Báo cáo khoa học: High resolution structure and catalysis of O-acetylserine sulfhydrylase isozyme B from Escherichia coli pot

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High resolution structure and catalysis of O-acetylserinesulfhydrylase isozyme B from Escherichia coliGeorg Zocher, Ulrich Wiesand and Georg E. SchulzInstitut fu¨r Organische Chemie und Biochemie, Albert-Ludwigs-Universita¨t, Freiburg im Breisgau, GermanyIn bacteria, archaea and plants, the biosynthesis ofl-cysteine involves l-serine and inorganic sulfur com-pounds [1–5]. In higher animals, however, l-cysteineis derived from l-methionine [1]. The bacterial path-way starts with a transferase that uses acetyl-CoA tomodify serine. The resulting O-acetylserine (OAS) isthen converted to cysteine by a sulfhydrylase (OASS,EC 2.5.1.47), which in general uses hydrogen sulfide.In a number of bacteria, the second step of synthesisis performed by the two isozymes A and B, namedCysK and CysM, respectively. CysK uses mostlyhydrogen sulfide, which is produced in a reductionpathway that begins with sulfate and requires dioxy-gen. In contrast, CysM has a characteristic mainchain variation around position 210 that opens theactive center for larger thiol-carrying compounds, inparticular for thiosulfate [2,6]. The reaction with thio-sulfate results in S-sulfo-cysteine, which can be easilyconverted to cysteine and sulfate. Consequently, theuse of thiosulfate is of particular importance in ananaerobic environment, because it does not requiredioxygen for the reduction of sulfate to hydrogensulfide. The isozyme CysM is of technical interestbecause it processes compounds much larger thanhydrogen sulfide, and is therefore a promising candi-date for the production of novel b-substitutedl-amino acids as building blocks for the synthesis ofpharmaceuticals and agrochemicals [7–9].Keywordsbiosynthesis ofL-cysteine; enzymatic assay;homodimer asymmetry; nonstandardL-amino acids; X-ray diffractionCorrespondenceG. E. Schulz, Institut fu¨r Organische Chemieund Biochemie, Albert-Ludwigs-Universita¨t,Albertstr. 21, 79104 Freiburg im Breisgau,GermanyFax: +49 761 203 6161Tel: +49 761 203 6058E-mail: georg.schulz@ocbc.uni-freiburg.deWebsite: http://www.structbio.uni-freiburg.de(Received 24 July 2007, revised 22 August2007, accepted 23 August 2007)doi:10.1111/j.1742-4658.2007.06063.xThe crystal structure of the dimeric O-acetylserine sulfhydrylase isozyme Bfrom Escherichia coli (CysM), complexed with the substrate analog citrate,has been determined at 1.33 A˚resolution by X-ray diffraction analysis.The C1-carboxylate of citrate was bound at the carboxylate position ofO-acetylserine, whereas the C6-carboxylate adopted two conformations.The activity of the enzyme and of several active center mutants was deter-mined using an assay based on O-acetylserine and thio-nitrobenzoate(TNB). The unnatural substrate TNB was modeled into the reported struc-ture. The substrate model and the observed mutant activities may facilitatefuture protein engineering attempts designed to broaden the substrate spec-trum of the enzyme. A comparison of the reported structure with previ-ously published CysM structures revealed large conformational changes.One of the crystal forms contained two dimers, each of which comprisedone subunit in a closed and one in an open conformation. Although thehomodimer asymmetry was most probably caused by crystal packing, itindicates that the enzyme can adopt such a state in solution, which may berelevant for the catalytic reaction.AbbreviationsCysK, O-acetylserine sulfhydrylase (EC 2.5.1.47) isozyme A; CysM, O-acetylserine sulfhydrylase (EC 2.5.1.47) isozyme B from Escherichiacoli; CysM(K268A), surface mutant K268A of CysM; CysM(RKE), triple surface mutant E57R-Y148K-R184E of CysM; CysM(salmo),isozyme B from Salmonella typhimurium; DTNB, S,S¢-bis(5-thio-2-nitrobenzoate); TNB, thio-nitrobenzoate; OAS, O-acetylserine; OASS,O-acetylserine sulfhydrylase; PLP, pyridoxal 5¢-phosphate.5382 FEBS Journal 274 (2007) 5382–5389 ª 2007 The Authors Journal compilation ª 2007 FEBSFive structures of CysK-type enzymes from bacteria[10–14], archaea [15] and plants [16,17], and two struc-tures of bacterial CysM [6,18], have been published.The differences between the isozymes CysK and CysMhave been described [6,18]. In this article, we presentthe