Active-Site Mobility Revealed by the Crystal Structure of Arylmalonate Decarboxylase from Bordetella bronchiseptica

J. Mol. Biol. (2008) 377, 386–394

doi:10.1016/j.jmb.2007.12.069

Available online at www.sciencedirect.com

Active-Site Mobility Revealed by the Crystal Structure of Arylmalonate Decarboxylase from Bordetella bronchiseptica E. Bartholomeus Kuettner 1 , Antje Keim 1 , Markus Kircher 2 , Susann Rosmus 2 and Norbert Sträter 1 ⁎ 1

Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany 2

BioSpring GmbH, Alt Fechenheim 34, 60386 Frankfurt am Main, Germany Received 29 October 2007; received in revised form 18 December 2007; accepted 26 December 2007 Available online 5 January 2008 Edited by G. Schulz

Arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica catalyzes the enantioselective decarboxylation of arylmethylmalonates without the need for an organic cofactor or metal ion. The decarboxylation reaction is of interest for the synthesis of fine chemicals. As basis for an analysis of the catalytic mechanism of AMDase and for a rational enzyme design, we determined the X-ray structure of the enzyme up to 1.9 Å resolution. Like the distantly related aspartate or glutamate racemases, AMDase has an aspartate transcarbamoylase fold consisting of two α/β domains related by a pseudo dyad. However, the domain orientation of AMDase differs by about 30° from that of the glutamate racemases, and also significant differences in active-site structures are observed. In the crystals, four independent subunits showing different conformations of active-site loops are present. This finding is likely to reflect the active-site mobility necessary for catalytic activity. © 2008 Elsevier Ltd. All rights reserved.

Keywords: AMDase; decarboxylation; X-ray crystallography; protein dynamics; biocatalysis

Introduction Arylmalonate decarboxylase (AMDase; E.C. 4.1.1.76) of Bordetella bronchiseptica catalyzes the enantioselective decarboxylation of α-aryl-α-methylmalonate to α-arylpropionate (Fig. 1).2 The 24.7-kDa (240-residue) monomeric enzyme is of biotechnological interest for its use in the synthesis of fine chemicals. Substrates with phenyl, 4-chlorophenyl or 6-methoxy-2-naphthyl substituents are readily turned over, whereas no decarboxylation is observed when the aryl group is 2-chlorophenyl, 1-naphthyl *Corresponding author. E-mail address: [email protected]. Abbreviations used: AMDase, arylmalonate decarboxylase; AspR, aspartate racemase; PDB, Protein Data Bank; ESRF, European Synchrotron Radiation Facility; BESSY, Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung.

or benzyl. It is yet unclear as to what extent the enzyme's substrate specificity or electronic requirements for stabilization of the transition state determine the influence of the aryl group. For the alkyl substituent, only a methyl group is accepted, whereas substrates with an ethyl group at the α carbon atom are not turned over. It is hoped that the crystal structure of AMDase will facilitate the rational design of variants with broader or different substrate specificities for biotechnological use. In contrast to many other decarboxylases, AMDase requires neither a metal ion nor an organic cofactor for activity. Information on the catalytic mechanism of AMDase is mainly derived from its very distant homology to aspartate and glutamate racemases and from a number of studies.2–8 The racemases catalyze the racemization and isomerization of α-amino acids via a two-base mechanism using two cysteine residues.9 Two domains of glutamate racemase are related by a pseudo-2-fold symmetry, which superimposes the two catalytic cysteines at equivalent positions on either side of the substrate's α carbon

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Crystal Structure of Arylmalonate Decarboxylase

387

Fig. 1. Reaction catalyzed by AMDase (adapted from Miyamoto et al.1). See the text for details.

