Báo cáo khoa học: Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens potx

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Báo cáo khoa học: Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens potx

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Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens Tanja G. Mosbacher 1 , Michael Mueller 2 and Georg E. Schulz 1 1 Institut fu ¨ r Organische Chemie und Biochemie, Albert-Ludwigs-Universita ¨ t, Freiburg im Breisgau, Germany 2 Institut fu ¨ r Pharmazeutische Wissenschaften, Albert-Ludwigs-Universita ¨ t, Freiburg im Breisgau, Germany Thiamine diphosphate (ThDP)-dependent enzymes participate in numerous biosynthetic pathways and catalyse a broad range of reactions mainly involving the cleavage and the formation of C–C-bonds. For instance, they catalyse nonoxidative and oxidative de- carboxylations of 2-ketoacids, produce 2-hydroxy- ketones, and transfer activated aldehydes to a variety of acceptors. However, they can also form C–N, C–O and C–S bonds [1,2]. The ThDP-dependent benzalde- hyde lyase (BAL, EC 4.1.2.38, suggested systematic name: 2-hydroxy-1,2-diphenylethanone benzaldehyde- lyase, i.e. benzoin benzaldehyde-lyase) catalyses the reversible ligation of two aromatic aldehydes to yield an (R)-2-hydroxyketone (Fig. 1). BAL was discovered by Gonzales and Vicuna who isolated it from the strain Pseudomonas fluorescens Biovar I, which was Keywords acyloin condensation; carbon–carbon ligation; crystal structure; seleno-methionine MAD Correspondence G. E. Schulz, Institut fu ¨ r Organische Chemie und Biochemie, Albertstr. 21, Freiburg im Breisgau, Germany 79104 Tel: +49 761 203 6058 Fax: +49 761 203 6161 Email: georg.schulz@ocbc.uni-freiburg.de Note After submission of this manuscript, we received a preprint of the following paper reporting that the mutation of His29 to alan- ine reduces the BAL activity to 5%. Kneen MM, Pogozheva ID, Kenyon GL & McLeish MJ (2005) Exploring the active site of benz- aldehyde lyase by modeling and mutagen- esis. Biochim Biophys Acta: Proteins and Proteomics, doi:10.1016/j.bbapap2005. 08.025 (Received 5 August 2005, revised 22 September 2005, accepted 29 September 2005) doi:10.1111/j.1742-4658.2005.04998.x Pseudomonas fluorescens is able to grow on R-benzoin as the sole carbon and energy source because it harbours the enzyme benzaldehyde lyase that cleaves the acyloin linkage using thiamine diphosphate (ThDP) as a cofac- tor. In the reverse reaction, this lyase catalyses the carboligation of two aldehydes with high substrate and stereospecificity. The enzyme structure was determined by X-ray diffraction at 2.6 A ˚ resolution. A structure-based comparison with other proteins showed that benzaldehyde lyase belongs to a group of closely related ThDP-dependent enzymes. The ThDP cofactors of these enzymes are fixed at their two ends in separate domains, suspend- ing a comparatively mobile thiazolium ring between them. While the resi- dues binding the two ends of ThDP are well conserved, the lining of the active centre pocket around the thiazolium moiety varies greatly within the group. Accounting for the known reaction chemistry, the natural substrate R-benzoin was modelled unambiguously into the active centre of the repor- ted benzaldehyde lyase. Due to its substrate spectrum and stereospecificity, the enzyme extends the synthetic potential for carboligations appreciably. Abbreviations AHAS, acetohydroxy acid synthase; ALS, acetolactate synthase; BAL, benzaldehyde lyase; BFD, benzoylformate decarboxylase; CEAS, carboxyethylarginine synthase; IPDC, indolepyruvate decarboxylase; MAD, multiwavelength anomalous diffraction; PDC, pyruvate decarboxylase; POX, pyruvate oxidase; SeMet, seleno- L-methionine; ThDP, thiamine diphosphate. FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS 6067 found in wood scraps in a cellulose factory [3]. They showed that this strain can grow on lignin-like substrates, because the endogenous BAL can cleave the acyloin linkage of R-benzoin and R-anisoin to use these compounds as a carbon and energy source [4]. BAL is a valuable tool for chemo-enzymatic synthe- ses because it generates various enantiomerically pure 2-hydroxyketones through aldehyde ligation or by partial decomposition of racemic mixtures [5–8]. The