structure of CysM complexed with the substrateanalog citrate at high resolution, together with enzy-matic activity data of several mutants. Moreover, weprovide a model of the substrate thio-nitrobenzoate(TNB) bound at the active center, which may be aguide for future enzyme engineering studies.Results and DiscussionCysM structuresIn solution, CysM from E. coli is a dimer of2 · 32 893 Da consisting of 303 amino acid residuesper subunit. An earlier study [6] yielded a mediumquality structure of the wild-type enzyme in crystalform I at 2.7 A˚resolution [P6522, four subunits perasymmetric unit; reservoir: 0.1 m ammonium sulfate,0.1 m citrate pH 5.6 with poly(ethyleneglycol)]. Animproved structure was derived from crystal form II ofthe triple surface mutant CysM(RKE) that diffractedto 2.1 A˚resolution, but was completely twinned,decreasing the effective resolution [I41, four subun-its per asymmetric unit; reservoir: 0.15 m CaCl2, 0.1 mHepes pH 7.6 with poly(ethyleneglycol)] [6]. In thisarticle, we report the structure of the surface mutantCysM(K268A) at 1.33 A˚resolution in crystal form III(Table 1). Crystal form III was grown essentially underthe same conditions as form I, except for the absenceof ammonium sulfate. The surface mutation K268Awas at the rim of a packing contact and was notrequired for crystallization, but was essential for thesuperior packing order and for reproducible crystalgrowth.The structure of crystal form III was determined bythe molecular replacement method. In contrast withthe other crystal forms, form III contained only onesubunit per asymmetric unit and a lower solventcontent, both of which are typical prerequisites forhigh resolution X-ray diffraction (Table 1). Althoughcrystal forms I and III were grown from the samecitrate buffer, only form III showed a citrate moleculebound to the active center. Apparently, the high ionicstrength of ammonium sulfate prevented citratebinding in form I. The structure of CysM in crystalform III is shown in Fig. 1. Citrate was bound intwo conformations with occupancies of 60% and40%, as revealed by the electron density depicted inFig. 2. Binding in multiple conformations indicateslow affinity, which, in turn, agrees with our observa-tion that citrate does not inhibit the enzyme (seebelow).In order to identify established structures of relatedenzymes, we searched the Protein Data Bank forsequence homologs and detected 11 entries withsequence identity above 30%, all of which wereOASSs. Lowering the threshold, the next entries weretwo cystathione b-synthases with 29% and 24% iden-tity. Ten of the entries were CysK-type enzymes, whichshowed around 40% sequence identity with isozymeCysM and are not considered in the following analysis.One entry was CysM from Salmonella typhimurium[CysM(salmo)] [18], which has 94% sequence identityand is closely related to the enzyme CysM from E. colipresented here.Enzymatic activity and reaction geometryIn order to obtain data on enzyme engineering for thesynthesis of novel compounds [7–9], we producedactive center mutants and determined their catalyticactivity using TNB as the nucleophile. TNB seems tobe most appropriate for guiding enzyme engineeringintended for the synthesis of compounds of similarsize. The activities of wild-type CysM and of the crys-tallized mutant K268A were identical, and only thewild-type value is given in Table 2. This agreementTable 1. Structure analysis. Values in parentheses are for the high-est resolution shell. The data were collected at 0.9050 A˚wave-length at beamline PX-II of the Swiss Light Source (SLS, Villigen,Switzerland). The crystal belonged to space group P6522 with unitcell axes a ¼ b ¼ 76.6 A˚and c ¼ 209.8 A˚containing one CysMsubunit per asymmetric unit and 55% solvent.Data collectionResolution (A˚) 63–1.33 (1.37–1.33)Unique reflections 83156 (6220)Completeness (%) 98.6 (87.9)Multiplicity 7.4 (7.9)Rsym-I(%) 5.9 (35)Average I ⁄ rI21.4 (3.5)RefinementNumber of atoms, protein(residues 1–294)2290Number of atoms, glycerol ⁄ citrate 12 ⁄ 26Number of water molecules 329Rcryst⁄ Rfree(2% test set) 0.158 ⁄ 0.172Average isotropic B-factors (A˚2)main chain ⁄ side chains 16.6 ⁄ 20.4glycerol ⁄ citrate ⁄ water 24.2 ⁄ 16.6 ⁄ 33.0Rmsdbond lengths (A˚) ⁄ angles (°) 0.016 ⁄ 1.68Ramachandran: mostfavorable ⁄ allowed (%)98.0 ⁄ 2.0G. Zocher et al. Structure of the O-acetylserine