atom.10–13 One cysteinate abstracts the α proton, and an enolate intermediate is formed. The second cysteine then protonates the carbon atom of the enolate from the other side under the formation of the enantiomeric amino acid. In AMDase, the two cysteine positions would correspond to residues 74 and 188. However, whereas Cys188 is conserved in the decarboxylase, the other cysteine is replaced by a glycine residue.2 Interestingly, the AMDase variant G74C has racemase activity,3 and the G74C/C188S double mutant is a decarboxylase that gives an enantiomer opposite to that of the wild-type enzyme.5 Whereas initial experiments were interpreted with a mechanism in which Cys188 forms a covalent intermediate with the substrate,14–16 it is now assumed that decarboxylation of the malonate substrate yields an enolate intermediate and that Cys188 is involved in the enantiospecific protonation step.1

Results and Discussion Crystallization and structure determination Crystals of AMDase could only be obtained after storage of the protein stock solution for about 4– 5 weeks at 277 K. Electron spray ionization mass spectrometry measurement of a crystallizable AMDase sample showed two main components with molecular masses of 26,025 (32%) and 26,101 Da (68%), which are 83 and 159 Da higher than that of freshly purified AMDase (25,942 Da), respectively. The calculated molecular mass is 25,945 Da, including the C-terminal His6-tag. These findings indicate the oxidation of one or two cysteine residues by formation of a disulfide linkage with 2-mercaptoethanol (78.1 Da), which was present in protein buffer. The structure of AMDase was determined by single isomorphous replacement with anomalous

scattering techniques up to 1.92 Å resolution (Tables 1 and 2). The final model contains 4 AMDase monomers, as well as 4 ethylene glycol molecules, 7 phosphate molecules, and 446 water molecules in the asymmetric unit. In all four protein chains, the five amino-terminal residues are not visible in electron density maps. Chains B and C contain additional unstructured (flexible) regions (Table 2). In the electron density map, cysteine residues 148 and 188 are modified by disulfide formation with 2mercaptoethanol in all four protein chains (Fig. 2). The modified cysteines 148 of chains A and D, as well as the modified cysteine 188 of chain A, are involved in crystal contacts, explaining why these crystals do not form from freshly prepared protein. Protein fold and flexibility AMDase has an aspartate transcarbamoylase-like fold, which consists of two similar domains related Table 1. Data collection and phasing statistics of AMDase crystals Crystal Wavelength (Å) Unit cell constants (Å) a b c Beamline Resolution (Å) Completeness (%) Unique reflections Multiplicity Rsym (%) Average I/σ Wilson B-factor (Å2) SigAnoc

Native

K2[Pt(CN)4]

0.931

1.07198

83.2 101.0 139.6 ID14.3 (ESRF) 30–1.92 (2.02–1.92)a 98.9 (93.7) 89,247 4.6 (2.2) 5.5 (43.4) 15.3 (2.2) 36.5

82.6 100.9 138.9 BL14.1 (BESSY) 30–2.4 (2.5–2.4)a 98.7 (90.4)b 86,562b 3.8 (3.5)b 5.4 (49.9)b 13.7 (2.7)b 59.2 1.72

a Values in parentheses correspond to the highest-resolution shell. b Friedel pair reflections were not merged. c SigAno = ∣F(+) − F(−)∣/∑(F(+),F(−)) (program XDS).20

Crystal Structure of Arylmalonate Decarboxylase

388 Table 2. Refinement statistics Resolution range (Å) Number of protein atoms Protein chains refined A B C D Number of nonprotein atoms Rwork (%) Rfree (%) Average B-factor (Å2) Main-chain A Main-chain B Main-chain C Main-chain D Solventa r.m.s.d. from ideal Bond length (Å) Bond angle (°) Ramachandran plot (%) Most favored Additionally allowed Generously allowed Disallowed