enzyme generates activated aldehydes either via direct aldehyde addition to ThDP or via cleavage of 2-hyd- roxyketones but is not involved in decarboxylation reactions [9]. BAL accepts a broad spectrum of aro- matic donor substrates, among them ortho-substituted benzaldehydes, and processes substituted acetaldehydes resulting in functionalized derivatives of (R)-2-hy- droxypropiophenone [10]. The enzyme also ligates two aliphatic aldehydes resulting in highly enantio-enriched acyloins [5]. In all reactions, BAL shows a high stereo- specificity for the R-configuration of the acyloin link- age [10]. Starting from the assumption that aldehydes which are not accepted as donor substrates may still be acceptor substrates and vice versa, a biocatalytic system for the asymmetric cross-carboligation of aromatic aldehydes has been developed [11]. Taken together, BAL broadens appreciably the substrate spectrum of the related enzymes benzoylformate decarboxylase (BFD) [12,13] and pyruvate decarboxy- lase (PDC) [14–16] used for similar syntheses. Here we report the crystal structure of BAL with bound cofac- tor ThDP at 2.6 A ˚ resolution, suggest the geometry of the reaction and explain the substrate specificity in structural terms. Results and Discussion Structure determination and description BAL is a homotetramer of 4 · 563 amino acid residues corresponding to a molecular mass of 4 · 58 919 Da. Each subunit binds one ThDP molecule using one Mg 2+ ion. The obtained crystals belong to spacegroup P3 1 21 with one tetrameric BAL molecule (wild-type plus invisible C-terminal His-tags) per crystallographi- cally asymmetric unit (Table 1). The crystal structure was determined by the incorporation of seleno- l-methionine (SeMet) and subsequent phasing with multiwavelength anomalous diffraction (MAD). Met1 was cleaved off during protein production as indicated by electrospray ionization mass spectroscopy (ESI- MS). The complete replacement of the 12 remaining methionines per subunit was demonstrated by ESI-MS, which showed a single peak at a mass of 562 ± 5 Da (expected 563 Da) higher than the mass of the wild- type. The SeMet diffraction data contained a good anom- alous signal to 3.0 A ˚ resolution. Among the expected 4 · 12 selenium sites, 4 · 11 were found and used for the initial phasing. The 4 · 1 missing SeMet positions were located in the mobile and therefore invisible C-terminal ends. After phase improvement, a model of Table 1. Data collection statistics. All crystals belong to spacegroup P3 1 21. The unit cell dimensions of the SeMet-labeled crystal were a ¼ b ¼ 150.3 A ˚ and c ¼ 195.8 A ˚ . Those of the wild-type BAL crystal were a ¼ b ¼ 154.7 A ˚ ,c¼ 200.7 A ˚ . The corresponding packing parame- ters were 2.69 A ˚ 3 ⁄ Da and 2.92 A ˚ 3 ⁄ Da, respectively. Values in parentheses refer to the highest resolution shells, which comprised 2.70– 2.58 A ˚ in all data sets. Data set SeMet-labeled BAL Wild-type peak inflection remote Wavelength [A ˚ ] 0.9793 0.9801 0.9393 0.9801 Resolution [A ˚ ] 20–2.6 20–2.6 20–2.6 20–2.6 Observables 598 889 598 513 429 583 324 789 Unique reflections a 156 252 (17 815) 156 058 (17 756) 143 633 (16 819) 79 796 (10 122) Completeness [%] 99.5 (96) 99.2 (95) 98.2 (94) 98.8 (99) R sym-I [%] 8.2 (39) 7.4 (38) 7.9 (38) 11.1 (37) Multiplicity 3.8 (3.5) 3.8 (3.5) 3.0 (2.8) 8.1 (7.9) Average I ⁄ r I 12.5 (3.4) 13.9 (3.8) 12.8 (3.6) 13.3 (5.5) a For the MAD data sets Friedel pairs are treated as independent reflections. O O H 2 BAL benzaldehyde R-benzoin OH Fig. 1. Benzaldehyde lyase (BAL) catalyzes cleavage and formation of R-benzoin. BAL is known to accept several other substituted aro- matic or aliphatic acyl-acceptors as substrates for the formation of an acyloin [28]. ThDP-depending benzaldehyde lyase T. G. Mosbacher et al. 6068 FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS SeMet-labeled BAL was built and refined to 2.6 A ˚ resolution. This model served as a template for the wild-type BAL structure, which was determined by molecular replacement. The structure of wild-type BAL was refined to 2.6 A ˚ resolution resulting in a model closely similar to that of SeMet-BAL. It included residues 2–555 of each sub- unit as well as four molecules of ThDP and four Mg 2+ ions. The eight C-terminal residues were disor- dered