered by the variations of the structures in the helical domain,although the effects of crystal packing on the positional relation-ships of two subunits cannot be ignored.

Active site of DcsE. An �/�-hydrolase superfamily which in-cludes HATs has been well studied for over 20 years (31). Thecanonical fold consists of an eight-stranded, mainly parallel �sheet which is surrounded by a total of six � helices (32). Thesecond strand of the fold is oriented in the antiparallel direction.The amino acid sequence similarity among the various membersof this superfamily is effectively nonexistent, suggesting substan-tial evolutionary divergence (33). This divergence is also reflectedin the plethora of chemical transformations that are catalyzed by�/�-hydrolase superfamily members. The catalytic functions,such as hydrolase, thioesterase, haloperoxidase, dehalogenase,and COC bond-breaking activities, have been observed in mem-bers of this superfamily (34). However, the domain seen in thesuperfamily protein possesses a conserved structural feature,called a catalytic triad, which is constituted by Ser, Asp, and Hisresidues.

The residues present in the active site are disordered in thecrystal structure of the L. interrogans HAT (30), whereas they areordered in the crystal structures of DcsE and the H. influenzaeHAT (24). In addition, a substrate-binding pocket in the L. inter-rogans HAT is more open than that in DcsE and the H. influenzae

FIG 2 Overall structure of DcsE. A stereo view of a monomeric form of DcsE is shown in a ribbon representation. The central � sheet is colored in cyan. The �helices in the �/�-hydrolase domain are colored in magenta, and those in the helical domain are in green. The side chains of the residues forming a catalytic triad(Ser149, Asp319, and His352) are shown as sticks, of which the carbon atoms are shown in yellow. The figure was drawn by PyMOL (29).

FIG 3 Structural comparison of DcsE with two HATs. The monomeric anddimeric structures of DcsE (red) are superimposed on those of HAT from H.influenzae (blue in panels A and C, respectively) and those from L. interrogans(green in panels B and D, respectively). The direction of the view in A and B isthe same as that in Fig. 2. The C-� atoms in the �/�-hydrolase domain weremaximally fitted in panels A and B, while those in one monomer were maxi-mally fitted in panels C and D. Structural superposition was performed usingthe align command in PyMOL (29).

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HAT. Since the crystal structures of DcsE and the H. influenzaeHAT may better reflect the enzymatic active form compared withthe crystal structure of the L. interrogans HAT, we discuss theactive-site structure of DcsE by comparing it with that of the H.influenzae HAT, as described below: on the bottom of the tunnelof DcsE, Ser149, Asp319, and His352 residues form a catalytic triad(Fig. 2 and 4A), which correspond to Ser143, Asp304, and His337 inthe H. influenzae HAT, respectively (Fig. 4B). It should be notedthat the torsion angles of the active-site Ser149 residue in DcsE arelocated in the generously allowed region in the Ramachandranplot, but the electron density is clear, like those of the catalytic Serresidues of the enzymes in the �/�-hydrolase superfamily. Theenzymatic reaction of DcsE would be progressed through theacetylated enzyme on the basis of the proposed reaction mecha-nism of HAT (20, 35, 36); entrance of the first substrate acetyl-CoA into the active site is followed by initiation of a nucleophilicattack of the hydroxyl moiety of Ser149 on the thioester bond ofacetyl-CoA. The predicted tetrahedral transition state is stabilizedby means of an oxyanion hole formed by the backbone amidenitrogens of Leu53 and Met150. Met150 is situated at the N terminusof the �2 helix, which has an additional positive charge via a helicaldipole effect. After the collapse of the tetrahedral transition state,an acetylated enzyme intermediate in which the Ser149 residue isacetylated is formed, together with the release of CoA. Next, theentrance of L-serine into the active site is followed by a nucleo-philic attack of the hydroxyl moiety on the carbonyl carbon of theacetyl group through a second tetrahedral transition state, againstabilized by the same oxyanion hole.