30–1.92 6688 6–240 6–39, 48–154, 163–238 6–241 6–39, 45–240 533 17.8 20.7 39.4 61.9 44.1 36.3 49.9 0.018 1.403 93.6 6.1 0.3 (2)b 0

a

Water, ethylene glycol, and phosphate molecules. A158 (chain A) and T146 (chain C) are well defined on the electron density maps. b

by a pseudo dyad. Each domain contains a fourstranded parallel β-sheet (strand order: 2, 1, 3, 4) covered by α-helices on both sides (Fig. 3a and b). The active site is located between the two domains. A superposition of the four monomers in the asymmetric unit reveals significant deviations of the main-chain trace near the domain interface and the active-site cysteine 188 (Fig. 3c). In detail, the loops between β1 and αA (residues 14–22), β2 and αB (residues 39–48), β3 and αC (residues 73–75), β5 and αF (residues 123–133, partly including this helix), and β6 and αH (residues 151–162), as well as between β7 and αI (residues 187–193), show deviations of N 2 Å in the Cα positions. The largest main-chain differences are observed for residues 14– 22, 39–48, and 151–162. Whereas the loop conformations in chain C (residues 14–22, 39–48, 73–75, and 151–162) and chain D (residues 39–48, 151–162, and 123–133) may be influenced by crystal packing interactions, chains A and B, as well as the other mentioned loops of chains C and D, are not involved in direct crystal contacts. Monomers A and B have a similar conformation (r.m.s.d. = 0.54 Å). The flexibility of two of the abovementioned loops is also demonstrated by their disorder in monomers B and D. Residues 40–47 and 155–162 of chain B and residues 40–44 of chain D are not visible on the electron density map. The C188 loop in chains A and B adopts a conformation strikingly different from that of monomers C and D. In chains A and B, the loop containing the catalytic cysteine is solvent-exposed in the active-site cleft, similar to the situation in the aspartate or glutamate racemases. This is assumed to be the catalytically competent conformation. In monomers C and D, Cys188 folds into the hydro-

phobic core of the C-terminal domain. It is not yet clear whether this conformation is of relevance to the catalytic function of the enzyme. Most likely, it only reflects the general flexibility of active-site loops and is caused by the more hydrophobic nature or the larger size of the modified Cys188 side chain. We currently do not assume that this conformation plays a role in a putative mechanism in which Cys188 forms a covalent intermediate with the substrate. The flexibility in the active-site loop conformations suggests that the enzyme might undergo a conformational change upon substrate binding. In the conformers present in our crystals, a rather large and deep active-site cleft is formed. We assume that the flexible loops close upon substrate binding to form a catalytically competent arrangement and to exclude water from the substrate-binding pocket. A substrate-induced closure motion has also been discussed for glutamate racemase.12 A comparison of the structures of the two glutamate racemases MurI (1B73) and RacE (1ZUW) showed that the two domains of the enzyme reorient, and a substantial reorganization of the residues in the active-site region is observed, with a maximum displacement of 13 Å. In the closed conformation observed in the RacE–glutamate complex, the substrate is completely buried from the solvent. In addition to the domain movement, the region that corresponded to the flexible loop of residues 151–162 in AMDase (between β6 and α8) also changed conformation in the glutamate racemase. In light of the (albeit distantly) related structures and catalytic mechanisms, these findings support the hypothesized substrate-induced closure motion in AMDase involving the active-site loop regions and, possibly, domain reorientation.

Fig. 2. Modification of Cys148 in chain D by formation of a disulfide bridge to β-mercaptoethanol. An (2mFo − DFc) electron density map contoured at the 1σ level is shown in blue, and an (mFo − DFc) omit electron density map (3σ) for modification is shown in green. Hydrogen bonds (b 3.5 Å) and van der Waals contacts (b 4 Å) are depicted as dotted lines. Residues of a symmetry-related protein molecule in the crystal are presented as carbon atoms in magenta.