in both structures. Data collection and refine- ment statistics are given in Tables 1 and 2. The crystals of SeMet-labelled BAL and wild-type BAL grew under almost identical conditions and showed the same packing scheme but quite different unit cell axes. Since the B-factors were lower and the refinement results better for the wild-type crystals than for SeMet- labelled crystals, we refer in the following to the wild- type structure (Fig. 2). The BAL homotetramer has an overall size of approximately 95 · 95 · 75 A ˚ 3 . No significant struc- tural differences were found between the four crystallo- graphically independent subunits of the tetramer (Fig. 3). Each subunit consists of the three domains Dom-a, Dom-b and Dom-c (Fig. 2), named as in pre- vious annotations. All three domains consist of a cen- tral six-stranded parallel b-sheet flanked by a varying number of a-helices. Residues involved in binding of the cofactor ThDP are located at the C-terminal ends of the b-strands of Dom-c (diphosphates and Mg 2+ ) and of Dom-a¢ of a neighbouring subunit (pyrimidine moiety). The active centre is defined by the thiazolium ring of ThDP, which sits in a deep pocket opening to the outer surface of the tetramer. The four subunits A, B, C and D form the two tight dimers A–B and C–D around the molecular axis P (Fig. 3), in which each subunit buries a solvent-access- ible surface area of 3270 A ˚ 2 . The two tight dimers are associated much less tightly around the molecular axes Q and R to form a D 2 -symmetric homotetramer. These secondary interfaces bury 1790 A ˚ 2 per subunit. The tight contact is formed by Dom-a and Dom-c of subunit A with their counterparts in subunit B. It is stabilized by a large number of hydrogen bonds. The weaker contact results from an association of Dom-a and Dom-b of subunit A with the respective domains of subunit D. It contains only few hydrogen bonds. A Fig. 2. Stereo ribbon plot of a BAL subunit composed of the three domains Dom-a (residues 1–183, blue), Dom-b (residues 184–363, orange) and Dom-c (residues 364–563, green). The cofactor ThDP is shown as a ball-and-stick model and Mg 2+ as a pink sphere. The secon- dary structures are labeled. Table 2. Refinement statistics. Data set SeMet-labeled BAL peak data set Wild-type Resolution range [A ˚ ] 20–2.6 20–2.6 Structured peptide (all four subunits) 3–555 2–555 Water molecules 404 477 Average B-factors [A ˚ 2 ] (mainchain ⁄ total) 43.3 ⁄ 43.8 34.7 ⁄ 35.6 R cryst [%] 21.2 19.9 R free [%] 24.5 22.0 R.m.s.d. bond lengths [A ˚ ] ⁄ angles [degr.] 0.012 ⁄ 1.40 0.012 ⁄ 1.46 Ramachandran angles in favored region [%] 96.5 96.8 Ramachandran angles in allowed region [%] 3.5 3.2 T. G. Mosbacher et al. ThDP-depending benzaldehyde lyase FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS 6069 large cavity lined by the four Dom-a is located at the centre of the tetramer. It contains a considerable number of crystallographic water molecules and is not connected to the active centre pocket. To detect possible conformational changes of the tetramer, we compared the wild-type and the SeMet- labelled structures of BAL. A chainfold superposition of the four central Dom-a showed a good agreement in these domains but a radial contraction bringing the outer Dom-b and Dom-c of SeMet-BAL up to 1.4 A ˚ closer to the centre when compared with the wild-type. Moreover, the shrinkage of SeMet-BAL involves a 0.5-A ˚ approach of Dom-c (fixing the diphosphate of ThDP) towards Dom-a¢ (binding the pyrimidine moi- ety), which may affect the thiazolium ring suspended between the two fix points. This observed contraction reveals possible domain rearrangements and agrees with the crystal unit cell changes stated in Table 1. According to the crystallization conditions, the contraction seems to be caused by an increase of the PEG 200 concentration from 50% to 55%, removing water from the protein. Comparison with related proteins To find related proteins in the Protein Data Bank, we performed a general search using program dali [17]. This search identified a number of closely related structures all of which were ThDP-dependent enzymes involved in important metabolic pathways. The Z-scores ranged from 39.6 to 29.6 indicating close rela- tionships (Table 3). The related proteins are acetolac- tate