sulfhydrylase CysMFEBS Journal 274 (2007) 5382–5389 ª 2007 The Authors Journal compilation ª 2007 FEBS 5383was expected because position 268 is at the surface dis-tant from the active center and from the dimer inter-face (Fig. 1).The reported CysM structure contains a substrate-like active center ligand, which is the bound citratemolecule depicted in Fig. 2. A comparison with thefour known external aldimine complexes of CysK-typeenzymes [11,13,14,16] showed clearly that the C1-car-boxylate of citrate occupies the binding site of the car-boxylate of OAS. Whereas the C1-carboxylate is wellfixed at loop 69 (residues 68–72), the distal C6-carbox-ylate of citrate adopts two conformations. The hydro-xyl group of citrate points towards the internalaldimine, as is expected for the amino group of OAS(Fig. 2). In view of the bound citrate molecule, wedetermined the enzyme activity in the presence of upto 25 mm citrate, but observed no change. Therefore,citrate is not an inhibitor. This agrees with the twoobserved citrate conformations, because multiple bind-ing is usually weak.The observed kinetic parameters of wild-type CysMfrom E. coli are in general agreement with those of thehomolog CysM(salmo) [18,19]. Of the active centermutants produced, the deletion of a methyl group nearFig. 1. Stereo ribbon plot of the high resolu-tion structure of the CysM dimer, includingthe molecular twofold axis (black), which iscrystallographic. The position of the surfacemutation K268A is shown as a yellowsphere 25 A˚away from the active center.The cofactor PLP covalently linked to Lys41,the bound citrate molecule in its major con-formation and the mutated residues Thr68,Gln140 and Arg210 in the active center aredepicted as ball-and-stick models. The sub-units have different colors. The mobile loopsdefined in Fig. 5 are labeled using grayspheres. The active center pocket openingis indicated by a yellow stick.Fig. 2. Detailed stereoview of the activecenter of CysM. The covalently bound PLPand the associated citrate are shown inorange. Citrate was bound with 100% occu-pancy. The minor conformation of citrate isgray. The (Fo) Fc) electron density map ofcitrate is outlined at the 3.0 r contour level.The mutated residues are cyan. Hydrogenbonds to the citrate molecule are indicatedby broken lines. Chain cuts are marked byhalos.Table 2. Enzymatic activity of CysM from Escherichia coli. The esti-mated relative errors are about 20%. The OAS concentration wasalways 10 mM; the TNB concentration varied from 10 to 1000 lM.The temperature was 37 °C. The values in parentheses were mea-sured at 25 °C.kcat(s)1)KM(TNB)(mM)kcat⁄ KM(TNB)(%)TemperaturedependenceaWild-type 24 0.7 100b(41)c2.4T68S 11 0.6 55 (26) 2.1R210A – – 2 (0.8) 2.5Q140A – – 0.4 (0.1) 4T68A – – 0.1 (0.01) 10Q140E – – Inactive –aThe temperature dependence is defined here as kcat⁄ KM(TNB)measured at 37 °C relative to the value measured at 25 °C.bTheabsolute kcat⁄ KM(TNB) value at 37 °C was 3.5 · 104M)1Æs)1. Thisvalue was set to 100%.cThe absolute kcat⁄ KM(TNB) value at 25 °Cwas 1.4 · 104M)1Æs)1.Structure of the O-acetylserine sulfhydrylase CysM G. Zocher et al.5384 FEBS Journal 274 (2007) 5382–5389 ª 2007 The Authors Journal compilation ª 2007 FEBSpyridoxal 5¢-phosphate (PLP) in mutant T68S causedthe smallest disturbance (Table 2). Given the highactivity of this mutant, we determined the KM(TNB)value, which was essentially identical to that of thewild-type (Table 2). We conclude that the missingmethyl group of T68S decreases the activity onlyslightly and does not affect TNB binding. A decisivedecrease to merely 2% catalytic efficiency wasobserved with mutant R210A. Even stronger decreaseswere caused by the removal of a carboxamide inmutant Q140A, and by the deletion of a hydroxylgroup in mutant T68A. The enzyme was inactive whena carboxylate was introduced at position 140 (Q140E).The moderate activity reduction of T68S and thestrong effects of mutations Q140A, T68A and Q140Eagree well with the data derived for the correspondingmutants of the CysK-type enzyme from Arabidopsisthaliana [16].In a second series of experiments, we determined thekcat⁄ KM(TNB) values at 25 °C. The results were similarto those at 37 °C, except for a 2.3-fold decrease for thewild-type and for mutants T68S and R210A (Table 2).The 2.3-fold decrease relates well to the decrease