Crystal Structure of Arylmalonate Decarboxylase

389

Fig. 3. Structure of AMDase. (a) Stereo ribbon plot of monomer A: domain 1 (residues 6–103 and 211–227) is shown in red, blue, and black for helices, β strands, and loops, respectively, whereas domain 2 (residues 104–210 and 228–240) is shown in yellow, purple, and gray. Cysteine 188 is shown in green. (b) Topology plot of monomer A. Strands are depicted as triangles, and helices are depicted as spheres. The numbers correspond to the first and the last residues of the secondary structure element. (c) Stereo diagram of the superposition of the Cα traces of the four monomers in the asymmetric unit (A, black; B, blue; C, red; D, yellow). Regions with N2 Å deviation are numbered and shown in darker colors. Cysteines 188 are depicted as sticks.

Comparison with related protein structures A search with the DALI server18 revealed aspartate racemase (AspR) from Pyrococcus horikoshii19 as the closest known structural relative, with a Z-score of ∼15. However, a manual protein database20 survey showed that the more recently deposited structure of the protein ST0656 from Sulfolobus tokodaii [Protein Data Bank (PDB) ID 2DGD] is an even closer homolog (DALI Z-score is ∼23). ST0656 is a biochemically uncharacterized protein that is listed as a putative AMDase based on the homology to AMDase. When compared to AMDase, ST0656 superimposes with an r.m.s.d. of 1.9 Å (196 residues of the most similar chain A of AMDase and chain A of ST0656; 19% sequence identity for 222 residues), whereas AspR displays an r.m.s.d. of 2.8 Å (186 residues of chain C of AMDase and chain A of AspR; 14% identity for 235 residues) (Fig. 4a). All three enzymes differ in the relative orientation of their two domains. Compared to AMDase, the domain orientation in ST0656 differs by about 14°, whereas the rotational difference from AspR is 30° (Fig. 4b and c). Interdomain rotation axes are not collinear (i.e., the structure of ST0656 is, with respect to the domain orientation, not an intermediate between AspR and AMDase via a rotation around one axis). The individual domains of ST0656 superimpose onto AMDase with r.m.s.d. values of ≥ 1.6 Å, whereas values of ≥ 2.1 Å are obtained for the

comparison with AspR. Thus, in addition to the differences in the domain orientation, there are also significant differences in the domain structure, including considerable differences in helix structures (Fig. 4a). In particular, helices A, D, F, G, H, and I differ in the length between AMDase and at least one of the two relatives. Furthermore, additional or missing helices are present. The significant differences between the three enzymes also extend to their active-site structures. Although the aspartate and glutamate racemases have a catalytic reactivity different from that of AMDase, the catalytic activities of the G74C and G74C/C188S variants, as described in the Introduction, convincingly indicate a relationship at least at the second step of the AMDase reaction (i.e., the protonation of an enolate intermediate via the catalytic cysteine). The rather large differences in the active-site structures are thus surprising (Fig. 5). The distance between the two catalytic cysteines in glutamate racemase (6.9 Å; Cα atoms; 1ZUW) is much shorter than the corresponding distance between Cys188 and Gly74 (10.1 Å). Another difference is seen in the conformation of the loop between strand 2 and helix B, which is, in AMDase, much farther away from the substrate than the corresponding loop in the racemases. In the latter enzyme, the loop folds over the substrate and interacts with the carboxylate side chain of the glutamate via two hydrogen bonds from main-chain

390

Crystal Structure of Arylmalonate Decarboxylase

Fig. 4. Comparison of the structural relatives of AMDase. (a) Ribbon plots of AMDase: ST0656 from S. tokodaii (PDB ID 2DGD) and AspR from P. horikoshii (PDB ID 1JFL19). The secondary structure elements are labeled according to the AMDase structure. Additional helices before (“B −”) or after (“F+” and “H +”) the corresponding helices in AMDase are marked. (b) Superposition of monomer A of AMDase (N-terminal domain in red; C-terminal domain in yellow) and of monomer A of ST0656 (gray), with their N-terminal domains aligned. (c) Superposition of monomer C of AMDase and of monomer A of AspR (gray), with their N-terminal domains aligned. The interdomain rotation axes are depicted as blue lines. The orientation of AMDase in the superpositions is the same as in (a).