synthase (ALS) [18], acetohydroxy acid synthase (AHAS) [19], indolepyruvate decarboxylase (IPDC) [20], benzoylformate decarboxylase (BFD) [12], carb- oxyethylarginine synthase (CEAS) [21], pyruvate oxi- dase (POX) [22] and pyruvate decarboxylase (PDC) [14,15]. The overall sequence identity among these seven enzymes ranges from 19% to 29% with an aver- age of 24%. A comparison of the relative domain posi- tions in the D 2 -symmetric tetramer showed in general a good equivalence with deviations around 2 A ˚ . All enzymes are especially similar with respect to the tight dimer formed by Dom-a and Dom-c. Given the high dali scores of Table 3 and the drastic drop to the next lower score, these enzymes form a separate subset among the ThDP-dependent enzymes, which we name ‘POX group’ after the first established structure [22]. Fig. 3. Stereo ribbon plot of the D 2 -symmetric BAL tetramer with the three molecular twofold axes P, Q and R using the colors of Fig. 2. The tetramer should be described as a dimer of dimers. The tightest interfaces are around axis P. Each tight dimer contains two active cen- tres at its interface. ThDP is shown as a ball-and-stick model. Table 3. Superposition of BAL with related proteins using DALI [17]. Protein a PDB code FAD Chain length b Number of aligned residues Z-score c ALS d 1OZG None 565 419 39.6 AHAS 1JSC Structural 630 416 39.6 IPDC 1OVM None 554 495 34.4 BFD 1MCZ None 528 398 33.7 CEAS 1UPA None 573 439 32.0 POX 1POX Redox 585 356 31.7 PDC e 1ZPD None 567 397 29.3 a In all cases we used subunit A of the PDB file except for BAL where we used subunit D. b BAL contains 563 residues total. c The next lower Z-score was 16.7 indicating that the eight enzymes form a separated, closely related group. The ThDP-dependent transketo- lase [20] showed a Z-score of 13.1. d PDB file 1OZF of ALS yields the same values. e The PDC structure is that of Zymomonas mobilis. ThDP-depending benzaldehyde lyase T. G. Mosbacher et al. 6070 FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS Within this group the enzymes BFD and PDC are best characterized with respect to their function and therefore most relevant in organic synthesis. A struc- ture-based sequence alignment of BAL with BFD and PDC is shown in Fig. 4. The alignment assigns the residue equivalences at the active centres and it pre- sents the sequence of BAL in relation to its secondary structures. AHAS and POX contain FAD as a further cofactor besides ThDP which, however, plays merely a secondary role (Table 3). The FAD of POX accepts two electrons from the substrate and transfers them to dioxygen, whereas the FAD of AHAS is only required for structural integrity indicating that it is a relic of evolutionary development. The POX group shows very similar binding loca- tions for ThDP which also correspond to those of other ThDP-dependent enzymes [23]. In all enzymes ThDP assumes a V-conformation resulting in a close approach between the C2 atom of the thiazolium ring and the N4¢ atom of the pyrimidine moiety. A super- position of the cofactors is depicted in Fig. 5 revealing a remarkable conformational similarity. The diphos- phates are tightly bound to the polypeptide of Dom-c using Mg 2+ as a mediator. The Mg 2+ ion is octahed- rally coordinated to the sidechains of Asp448 and Asn475, to the backbone carbonyl of Ser477, to the diphosphate as well as to a water molecule (Fig. 6). This binding motif is present in all ThDP-dependent enzymes. In evolutionary terms the diphosphate-bind- ing site is the most important fix point of ThDP because it is best conserved as demonstrated by the diphosphate-binding sequence fingerprint G-D-G- X24-N-N that was detected long before any structure was known [24]. At the other end of ThDP, the pyrim- idine is well fixed in Dom-a¢ of another subunit: its N1¢ atom forms a strong hydrogen bond to a glutamic acid (Glu50 in BAL). A comparison of the relative B-factors along the ThDP molecules shows that the Fig. 4. Structure-based sequence alignment of BAL, BFD and PDC all of which are used in organic synthesis. The secondary structure of BAL is given for reference. The underlined segments are structurally aligned with BAL within the usual 3 A ˚ cutoff criterion. Lower case indi- cates lack of structure. The domain borders are indicated by