in kcatexpected from the ‘rule-of-thumb’ factor of two for a10 K temperature drop [20], showing that the activa-tion energy of the catalyzed reaction lies in the usualrange and does not change for T68S and R210A. Incontrast, mutants Q140A and T68A showed muchhigher temperature dependence factors, correspondingto an appreciable increase in the activation energy [20].We conclude that Q140A and T68A, which are closeto PLP, directly affect the reaction. In contrast, theactivity decrease of R210A, which is rather distant fromPLP, is probably a result of inefficient TNB binding,causing a large increase in KM(TNB). The proposedbinding deficiency agrees with our TNB model (seebelow) and also with an earlier thiosulfate model [6].The mutants were also checked with respect to theirA280⁄ A412ratio. A photometric measurement ofCysM(K268A) yielded a ratio of 4.3, which agrees wellwith the ratio of 4.0–4.2 established for the closelyhomologous CysM(salmo) [18]. It also agrees with thetheoretical value calculated from the absorption spec-tra of the tryptophans, tyrosines and PLP. Themutants showed A280⁄ A412ratios in the range 4.3–4.5,except for mutant Q140E with a ratio of 5.5. Thisdeviation was significant. It corresponds to a PLPoccupancy of about 75%. Mutant Q140E showed noenzymatic activity (Table 2). It is conceivable that thenewly introduced glutamate adjacent to PLP made asalt bridge to Lys41, prohibiting the formation of theinternal aldimine (see Fig. 2).In order to model the reaction geometry, we usedthe established external aldimine structure of a relatedCysK structure [11] and transferred it to CysM, whereit could be accommodated without steric collision(Fig. 3). The expected reaction geometry at the exter-nal aldimine intermediate [11] defines the thiol positionof TNB to a small region above the plane of the acry-late double bond. As a result of this constraint and ofthe spacious active center pocket of CysM, TNB wasplaced rather easily. In our model, the carboxylate ofTNB is fixed by Arg210 and the nitro group points tothe solvent (Fig. 3). The thiolate is located above theFig. 3. Stereoview of the reaction geometry based on the structure of CysM(K268A). The observed internal aldimine with Lys41 is given in atransparent mode (gray). The external aldimine structure has been transferred from a CysK-type enzyme [11]. It is shown together with amanually placed model of the bound substrate TNB, the carboxylate of which is fastened to Arg210. The thiolate of TNB is approximately atthe same position as the attacking sulfur of thiosulfate in a previous model [6], which is well suited for the nucleophilic attack (red dottedline) on the amino acrylate double bond (green spheres). Hydrogen bonds are given as black broken lines. All van der Waals distancesbetween TNB and its environment are above 3.0 A˚. The two shortest contacts are marked by green dotted lines.G. Zocher et al. Structure of the O-acetylserine sulfhydrylase CysMFEBS Journal 274 (2007) 5382–5389 ª 2007 The Authors Journal compilation ª 2007 FEBS 5385acrylate plane forming an S–Cb–Ca angle of about90°. This is an ideal position for attacking the doublebond. In summary, our negative experience with muta-tions close to PLP suggests that this region should notbe touched when trying to produce novel l-aminoacids [7–9]. Rather, such engineering attempts shouldfollow the TNB model, which suggests residuesMet119, Phe141, Thr175, Pro207 and Arg210 as themain targets.Induced fitA comparison between the E. coli CysM structures inthe three crystal forms revealed several characteristicfeatures, which are also valid for the crystal structureof CysM(salmo) [18]. In order to establish the intrinsicmechanical properties of CysM, we superimposed theobserved chain folds in Fig. 4. Deviations occurred atthe N- and C-termini and at the four surface loops atpositions 21, 60, 190 and 271, far away from the activecenter and also from the dimer interface. These dif-ferences are of low significance, because they are atpositions that are usually mobile. More interestingvariations occurred near the active center.As shown in Fig. 1, the opening of the active centerpocket to the solvent is rather distant from the dimerinterface. The opening can be considered as a mouthwith two lips. One lip consists of loops 69, 94 andhelix a4(118–132), and the other