nitrogens. In AMDase, this loop contains residues 39–48, which show conformational flexibility in the comparison of the four chains in the crystal (Fig. 3c). Interestingly, the mutation S36N decreased the activity of the wild-type enzyme by about 10-fold, whereas the activity of the G74C/C188S mutant was increased by ∼ 10-fold.4 This serine side chain is located in strand 2, and it points away from the active site interacting with the side chain of His58 of helix B (Fig. 5). Since the side chain of residue 74 cannot directly influence the active-site structure, it appears likely that the mutation of this residue influences the dynamics or the conformation of the loop between strand 2 and helix B. ST0656 contains only one active-site cysteine corresponding to Cys188 in AMDase. It is thus likely to be a decarboxylase rather than a racemase. However, the structural differences from AMDase, including the active-site region, are relatively large, and the sequence identity (19%) is quite low. Since ST0656 is not biochemically characterized, it remains unclear whether the structural differences

reflect a different substrate specificity or catalytic activity, or whether it is also a result of the conformational flexibility of AMDases. Active-site structure and catalytic mechanism The differences and variability in the active sites observed in the four molecules in the asymmetric unit of AMDase complicate a docking of substrates to the catalytic pocket. However, based on the experiments of various authors, 1–6,14,15 several constraints concerning the substrate-binding mode are known. By 13C labeling of either of the two carboxylate groups, it was shown that the decarboxylation reaction proceeds under the inversion of the configuration of the Cα carbon atom and that the pro-R carboxyl group is removed as carbon dioxide (Fig. 1).1 In analogy to the two-base mechanism of the aspartate and glutamate racemases and based on the change in the stereochemical outcome of the reaction for the G74C/C188S mutant, it is likely that the cysteines protonate the enolate intermediate and

Crystal Structure of Arylmalonate Decarboxylase

391

Fig. 5. Glutamate racemase RacE (gray) in complex with the glutamate substrate (yellow) superimposed onto AMDase (red). The two structures have been structurally aligned using both domains. The catalytic cysteines are shown for both structures, and the position of Gly74 of AMDase is marked in green. The hydrogen-bonding interactions of the glutamate with RacE are represented as dotted lines.

control the stereochemical outcome of the reaction.1 As the wild-type enzyme, this mutant catalyzes the elimination of the pro-R carboxylate group, showing that Cys188 does not play a major role in the decarboxylation reaction to the enolate intermediate. If the intermediate does not undergo a coordinated rearrangement in the active site (i.e., if it is oriented similarly to the substrate), then Cys188 should be located close to the pro-S carboxylate. On the other side, the pro-R carboxylate should face Gly74, since, in the G74C/C188S mutant, Cys74 protonates the enolate intermediate under configuration retention (Fig. 1). These constraints and the relationship with the racemases are the bases for the

positioning of a substrate in the active site of AMDase, as presented in Fig. 6. An analysis of the substrate specificity of AMDase showed that one α-substituent must be an aryl group, whereas the second α-substituent can be a hydrogen atom, a hydroxyl group, or a methyl group, but not a larger substituent such as an ethyl group.2 Obviously, the presence of larger groups sterically hinders productive substrate binding. In Fig. 6, a methylmalonate substrate has been placed in the AMDase active site such that the proS carboxylate group faces Cys188, whereas the proR carboxylate group points toward Gly74. Residues 74 and 188 are located on different sides of the

Fig. 6. Substrate-binding pocket at the interface between the N-terminal domain (green) and the C-terminal domain (red). A model for an α-methyl-α-phenylmalonate substrate is shown in yellow. This model has been positioned as described in the main text. In particular, the precise binding site and interactions of the aryl and alkyl substituents are unclear.