triangles. The crystallized BAL lacked Met1 and carried a C-terminal His-tag with the sequence 561 pfgshhhhhh which was disordered and therefore invisible in the crystal structure. The sequence of PDC continues with 562 KPVNKv (r). T. G. Mosbacher et al. ThDP-depending benzaldehyde lyase FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS 6071 thiazolium ring and the ethylene bridge have generally the highest mobility, which corresponds to the largest positional differences of these parts observed in Fig. 5. Active centre and reaction geometry While the overall polypeptide architecture as well as the binding mode of ThDP are quite similar within the POX group, the active centre is not well conserved. The active centre pocket of BAL is lined by nonpolar alipha- tic and aromatic but only few polar residues. In this respect, BAL is most closely related to BFD [12]. In both crystal structures of BAL a water molecule was identified at a distance of about 3.6 A ˚ from the C2 atom of ThDP. This water molecule forms hydrogen bonds with Gln113 and His29, among which Gln113 is known to play an important role in catalysis [25]. The structures of all group members show ThDP in the V-conformation that brings the C2 atom of thiazo- lium in close proximity to the N4¢ atom of the pyrimidine moiety. Moreover, one of the reported structures of ALS [18] contains an inhibitor that fixes the C2 to N4¢ approach through a covalent bond as shown in Fig. 5. Moreover, all group members have a glutamic acid forming a short hydrogen bond to the N1¢ atom of the pyrimidine ring, which was suggested to induce the 1¢,4¢-imino tautomer [22]. The actual presence of this tautomer was later demonstrated [26,27]. As shown in Fig. 6, the imino group is hydro- gen bonded to the carbonyl of Gly419 so that its lone electron pair points to the C2 atom of ThDP. It is therefore most likely that the catalytic cycle starts by transferring a proton from C2 to the imine. The result- ing C2 carbanion may then attack the carbonyl carbon of the substrate yielding a covalent ThDP-substrate intermediate. During acyloin cleavage, the next step is supposedly the deprotonation of the hydroxyl by His29 followed by the dissociation of the first aldehyde. The remaining activated aldehyde is then protonated and also released. The protonation is probably performed by the water attached to His29. During acyloin synthesis, on the Fig. 5. Superposition of the ThDP cofactors of BAL (grey), ALS (cyan), AHAS (green), IPDC (purple), BFD (yellow), CEAS (blue), POX (pink) and PDC (red) together with some residues important for cofactor binding and catalysis. The BAL residues are labeled and the BAL hydrogen bonds are displayed. The yellow and red residues at the top are His70 of BFD and Asn28 of PDC, respectively. For ALS we show an inhib- itor with a modified C2 atom (PDB file 1OZG) instead of ThDP available from 1OZF. This inhibitor contains an additional covalent bond between the C2 and N4¢ atoms and carries an isopropyl substituent. Fig. 6. Stereoview of ThDP-binding at BAL showing the initial (Fo-Fc)-electron density map of ThDP and Mg 2+ at the 3 r contour level. The cofactor binds in the typical V-conformation required for catalysis. BAL residues lining the active centre pocket and interacting with the co- factor are shown in blue and orange, corresponding to the two domains they belong to (Fig. 2). Hydrogen bonds are given as dotted lines. ThDP-depending benzaldehyde lyase T. G. Mosbacher et al. 6072 FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS other hand, the intermediate is an activated aldehyde that is going to attack an acceptor aldehyde suitably positioned in the active centre. Again, His29 is likely to participate in the reaction by forming a hydrogen bond to the oxygen of the acceptor aldehyde, which is eventu- ally converted to a hydroxyl group of the condensation product by deprotonating His29. Proton handling by His29-Nd1 is facilitated by the contact of the Ne2 atom to the bulk solvent (Note). All steps seem to involve small displacements of the thiazolium ring, which are possible because this ring is relatively mobile (Fig. 5). It should be noted that ThDP is suspended between Dom-c and Dom-a¢ which in a direct comparison between the wild-type and the SeMet structures