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Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden
Reprint requests to: T. Alwyn Jones or Kristina Bäckbro, Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden; e-mail: alwyn{at}xray.bmc.uu.se or nina{at}xray.bmc.uu.se; fax: +46-18-536971.
(RECEIVED March 4, 2005; FINAL REVISION April 22, 2005; ACCEPTED April 22, 2005)
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Keywords: Mycobacterium tuberculosis; Rv0216; hotdog fold; protein structure; crystallography
Abbreviations: ACP, acyl carrier protein • CoA, coenzyme A • COG, cluster of orthologous groups • DHD, double hotdog • FAS, fatty acid synthase • PDB, Protein Data Bank • PMSF, phenylmethane sulfonyl fluoride • RMSD, root-mean-square distance • SeMet, seleno-methionine • SHD, single hotdog • TB, tuberculosis.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051442305.
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Introduction |
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Three-dimensional structural information on Rv0216 might cast light on its biological function. Indeed, our crystallographic studies show that Rv0216 exhibits overall structural similarities to a group of enzymes that use thiol esters as substrates. The greatest similarity is to bacterial and eukaryotic R-specific enoyl hydratases, but Rv0216 is distinct from these enzymes in the composition of its putative catalytic residues. The new structure allows us to re-evaluate the structure–function relationship of a group of M. tuberculosis proteins that are likely to have structures similar to Rv0216. Half of these proteins have been shown to be essential for bacterial survival during infection (Sassetti and Rubin 2003) and/or growth (Sassetti et al. 2003).
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, 2). The somewhat elongated molecule is ~35x45x55 Å and is built up primarily of an extended anti-parallel 
-sheet of 10 strands and two long 
-helices (3 and 6) (Fig. 1). Two similar structural repeats of ~140 residues are related by an approximate twofold rotation axis. Each repeat consists of a domain that is built up from a -sheet of five strands (ordered 1-3-4-5-2) that wraps around a long -helix. The -sheets pack around the local dyad axis such that the second strand in each domain abuts to create the extended sheet. The helix packs onto the concave surface of the sheet and is located in the sequence between the first two -strands of each domain. It is linked to the end of the first -strand by an ~45-residue stretch that folds to form a lid that interacts with the long helix of the other domain (Fig. 1). The lid of the first domain contains two short -helices, while the lid of the second domain has only one. The last strand of the first domain is connected to the first strand of the second by a 40-residue loop that extends over the entire convex surface of the extended -sheet. Each domain has the so-called hotdog topology first seen in Escherichia coli -hydroxydecanoyl thiol ester dehydrase (Leesong et al. 1996). This enzyme, also called FabA, catalyses two reactions (dehydration and isomerization) on fatty acid thiol esters of acyl carrier protein (ACP) (Bloch 1971). It is a homodimer with a quaternary structure similar to Rv0216. The double hotdog (DHD) fold of Rv0216 has also been previously observed; the first report of it was in E. coli thioesterase II (Li et al. 2000).
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). At the amino acid level this structural alignment shows 24 identical residues, corresponding to a sequence identity of ~16%. The longest conserved stretch of amino acids is the pentapeptide G106-DTLY/G286-DTLY starting at the short loop connecting the second and third -strands of each domain. This is an interesting peptide stretch that crosses over the -meander made up from the fourth and fifth strands, on the concave face of the sheet (Fig. 2A). The side-chain of D107 helps stabilize this loop by forming a hydrogen bond to the main-chain amide of 104. One other conserved side-chain, Q80, interacts via hydrogen bonds to the main chain of this pentapeptide. Two other conserved clusters occur along strands 4 and 5 (G134-LR/G308-LR and D144RxVLD/D324RxVLD), but only one set of conserved side-chain interactions is retained: the salt link across the strands between R136 and D149.