We attempted to obtain crystals of DcsE bound to L-serine orL-homoserine by the soaking or cocrystallization experiment.However, no amino acid might be bound to DcsE in the crystals,judging from the electron density map. The crystal structures of

the structurally defined HATs bound with a substrate or inhibitorhave not yet been reported (24, 30). In the H. influenzae HAT, sidechains of Arg212 and Asp338, which correspond to Arg218 andAsp353 in DcsE (Fig. 4A and B), respectively, are proposed to in-teract with the carboxyl and amino groups of L-homoserine, re-spectively (24). Since the side chain of L-serine, to which DcsEbinds favorably, is one carbon unit shorter than that of L-homo-serine, the substrate-binding pocket of DcsE might be more com-pact than that of HAT. However, the relative positions of the im-portant residues for the catalysis and the binding of the secondsubstrate are nearly identical in DcsE and the H. influenzae HAT.Additionally, it is worth noting that the residue that would bind tothe carboxyl group of the second substrate (Arg218 of DcsE orArg212 of the H. influenzae HAT) is derived from the helical do-main, whereas the other important residues are from the �/�-hydrolase domain. This indicates that the size of the substrate-binding pocket may be varied during the binding of the secondsubstrate.

Interestingly, a turn region containing Leu53 of DcsE, whichmay be responsible for the formation of the oxyanion hole, isstructurally different from the counterparts of the structurallyknown HATs. The first and last residues in the turn of DcsE areGly52 and Pro55, respectively, while the corresponding residues ofHAT are Ala and Gly (Fig. 5A and B). In the HAT structure (Fig.5B), a hydrogen bond is formed between the carbonyl oxygen ofthe first residue and the backbone amide of the last residue in theturn. In contrast, a hydrogen bond is not formed in the corre-sponding turn of DcsE because the last Pro residue possesses noamide hydrogen (Fig. 5A). Additionally, due to the steric hin-drance of the side chain of the Pro55 residue, the region after theturn is slightly apart from the �3 strand immediately before the

FIG 4 Structures of active site in DcsE (A) and the H. influenzae HAT (B). Water molecules (indicated by the letter W with a number) are shown as red spheres,and the dotted lines represent hydrogen bonds.

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turn. The unusual turn in DcsE may be closely associated withthe enzymatic function of DcsE.

Furthermore, more water molecules are lined on one side ofthe turn of DcsE than in the case of HAT, and a robust hydrogen-bonding network is formed in DcsE (Fig. 5A and Table 3). Indetail, water molecule 1 (Wat1) forms hydrogen bonds with thebackbone amide and hydroxyl group of the Ser54 residue and withWat2 and Wat4. Moreover, five waters (Wat3 to Wat7) are locatedbetween the amide nitrogen of Gly52 and that of Asp56, althoughthe electron densities of Wat5 and Wat6 are slightly weak. Further-more, Wat4 can bind to Wat1 and Wat2, and Wat5 binds to thebackbone carbonyl oxygen of the Trp70 residue by the mediationof Wat8. In the crystal structure of the H. influenzae HAT, whichwas determined at a higher resolution (1.65 Å) than that of DcsE,two water molecules corresponding to Wat5 and Wat7 in DcsE arenot found (Fig. 5B and Table 3); only six water molecules arepresent on one side of the turn. As a result, the number of hydro-gen bonds in the H. influenzae HAT is lower than that in DcsE.Moreover, the positions of water molecules in the H. influenzae

HAT are somewhat different from those in DcsE. Namely, Wat2

and Wat4 in the H. influenzae HAT are apart from the correspond-ing positions in DcsE, and a hydrogen bond is not formed betweenthem. The positional differences of the water molecules betweenDcsE and the H. influenzae HAT are likely to be caused by theeffects of the side chains in the turn. That is, since the first residue(Gly52) in the turn of DcsE contains no side chain, Wat2 can existnear the residue in DcsE. In contrast, since the last residue (Pro55)in the turn of DcsE has a side chain, Wat4 must exist separatelyfrom the residue. These two constraints on the positions of Wat2

and Wat4 seem to make it possible to form a robust hydrogen-bonding network on the turn of DcsE with the help of two addedwaters.