is formed byloops 202 and 215 (Fig. 4). The lip positions varygreatly between the structures. Similar changes havebeen reported for the other isozyme, CysK, for whichthe extreme lip positions have been named ‘open’ and‘closed’ [11]. The chain fold of CysM(K268A) with thebound citrate is ‘half-closed’ (Fig. 4). Although thevariations in Fig. 4 are probably caused by more orless random crystal contacts, they still outline theavailable conformational space and, most probably,the induced fit motions during the reaction.The conformational changes are also reflected in theB-factor distributions that report the polypeptide chainmobility. As the B-factor level is strongly dependenton the quality of the crystal order, the B-factor distri-butions have been normalized by referring them to theaverage B-factors of the respective chains. They aredisplayed in Fig. 5. The distribution of CysM(K268A)shows nine characteristic mobility peaks. Of these, theloops at peak positions 94, 116, 132, 202 and 215 formthe lips of the mouth of the active center pocket(Fig. 1) and are therefore important for catalysis. Theother peaks correspond to loops at the surface that areusually mobile (Fig. 1). Interestingly, loop 69 is closeto PLP and not mobile (Fig. 5), although it partici-pates in the induced fit (Fig. 4).The mobility distributions of CysM(K268A), wild-type CysM and CysM(salmo), and those of subunits Band D of CysM(RKE), resemble each other closely(Fig. 5). However, a most surprising deviation of theB-factor distribution occurs in subunits A and C ofCysM(RKE) [6]. The CysM(RKE) crystal containsdimers A–B and C–D, providing four independent sub-unit structures. Dimer A–B is asymmetric with respectto mobility and also with respect to structure. TheB-factor distribution of subunit A is exceptional, as itshows almost no mobility peak. In contrast, therespective distribution of subunit B shows the commonmobility peaks, including those of the active center lips(Fig. 5). The same asymmetry is observed with sub-units C and D of the other dimer. As the three muta-tions of CysM(RKE) are all at the surface distantfrom the active center, they are unlikely to affect theinternal stability of the protein. Consequently, theFig. 4. Stereoview of a superposition of fivedistinct CysM chain folds showing wild-typeCysM in blue [6], CysM(RKE) subunit A ingreen, CysM(RKE) subunit D in orange [6],CysM(K268A) in red and CysM(salmo) ingray [18]. The highly mobile regions arelabeled using gray spheres (see Fig. 5).Structure of the O-acetylserine sulfhydrylase CysM G. Zocher et al.5386 FEBS Journal 274 (2007) 5382–5389 ª 2007 The Authors Journal compilation ª 2007 FEBSobserved asymmetry should reflect a general propertyof CysM.The asymmetry of CysM(RKE) is probably causedby crystal packing contacts. Such contacts are usuallyweak, so that they can only switch between conforma-tions that are connected via low energy barriers. As aconsequence, the observation of two independentasymmetric homodimers in a crystal indicates that thisasymmetric state can be easily adopted in solution.Therefore, it is conceivable that the ‘closed’ conforma-tion of subunit A corresponds to the CysM conforma-tion after substrate binding, whereas the ‘open’conformation of subunit B shows CysM when releas-ing the products after the reaction has taken place.Such a see-saw system is discussed as ‘half-site reactiv-ity’ [21]. We conclude that the observed asymmetrysuggests that CysM is a suitable candidate for explor-ing the half-site reactivity hypothesis.Experimental proceduresMutagenesis and activity assayThe mutants were produced with the QuikChange method(Stratagene, Heidelberg, Germany), verified by DNAsequencing (SeqLab, Go¨ttingen and GATC, Konstanz,Germany) and expressed and purified as described previ-ously [6]. They were stored at )20 °Cina12mgÆmL)1solution containing 10 mm Tris ⁄ HCl pH 8.0. For theassay, we incubated 950 lL of buffer A (100 mm HepespH 7.0, 10 mm OAS, 10–1000 lm TNB) at 37 °C (or25 °C) for 3 min, and started the reaction by adding50 lL of a solution containing 0.5–80 lg of the enzyme.The enzyme solution was always freshly prepared fromstored protein so that the exposure time to 37 °C (or25 °C) was minimized. This was important for the lowactivity mutants at positions 68 and 140 near PLP. TNBwas always freshly prepared in 