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-sheet. Structure alignment statistics are given in Table 3
and the superimposed structures are shown in Figure 3. The best matching structures can be aligned so that ~250 CA atoms have RMSDs of <2 Å while in the less similar structures the number of matching pairs is halved. The two most similar structures to Rv0216 are the (R)-specific enoyl-CoA hydratase from Aeromonas caviae (Hisano et al. 2003; PDB code 1IQ6
[PDB]
) and the so-called monoamine oxidase regulatory protein from Archaeoglobus fulgidus (A.A. Fedorov, E.V. Fedorov, R. Thirumuruhan, and T.A. Jones, unpubl.; PDB code 1Q6W
[PDB]
). In these three, the alignment of the -sheet and central helix are very well conserved, as is the lid section of Rv0216 linking the first strand in each domain to the hotdog helix. The reverse turn between strands 3 and 4 is truncated in both the 1IQ6 and 1Q6W structures. In the Archaeoglobus fulgidus SHD structure, the C terminus of the hotdog helix is extended by one turn. The structure-based sequence identity between Rv0216 and these two structures is 13% and 16%, respectively. In the least similar structure, the 4-hydroxybenzoyl-CoA thioesterase from Arthobacter sp. strain SU (Thoden et al. 2003; PDB code 1Q4T
[PDB]
), the Rv0216-like lid is much reduced and replaced by an N-terminal extension from a dimer-related chain. In this comparison, the central 10 -strands of Rv0216 align less well and the hotdog helix is displaced relative to the sheet. The 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas sp. strain CBS-3 (Thoden et al. 2002; PDB code 1LO7
[PDB]
) has more similar overall structure to Rv0216 and the main hotdog helices are well aligned. The lid connection in this structure, however, is much shorter so that one edge of the hotdog helix is solvent accessible. Of the DHD structures, the eukaryotic hydratase 2 enzyme (Koski et al. 2004) shows the best overall structural similarity, including a close fit to the lid structures of Rv0216. Although the lid from the second domain is particularly well conserved, the first hotdog helix is highly distorted. The conserved structure-based sequence identity with Rv0216 is only 8%. The superimposed structures in Figure 3B show that the -sheet is well conserved despite the numerous -bulges, which produce local distortions. The hotdog helices move as rigid bodies to adapt to changes in sequence, although the first hotdog helix is the least well conserved in structure (Fig. 3A). The largest variations in the hotdog motif occur in the lid and the loops connecting secondary structures. Only the crossover between 2 and 3 is a structurally well-conserved loop (Fig. 3C).
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). They appear to enter between the central two strands of the -sheet into a cleft formed by the N-terminal portion of the hotdog helix from one domain, and the C-terminal portion of the helix from the other domain. The lid loop, when present, folds on top of the helices to close off the active site. In at least one structure, eukaryotic hydratase 2, the active site is a tunnel rather than an invagination (Koski et al. 2004).
Putative active site of RV0216
A number of sequences, in a wide range of actino- and proteobacteria, were found to be homologous to Rv0216 using an iterative approach with WU-BLAST2 (Lopez et al. 2003). These Rv0216-like proteins have only a few conserved regions (Fig. 4). Although separated in sequence, these conserved regions cluster in the same neighborhood of a deep pocket, sequestered from the bulk solvent. This pocket coincides with the active site of the enzymes given in Table 3. For three of these hotdog-fold enzymes, a histidine residue in the lid region (H237 in Rv0216) has been implicated in catalysis. In E. coli FabA (Leesong et al. 1996) this histidine (residue 70) is the target for a mechanism-based, suicide inhibitor. Together with a neighboring aspartyl side-chain from the twofold related hotdog helix (residue 84), these residues are proposed to catalyze both the dehydration and isomerization reactions performed by the enzyme. The other two enzymes are bacterial and eukaryotic hydratases (Hisano et al. 2003; Koski et al. 2004) that catalyse the R-specific hydration of 2-enoyl coenzyme A (CoA) intermediates to 3-hydroxyacyl-CoA molecules. It has been suggested that the catalytic residues are also a pair of histidine aspartyl residues. The bacterial hydratase is a SHD homodimer with two active sites, while the eukaryotic enzyme is a DHD monomer where only one active site has been conserved. The pair of catalytic residues is located on the same stretch of the lid region, separated by four intervening residues, and the lids in these enzymes have very similar structures. In the eukaryotic hydratase, this local sequence was identified as a highly conserved