392 substrate-binding pocket. The aryl group may than be oriented more toward the side of the pocket formed by the two tyrosines 80 and 126 or toward the prolines 14 and 15. Both sides of the binding pocket are relatively hydrophobic for binding of the aryl group. If the aryl group is oriented toward the tyrosines, the space for the methyl group is more restricted, in agreement with the results of the substrate specificity analysis.2 An interaction of the aryl group with the tyrosines is also in agreement with 19F and 31P NMR experiments on a complex of the enzyme, with an inhibitor covalently attached to a cysteine of AMDase, most likely Cys188.15 These experiments indicated that the substrate aryl group interacts with aromatic active-site residues. However, if the reaction mechanism does not proceed via a covalent intermediate bound to Cys188, as is now assumed, these data may not be relevant to the catalytic mechanism. We suggest that the pro-R carboxylate group is bound by the two main-chain nitrogen atoms of residues 75 and 76, similar to the binding of the glutamate substrate's α-carboxylate group in glutamate racemase (e.g., to residues 93 and 94 in the Escherichia coli enzyme). After domain reorientation upon substrate binding, it appears possible that the second carboxylate group binds to the amide nitrogens of residues 188 and 189. The side chains of Thr75 and Ser76 may participate in the catalysis of the decarboxylation step. However, a detailed analysis of this mechanism must await experimental studies on the substrate- or intermediate-binding modes or preparation of mutant enzymes. In summary, we have determined the crystal structure of AMDase showing considerable mobility of the active-site loops, most likely supporting the formation of an active complex in a substrateinduced closure motion. Despite the relationship between catalytic mechanisms and sequences, the domain orientation and active-site structures of the racemases and AMDase are surprisingly different. In conjunction with biochemical data, we obtained a first model for the substrate-binding mode that guides further studies on catalytic mechanism and changes in substrate specificity via site-directed mutagenesis.

Materials and Methods Enzyme preparation The AMDase gene was chemically synthesized with an affinity-tag sequence coding for six C-terminal histidines. The complete gene was cloned into pBAD His A vector (Invitrogen), and E. coli Top10 cells (Invitrogen) were transformed. For expression of the enzyme, an overnight culture of these cells in LB medium containing 200 μg/ml carbenicillin was inoculated. On the next morning, 1 ml of the cells was used to inoculate 1 l of medium (incubation at 37 °C and 300 rpm). After an OD600 nm of 0.6 had been reached, a final concentration of 0.02% arabinose was

Crystal Structure of Arylmalonate Decarboxylase adjusted. After 8 h, the cells were harvested by centrifugation and sonified in 20 mM Tris–HCl (pH 8.0) on ice. Afterward, the solution was clarified by centrifugation (10,000g for 30 min). The supernatant was purified by NiNTA affinity chromatography (Qiagen column). Unspecifically bound proteins were eluted with 25 mM NaH2PO4, 350 mM NaCl, and 50 mM imidazole (pH 8.0). AMDase was eluted by using the same buffer containing 250 mM imidazole. This fraction (10 ml) was dialyzed three times against 5 l of 20 mM Tris–HCl (pH 8.0) and 0.5 mM β-mercaptoethanol. The final yield was about 20 mg protein/l culture medium. Crystallization Bipyramidal orthorhombic crystals (space group P212121) of AMDase (maximum size, about 0.25 mm × 0.17 mm × 0.08 mm) were grown at 292 K by the vapor-diffusion hanging-drop setup using equal volumes (1 μl each) of protein solution [7–8 mg/ml AMDase, 20 mM Tris–HCl (pH 8.0), and 0.5 mM β-mercaptoethanol] and reservoir solution [1.4–1.8 M sodium/potassium phosphate (pH 5.8– 6.4), 500 μl/well]. The protein solution had to be stored for about 4–5 weeks at 277 K prior to crystallization. After this time, aliquots of the enzyme were frozen in liquid nitrogen and stored at 193 K. Crystal preparation Crystals were stepwise transferred with a rayon loop (Hampton Research, Aliso Viejo, CA, USA) to cryoprotection solutions consisting of the reservoir solution with a sodium/potassium phosphate concentration of 2.4–2.8 M and a final ethylene glycol concentration of up to 20% (vol/vol). Afterward, crystals were plunged into liquid nitrogen and stored until data collection. Derivatization of crystals with 10 mM K2[Pt(CN)4]·3H2O (Jena BioScience, Jena, Germany) was performed during the cryosoaking procedure mentioned above. Data collection and processing Diffraction datasets of a native crystal and a platinumderivatized crystal were recorded at the synchrotrons European Synchrotron Radiation Facility (ESRF; Grenoble, France) and Berliner ElektronenspeicherringGesellschaft für Synchrotronstrahlung (BESSY; Berlin, Germany) at beamlines equipped with the detector system Q4 ADSC (Area Detector System Corporation, San Diego, CA, USA) or MAR225 CCD (MarResearch, Norderstedt, Germany), respectively. The resolution limit was chosen such that the signal-to-noise ratio was N2 and Rsym was lower than about 50% for the highest-resolution shell. Processing was performed with the XDS package.17 SCALEIT21 was used to scale the derivative and native datasets. Structure determination and refinement In anomalous Patterson maps, five platinum sites were located. Single isomorphous replacement with anomalous scattering phasing with MLPHARE22 yielded initial phases with a figure of merit of 0.33 and a phasing power of 0.98 for acentric reflections. The Cullis R-factor was 0.89 for both anomalous and isomorphous differences. The phases were improved and extended to 1.92 Å