of BAL underwent a relative dis- placement of 0.5 A ˚ . It is therefore conceivable that domain motions affect the positional freedom of thia- zolium and thus catalysis. Since such domain displace- ments may be caused by the Brownian motion, it is further possible that the enzymes channel thermal energy into the chemical reaction. Substrate specificity BAL shows a general preference for nonpolar sub- strates [8,28] and is highly stereospecific with respect to benzoin, cleaving only R-benzoin out of a racemic mixture [5]. Moreover, BAL reacts with benzaldehyde and acetaldehyde to yield (R)-2-hydroxypropiophenone [28], in contrast to BFD, which uses the same educts to produce the S-enantiomer [29]. In order to explore the geometry of the reaction catalyzed by BAL, R-ben- zoin was modeled into its active centre (Fig. 7). The resulting model accounts for a nucleophilic attack from the deprotonated C2 atom of the thiazolium ring under the expected Bu ¨ rgi-Dunitzangle of 103° ±3° onto the carbonyl carbon of R-benzoin [30]. Fulfil- ling this restraint, the substrate is uniquely defined with respect to general location and conformation because all alternatives met severe steric obstacles. In contrast to R-benzoin, any model of the S-enantio- mer gave rise to major sterical clashes, which explains the stereospecificity of BAL. In the resulting R-benzoin model the hydroxyl is located at the posi- tion of the water molecule observed in both crystal structures of BAL (Figs 6 and 7) as well as in the crystal structure of BFD [12]. We suggest that this applies for all acyloin cleavage reactions of BAL. During acyloin C–C-bond formation, on the other hand, this water site accommodates the oxygen of the acceptor aldehyde. All residues lining the active centre pocket are depic- ted in Fig. 7. A comparison with the functionally well- established enzymes BFD and PDC shows almost no conservation (Fig. 4). However, Ala480 and Phe484 are conserved between BAL and BFD. These residues were therefore mutated resulting in decreased activity, as to be expected from their location within the active centre [31]. In BAL, the chain around Phe484 is quite mobile with B-factors about 20 A ˚ 2 higher than average so that this side chain may close down on a bound substrate performing an induced-fit motion. Such a side chain displacement is supported by a comparison with BFD, where Phe484 points into the active centre as shown in Fig. 7. The established structure of BAL invites further efforts to identify the roles of the various catalytic residues through mutational and structural studies combined with enzyme kinetic measurements. This knowledge together with designed mutations are likely to expand the range of organic compounds that can be produced enzymatically. Fig. 7. Model of R-benzoin (green) bound in the active centre of BAL. The model allows for a nucleophilic attack of the deprotonated C2 atom of ThDP at the targeted carbonyl-group of R-benzoin under the expected Bu ¨ rgi–Dunitz angle [30] (dotted line, red). The suggested pro- ton transfer from C2 to N4¢ is indicated (dotted line, green). All residues lining the active centre pocket are given as ball-and-stick models in domain colors (Fig. 2). Their Ca atoms are black. The Phe484 conformation observed in the BFD crystals is yellow. The site of the water molecule bound to BAL is marked by a halo and occupied by the substrate hydroxyl group. T. G. Mosbacher et al. ThDP-depending benzaldehyde lyase FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS 6073 Experimental procedures Expression and purification Wild-type BAL with a C-terminal His-tag (Fig. 4) was obtained from Escherichia coli SG13009 cells following a previously described procedure [25]. Cells were grown at 37 °C and expression of BAL was induced with isopropyl- b-d-thiogalactopyranoside (IPTG). After cell lysis, the supernatant was applied to a Ni-chelate column. The enzyme was further purified on a gel permeation column (Superdex 200, Amersham-Pharmacia, Freiburg, Germany) using buffer A (25 mm Hepes pH 6.9, 200 mm NaCl, 2.5 mm MgCl 2 , 0.1 mm ThDP and 2 mm dithiothreitol). BAL-containing fractions were identified by SDS ⁄ PAGE, pooled and adjusted to a concentration of 20 mgÆmL )1 . The typical yield was 8 mg proteinÆg )1 cell pellet. SeMet- labelled