segment termed the hydratase 2 motif (Qin et al. 2000), part of which (D-X-N-P-[LIV]-H) contains the proposed active site residues. Their lids differ in structure from the lid in FabA, but the catalytic histidine residues are structurally well conserved (Fig. 2B). The catalytic aspartyl residue of the dehydrase is spatially close to the aspartyl residues of the hydratases, despite being from another subunit. In their discussion of the hydration reaction, both Leesong et al. (1996) and Hisano et al. (2003) favor a mechanism where the aspartic acid residue activates a water molecule, which attacks the C3 atom of a 2-enoyl substrate while the histidine residue donates a proton to the C2 atom of the substrate. In contrast, Koski et al. (2004) favor a mechanism where the catalytic water molecule is first coordinated by the conserved aspartyl, asparagine, and histidine side-chains of the hydratase motif, which then attacks the substrate via a concerted transition state where both the proton and hydroxyl group are derived from the water (Bahnson et al. 2002). In all these structures, the hydrogen bonds from the unpaired mainchain nitrogen atoms at the N terminus of the hotdog helix are likely to play a role in the interaction with the substrate. The FabA dehydrase is unique among these enzymes in having a second activity, namely, the isomerization of (E)-2-decenoyl-ACP to (Z)-3-decenoyl-ACP. Leesong et al. (1996) argue that this reaction is carried out by the same catalytic machinery.
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In the Rv0216 family, the proposed active site is an enclosed cavity rather than a tunnel, with a volume and shape similar to the bacterial hydratase (Fig. 5A). The residue lining the end of the cavity is a highly conserved arginine residue (R152) that forms hydrogen bonds to the side chains of N91, S81, and T85. The asparagine at residue 91 is conserved in almost all family members. In one member both the residues equivalent to R152 and N91 are changed, and in another the asparagine is changed to a glutamic acid and therefore capable of forming a salt link with the residue equivalent to R152. The side chains that are equivalent to residue 81 are very highly conserved S/T residues. At residue 85, there is a preference for only a threonine. This cluster of residues around the arginine side chain results in satisfying four of the five possible hydrogen bonds via protein side chains and the fifth via a water molecule.
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). However, since the binding pocket is closed by hotdog helix 6, in contrast to the eukaryotic enzyme, the four aliphatic carbons at the end of the acyl chain cannot be fitted without substantial conformational changes. The substrate, stretching up through the opening of the tunnel, is potentially interacting with a rim of well-conserved residues at the entrance to the proposed active site (including A90, L92, R119, K124, G131, L132, M155, L247, H279, and P282) (Fig. 5B). Based on the above structural similarities to enoyl-CoA hydratases, the enoyl-CoA hydratase activity of Rv0216 was assayed using the minimal substrate, 2-butenoyl CoA (crotonyl-CoA), as substrate, and found to be inactive.
Related M. tuberculosis proteins
Rv0216 has been assigned to Cluster of Orthologous Groups (COG) 2030 (Tatusov et al. 1997) based on its sequence similarity but it is not assigned to any Pfam family (Bateman et al. 2004). However, some members of COG 2030 are also classified as members of Pfam 0175, a family whose members contain a domain of ~130 amino acids. This domain corresponds to the SHD domain of the bacterial hydratase of Hisano et al. (2003) and the C-terminal hotdog domain of Rv0216. Both COG 2030 and Pfam 0175 include seven other M. tuberculosis proteins. These proteins vary widely in size, from 142 to 3069 amino acids, and in Pfam annotation (Table 4). Five of these proteins contain a hydratase 2 motif as described by Qin et al. (2000). In light of our crystallographic study on Rv0216, we have re-evaluated this group of proteins. As part of our standard bioinformatics evaluation of M. tuberculosis targets, we have made use of fold-recognition evaluations. Before solving the structure of Rv0216, the 3D-PSSM server (http://www.sbg.bio.ic.ac.uk/~3dpssm/) of Sternberg and coworkers (Kelley et al. 2000) indicated that the structure might contain a hotdog-domain fold, most similar to PDB code 1IQ6
[PDB]
. We have, therefore, also evaluated the sequences of the other seven M. tuberculosis proteins with the same fold recognition server (Table 4). Our analysis indicates that at least five of them (Rv2499c, Rv0130, Rv0636, Rv3389c, and Rv3538) are likely to be SHD dimers or DHDs that are related in structure to Rv0216. Two more (Rv0241c and Rv2524c) are likely to contain at least one SHD domain, but may be DHDs.