Crystal Structure of Arylmalonate Decarboxylase (program DM23). An initial model could be automatically built with ARP/wARP,24 which was manually improved by non-crystallographic symmetry mapping of already built model fragments using PyMOL† and Coot.25 This incomplete model was refined using REFMAC5,26 and a more complete model could be built based on the improved electron density maps. Refinement of four AMDase monomers per asymmetric unit was conducted using optimal TLS groups (four per monomer) as derived from the TLSMD Internet server.27 No non-crystallographic symmetry restraints were applied. Solvent and buffer molecules were manually added based upon (Fo − Fc) difference electron density maps. The programs Procheck28 and WHAT_CHECK29 were used for structure validation. Structure comparisons and visualizations A search for structural homologs was conducted with the DALI Internet server.18 Alignment of AMDase monomers was performed with the program ALIGN.30 Structural superimpositions, including structure-based sequence alignment of AMDase with related proteins [ST0656 from S. tokodaii (PDB ID 2DGD); AspR from P. horikoshii (1JFL19); and substrate–analog-bound glutamate racemases from Aquifex pyrophilus (1B7410), E. coli (2JFN11), Enterococcus faecalis (2JFP11), Enterococcus faecium (2JFV and 2JFW11), Helicobacter pylori (2JFX11), Bacillus subtilis (1ZUW12), Bacillus anthracis isoenzymes 1 + 2 (2GZM and 2DWU13), and Staphylococcus aureus (2JFQ11], were carried out with the program INDONESIA‡. Interdomain screw axes were calculated by the following procedure. First, one domain of both structures was superimposed using the program ALIGN,30 and superposition operators were applied to both domains using PDBSET.22 Then the interdomain screw operator was determined by superposition of the second domain via the program ALIGN.30 All molecular figures were created with the program PyMOL†. Protein Data Bank accession numbers Coordinates and structure factors for the structure of AMDase have been deposited with the RCSB Protein Data Bank with acession code 2VLB.

Acknowledgements Financial support for travel to ESRF was received from the European Community-Research Infrastructure Action under the FP6 “Structuring the European Research Area Program” (contract no. RII3/ CT/2004/5060008). We acknowledge access to synchrotron beamlines ID14.3 (ESRF) and BL-14.1 (BESSY) and local support by Olivia Sleator (ESRF) and Heike Richter and Jörg Schulz (BESSY). We thank Corinna Sykora and Prof. Ralf Hoffmann for mass spectrometry measurements.

† http://www.pymol.org ‡ http://xray.bmc.uu.se/dennis/

393

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Crystal Structure of Arylmalonate Decarboxylase

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