Substrate specificity of DcsE. As described above, the unusualturn at the active site of DcsE may play an important role in thesubstrate specificity. We examined the enzymatic properties of thesite-directed mutants of DcsE, in which the Gly52 and Pro55 resi-dues in the turn are replaced by Ala and Gly, respectively. Asshown in Table 2, a G52A mutant of DcsE has much lower activityfor the acetylation of L-serine but keeps the activities toward L-homoserine. A mutant of DcsE, P55G, can acetylate both L-serineand L-homoserine. In particular, the Km value of the mutant forL-homoserine decreased much more dramatically than that of thewild type. A double mutant, designated G52A-P55G, utilizes L-homoserine as a substrate for acetylation but scarcely utilizes L-serine. These results indicate that the turn structure is an impor-tant factor in deciding the substrate specificity.

To investigate the relationship of the turn structure on thesubstrate specificity, we determined the crystal structure of theG52A-P55G mutant of DcsE, which preferentially catalyzesthe O-acetylation of L-homoserine, revealing that the turn struc-ture in the G52A-P55G mutant (Fig. 5C) is similar to that in the H.influenzae HAT (Fig. 5B). Of the water molecules present on oneside of the turn in the G52A-P55G mutant, Wat2, Wat5, and Wat6

are lost in one subunit, while Wat2, Wat6, and Wat7 are lost in theother subunit (Table 3). In addition, the positions of the remain-ing water molecules in the mutant are different from those in the

FIG 5 Water molecules on one side of the turn in the active site. in wild-type DcsE (A), the H. influenzae HAT (B), and the G52A-P55G mutant of DcsE (C).

TABLE 3 B factors of water molecules lining one side of the turn region

Watermolecule

B factor (Å2)a

DcsE

H. influenzae HATbWild type G52A-P55G

Wat1 33.4 (30.4) 29.1 (29.0) 34.4Wat2 26.6 (32.2) — (—) 37.0Wat3 22.0 (14.5) 20.6 (15.8) 19.3Wat4 22.4 (19.6) 24.3 (22.6) 32.7Wat5 33.7 (34.0) — (30.2) —Wat6 35.3 (28.8) — (—) 35.9Wat7 18.5 (19.8) 31.2 (—) —Wat8 26.3 (25.3) 29.0 (23.8) 33.3a Values are B factors of waters bound to one monomer, whereas those in parenthesesare B factors of water molecules bound to the other monomer contained in anasymmetric unit. —, absence of the corresponding water molecule.b An asymmetric unit of the crystal of the H. influenzae HAT contains one monomer.

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wild type: Wat1 is apart from the turn, whereas Wat3, Wat4, andWat5 are closer to the turn in the G52A-P55G mutant. Further-more, a hydrogen bond is formed neither between Wat1 and Wat4

nor between Wat7 and the amide nitrogen of Asp56. As a result, therobust hydrogen-bonding network, which is found in the wildtype, is not formed in the G52A-P55G mutant. The average Bfactor of the atoms in residues 51 to 56 of the wild-type DcsE islower than that of all protein atoms by 6.1 Å2. However, the aver-age B factors of the atoms in the corresponding residues of theG52A-P55G mutant of DcsE and the H. influenzae HAT are lowerthan those of all protein atoms by 4.9 and 3.1 Å2, respectively. Thestability of the turn in the wild-type DcsE may occur because of therobust hydrogen-bonding network near the turn region. Further-more, the electron densities of the side chains of Arg218 and Asp353

in the DcsE mutant, which may be necessary for the binding of thesubstrate, are weaker than those in the wild type. The flexibility ofthe substrate-binding residues in the mutant may occur as a resultof the disappearance of the robust hydrogen-bonding networknear the turn region.