50 mm Hepes pH 7.0by adding 2 mm dithiothreitol and 0.5 mm S,S¢-bis(5-thio-2-nitrobenzoate) (DTNB) to yield 1 mm TNB. Theabsorption of TNB was monitored at 412 nm using e412¼13 600 m)1Æcm)1[19], as well as at 500 nm using e500¼970 m)1Æcm)1, which was established in a separate experi-ment. The measurement at 500 nm was necessary in orderto reach TNB concentrations beyond the Michaelis con-stant of 0.7 mm. The cysteine-nitrobenzoate produced hasits absorption maximum at 312 nm and does not absorblight at 412 nm. The values for kcatand KM(TNB) wereobtained from reciprocal plots; the values forkcat⁄ KM(TNB) were derived from linear plots.Crystallization, structure determination,refinement and modelingThe surface mutant K268A was produced and purified asdescribed previously [6] and then crystallized using thehanging drop method. The drops contained 2 lLofan8mgÆmL)1enzyme solution mixed with 2 lL of reservoirbuffer [100 mm sodium citrate pH 5.4, 18% (w ⁄ v) poly(eth-yleneglycol) 3000]. Crystals of CysM(K268A) grew withinabout 10 days at 20 °C to sizes of up to 1000 lm ·400 lm · 400 lm. The crystals were transferred in foursteps to 28% (v ⁄ v) glycerol in reservoir buffer and flash-frozen in a 100 K nitrogen gas stream.Fig. 5. Relative B-factor distributions of CysM subunits in four dif-ferent crystal forms. The B-factors were referred to the respectivesubunit averages in order to eliminate differences arising from crys-tal packing quality variations. All distributions were smoothed bysliding a three-residue-averaging window along the chain. The topdiagram K268A refers to the reported high resolution structure withlabels at nine high mobility peaks (see Figs 1 and 4). DistributionWT is an average of the four closely related subunit chains of thewild-type structure [6]. The distribution of CysM(salmo) is from sub-unit A, which is virtually the same as those of the other seven sub-units [18]. The two distributions at the bottom are from dimersA–B and C–D of CysM(RKE) [6] which, however, were split into anaverage of the closely related subunits B and D and the equallywell-related subunits A and C.G. Zocher et al. Structure of the O-acetylserine sulfhydrylase CysMFEBS Journal 274 (2007) 5382–5389 ª 2007 The Authors Journal compilation ª 2007 FEBS 5387The X-ray data were collected at the Swiss Light Source(Villigen, Switzerland) (Table 1) and processed with pro-grams xds and xscale [22]. Using phaser [23] and thewild-type CysM structure [6], the phases were establishedby molecular replacement. To avoid model bias, the CysMstructure and the water structure were completely rebuiltusing arp ⁄ warp [24]. The structure was manually com-pleted using coot [25] and then refined with refmac5 [26].Finally, we performed a translation libration screw refine-ment with refmac5 using the 12 translation libration screwgroups (1–22, 23–65, 66–84, 85–98, 99–114, 115–131, 132–164, 165–188, 189–208, 209–221, 222–249, 250–294) pro-posed by the program tlsmd [27]. The CysM structure wasvalidated with rampage [28]. The rigid TNB molecule waspositioned manually into the active center. Numerousoptions were checked visually using coot [25], and inter-preted with respect to the quality of all contacts. The short-est distance to the adjacent residues was maximized inorder to avoid steric hindrance as much as possible. Figureswere drawn using povscript+ [29] and povray (http://www.povray.org). The coordinates and structure factorshave been deposited in the Protein Data Bank under acces-sion code 2v03.AcknowledgementsWe thank the team of beamline PX-II at the SwissLight Source (Villigen, Switzerland) for their help withdata collection, and Wacker-Chemie (Munich, Ger-many) for support of the project.References1 Cooper AJL (1983) Biochemistry of sulfur-containingamino acids. Annu Rev Biochem 52, 187–222.2 Kredich NM (1996) Biosynthesis of cysteine. In Escheri-chia coli and Salmonella typhimurium: Cellular andMolecular Biology (Neidhard FC, ed.), pp. 514–527.ASM Press, Washington DC.3 Borup B & Ferry JG (2000) Cysteine biosynthesis in theArchaea: Methanosarcina thermophila utilizes O-acetyl-serine sulfhydrylase. FEMS Microbiol Lett 189, 205–210.4 Mino K & Ishikawa K (2003) Characterization of anovel thermostable O-acetylserine sulfhydrylase fromAeropyrum pernix K1. 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