BAL was obtained by introducing the expression vector into the methionine-auxotrophic E. coli strain B834(DE3). Cells were cultured in LeMaster medium [32] containing 25 mgÆL )1 seleno-l-methionine (Acros). Cultiva- tion and purification procedures were the same as for wild- type BAL. The yield of purified SeMet-labeled BAL was about 6 mgÆg )1 cell pellet. Full incorporation of SeMet was verified by ESI-MS. Crystallization and data collection The purified protein (wild-type and SeMet) was dialyzed for 12 h against buffer B (5 mm Hepes pH 6.9, 10 mm NaCl, 2 mm MgCl 2 and 2 mm dithiothreitol). The solution was then adjusted to a concentration of 12 mgÆmL )1 and crystallised by the hanging drop vapour diffusion method at 20 °C. The drops consisted of 1.8 lL protein in buffer B, 0.2 lL of an Agarose-LM solution (3%, 37 °C; Hampton Research, Alieso Veijo, CA, USA) and 2 lL buffer C (50% (v ⁄ v) PEG 200 for wild-type BAL or 55% (v ⁄ v) PEG 200 for SeMet-labelled BAL, 100 mm Mes pH 6.85), which was also used as the reservoir. The crystals appeared after about 3 days and reached maximum sizes of 300 · 80 · 80 lm 3 a week later. All crystals belonged to spacegroup P3 1 21. They were flash-frozen in liquid nitrogen without a further addi- tion of a cryo-protectant. Data collection of the wild-type crystals was carried out at beamline PX of the Swiss Light Source (Villigen, Switzerland). MAD data were collected from a single SeMet crystal using beamline BW7A at the EMBL-outstation (DESY Hamburg). All data were proc- essed and scaled with program XDS [33]. Structure determination and refinement Using the MAD data sets, the positions of the selenium atoms were established with solve [34]. The selenium sites were refined and used for initial phasing with sharp [35]. Density modification and initial model building was carried out using resolve [36]. The model was manually completed with XFIT [37] and subsequently refined with the Anneal and Minimize options of CNS [38] followed by a restrained refinement with refmac [39]. Water molecules were intro- duced using arp ⁄ warp [40]. They were confirmed wherever the (2Fo-Fc)-map showed a density above 0.8 r and the environment allowed the formation of hydrogen bonds. The procedure resulted in about 0.2 water molecules per residue. The refinement was completed with 10 cycles of tls ⁄ refmac [41] specifying each subunit of the tetramer as a TLS group. Non-crystallographic symmetry restraints were used throughout the refinement. Subsequently, the structure of wildtype BAL was established by molecular replacement using molrep [39]. It was refined in the same way starting from the model of SeMet-labeled BAL. Both structures were evaluated with procheck [42] and rampage [43]. Model building of R-benzoin in complex with BAL was performed by manually docking the substrate into the active centre, followed by energy minimization using the Anneal and Minimize options of CNS. For the structure similarity search we used dali [17]. It should be noted that the general search with dali failed to find the enzymes als and ipdc in the Protein Data Bank. Structural superposi- tions were performed with lsqman [39]. Figures were produced with povscript+ [44] and povray [http:// www.povray.org]. The coordinates and structure factors have been deposited in the Protein Data Bank under acces- sion codes 2AG0 and 2AG1. Acknowledgements We thank Martina Pohl for kindly providing us with the gene of the benzaldehyde lyase and for helpful discus- sions, the teams of the Swiss Light Source (Villigen ⁄ CH) and of the EMBL outstation Hamburg for their help with data collection and J. Wo ¨ rth for the ESI-MS meas- urements. Further, we thank M. J. McLeish for sending us a preprint of his paper (details in Note). The project was supported by the Deutsche Forschungsgemeinschaft under grants SFB-380 and SFB-388. References 1Mu ¨ ller M & Sprenger GA (2004) Thiamine-dependent enzymes as catalysts of C-C bonding reactions: the role of ‘orphan’ enzymes. In Thiamine: Catalytic Mechanisms in Normal and Disease States (Jordan, F & Patel, MS, eds), pp. 77–92. Marcel Dekker, London. 2 Jordan F (2003) Current mechanistic understanding of thiamin diphosphate-dependent enzymatic reactions. 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