DISCUSSION

HAT is an enzyme that catalyzes the transfer of the acetyl groupfrom acetyl-CoA to the hydroxyl group of L-homoserine in theL-methionine biosynthetic pathway. However, DcsE efficiently

catalyzes the transfer of the acetyl group from acetyl-CoA to thehydroxyl group of L-serine, despite being highly homologous toHAT. When determined in an in vitro experiment, the Km valuefor L-serine of DcsE is high (4.9 � 1.3 mM). However, we havefound that the DcsE-encoding gene is located in the DCS biosyn-thetic gene cluster (10), and other research groups (13, 15) havedemonstrated that the formation of O-acetyl-L-serine from L-ser-ine is inevitable as one step in DCS biosynthesis. In general, L-ser-ine O-acetyltransferase (SAT) is necessary as an enzyme to acety-late L-serine for the biosynthesis of L-cysteine.

On the basis of the proposed reaction mechanism of HAT (20,35, 36), DcsE transfers an acetyl group from acetyl-CoA onto theSer149 residue and then transfers the acetyl group from Ser149 ontoL-serine through the formation of the tetrahedral intermediate.Based on the crystal structure of DcsE, we constructed the pre-dicted model for the second transition state (Fig. 6A). The modelshows that the carboxyl and amino moieties of L-serine can bepositioned near the side chains of Arg218 and Asp353, respectively,to form salt bridges to the enzyme. In addition, the carboxyl groupof L-serine can further interact with the backbone amide nitrogenand the side chain hydroxyl of Ser54, and the � carbon of thesubstrate can be positioned near the Gly52 residue. Furthermore,the negative charge formed on the carbonyl oxygen from the acetylmoiety can be stabilized by the backbone amide nitrogens of Leu53

FIG 6 Predicted transition-state models of DcsE. The transition states, which may be formed after the binding of L-serine (A) or L-homoserine (C) to theacetylated DcsE intermediate, are modeled. (B and D) The L-serine (A) and L-homoserine (C) binding models viewed from another direction, respectively. Inthese models, Wat3 to Wat7 were inserted into the structures in panels A and C and Arg218, His352, and Asp353 were eliminated. (E) The model of the G52A mutantbound to L-serine, in which Gly52 in panel A was replaced by Ala. The carbon atoms from substrates and acetyl groups are shown in green.

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and Met150. On the basis of this binding model, since the twointeraction distances between the carboxyl group of L-serine andthe side chains of Arg218 (3.5 Å) and between the amino group ofL-serine and the side chains of Asp353 (3.5 Å) are somewhat long,the formed salt bridges are weak. However, in the actual enzymaticreaction, a conformational change may be induced in DcsE tobind L-serine more strongly. In addition, the binding of L-serinewould be accompanied by the disappearance of only Wat1 andWat2 (Fig. 6B).

We also constructed a predicted transition-state model boundto L-homoserine instead of L-serine (Fig. 6C). In contrast to theL-serine-bound model, the carboxyl and amino moieties maystrongly interact with the side chains of Arg218 and Asp353, respec-tively. It is also suggested that the carboxyl group of L-homoserinecan interact with the backbone amide and the side chain hydroxylof Ser54. In fact, the residue corresponding to Ser54 in DcsE is Seror Thr in HATs, indicating that the hydroxyl moiety of the thirdresidue in the turn of HAT is involved in the binding of L-homo-serine. However, on the basis of the predicted model, the � and �carbons of the substrate are too close to Wat4 (Fig. 6D). Therefore,in addition to Wat1 and Wat2, Wat4 must be removed from theactive site for the binding of L-homoserine to DcsE. The B factor ofWat4 is relatively low, indicating that the Wat4 molecule is inflex-ible (Table 3). The removal of Wat4 may be energetically difficult,since it accompanies the loss of hydrogen bonds with the sur-rounding water molecules. Due to the formation of a robust hy-drogen-bonding network on one side of the turn in DcsE, DcsE islikely to have a low affinity toward L-homoserine, leading to thelow reactivity toward L-homoserine. In contrast, HAT or theG52A-P55G mutant of DcsE, in which a robust hydrogen-bond-ing network is not formed on one side of the turn, can moreefficiently acetylate L-homoserine.

The G52A single mutant and G52A-P55G double mutanthardly acetylate L-serine (Table 2). We constructed the predictedtransition-state model between the G52A mutant and L-serine(Fig. 6E) by adding a methyl group to the � carbon of Gly52 in thetransition-state model between the wild-type DcsE and L-serine.In the model, the � carbon of L-serine is too close to the intro-duced methyl group of Ala52, suggesting the low affinity of theG52A mutant toward L-serine. This is in agreement with an exper-imental result. The kcat/Km values for both L-serine and L-homo-serine in the P55G single mutant were improved (Table 2). Thissuggests that the mutant is able to handle both substrates. Consid-ering the information obtained in this study, since a robust hydro-gen-bonding network may not be formed on one side of the turnnear the active site in the mutant, it recognizes L-homoserine as asubstrate. In addition, the mutant may be able to acetylate L-serinedue to the absence of the side chain at the Gly52 residue.

Whether O-acetyl-L-serine as an enzyme product by DcsE isinvolved in L-cysteine biosynthesis in DCS-producing S. lavendu-lae remains undetermined. According to available genome se-quence information, genes encoding proteins homologous toHAT or SAT are not found in all strains that belong to the genusStreptomyces. However, each streptomycete contains a few genesencoding a protein homologous to O-acetylserine sulfhydrylase,which catalyzes the synthesis of L-cysteine using O-acetyl-L-serineand sulfide. One of the O-acetylserine sulfhydrylase homologuesin each strain shows high sequence similarity with CysM fromMycobacterium tuberculosis. The enzyme shows low O-acetylser-ine sulfhydrylase activity but efficiently catalyzes the addition of

the Cys residue to the C terminus of CysO using O-phospho-L-serine and thiocarboxylated CysO (37–39). After the CysM reac-tion, L-cysteine is produced from CysO by the action of carboxy-peptidase, Mec�. In all of the streptomycete genomes alreadysequenced, three putative genes encoding CysM, CysO, and Mec�

are clustered. In addition, it was reported that streptomycetesshow a weak ability to produce sulfide from sulfate (40), althoughthey have genes responsible for the reduction of sulfate. Theseobservations indicate that L-cysteine is generated via O-phospho-L-serine, but not O-acetyl-L-serine, in DCS-producing S. lavendu-lae. O-Acetyl-L-serine, a reaction product of DcsE, may participateonly in the biosynthesis of DCS.

An in silico study to find proteins homologous to HAT sug-gested that an amino acid sequence of the turn region which isinvolved in the formation of the oxyanion hole is classified into thefollowing two sequences: Ala-(Leu/Phe)-(Ser/Thr)-Gly and Gly-Leu-Ser-(Pro/Ala). HAT homologues carrying the former se-quence may catalyze only the acetylation of L-homoserine,whereas those carrying the latter sequence, like DcsE, may prefer-entially acetylate L-serine. In bacteria, the latter enzymes exist onlyin the family Xanthomonadaceae, which includes the genera Pseu-doxanthomonas, Stenotrophomonas, Xanthomonas, and Xylella,known as plant-pathogenic bacteria. The genome informationsuggests that many fungi possess the two kinds of HAT describedabove. In most cases, these fungi have O-acetylserine sulfhydry-lase-encoding genes but not SAT-encoding genes, indicating thatDcsE-like HAT homologues may function in the L-cysteine bio-synthetic pathway in these microbes, as suggested in Aspergillusnidulans (21). On the basis of the tertiary structure of DcsE deter-mined in this study, if an inhibitor to the enzyme can be devel-oped, its use as an antifungal agent will be expected.

ACKNOWLEDGMENTS

We are grateful to the beam-line staff at SPring-8, Japan, for their kindhelp with X-ray data collection. We also thank Takemasa Sakaguchi forvaluable advice.

We also give thanks to the Japan Aerospace Exploration Agency(JAXA) for the crystallographic experiment and financial support.

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