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, however, Rv2499c appears to be an Rv0216-like SHD structure and hence likely to form a homodimer. This protein matches the sequence profile of Rv0216-like proteins in both hotdog domains, in particular the conserved residues in the proposed active site. Two additional proteins, Rv0130 and Rv0636, are more similar to the SHD bacterial R-specific 2-enoyl-CoA hydratase (Fig. 4; Table 4). These similarities include the active site, the hydrophobic patch lining the entrance of the substrate-binding pocket (L35, F47, I51, F83, 1IQ6 numbering), and the 2-3 crossover loop (F83-D91). Although the hydratase 2 motif defined by Qin et al. (2000) is not present in Rv0130, only two residues differ (Fig. 4). In regions where the existing SHD crystal structures show the largest variation, namely, the connection between the hotdog helix and the 2-strand, there is poor sequence identity. In the final two -strands, the two known structures show a preference for small, branched side chains pointing inward, which can be recognized in the M. tuberculosis sequences, but a correct alignment is difficult to achieve.
Two of the seven M. tuberculosis proteins, Rv3389c and Rv3538, are likely to be incorrectly classified as "possible" or "probable" dehydrogenases. This seems to have arisen because of sequence similarity to a multifunctional enzyme, where the second function is a dehydrogenase activity. We suggest that Rv3389c and Rv3538 are DHD enzymes, based on their structure-based sequence alignments (Fig. 4) and PSSM scores (Table 4). The second hotdog domains are very well conserved and clearly indicate R-specific enoyl hydratase-like active sites. The first hotdog domains show much lower overall sequence similarity but have local identity patterns that map to important areas of SHD structure. Residues L16–Y17 (1PN2 hydratase 2 numbering), on the first helix of the lid, close to the internal dyad axis, are positioned where there is a hydrophobic dependency as part of the dimer formation, while G22 terminates the helix at a Schellman turn (Schellman 1980). The P42-TF sequence is the start of the hotdog helix with F44 close to the side-chain of Y17. The next sequence-conserved region, L72-HGE, is located at the start of the 2 in the vicinity of the active site, while the P84-X-P peptide is at the important 2-3 crossover loop. The residues at the end of 3 and the reverse turn contain another conserved region, P102-KG, where the lysine residue interacts with one of the phosphates of the substrate in the eukaryotic DHD hydratase (Koski et al. 2004). Finally, R134 is the last residue in the final -strand of the first hotdog domain, and is the beginning of a remarkably well-conserved feature in the other proteins (RGxGGFGG). This begins the linker to the second hotdog domain, and starts at a highly conserved proline residue, P162.
Two other proteins, Rv0241c and Rv2524c, show indications of DHD hydratase-like structures. Rv0241c is large enough to be a DHD, its C-terminal half shows a good PSSM score to the bacterial R-specific enoyl hydratase, and its sequence contains a hydratase 2 motif. Its N-terminal region shows a poorer but still significant score to the same enzyme. Similarly, a 149-amino-acid segment (residues 1197–1345) in the much larger Rv2524c shows a high structural similarity to the bacterial R-specific enoyl hydratase and also contains a hydratase 2 motif. An equally long portion of the preceding sequence (residues 1038–1196) shows a much poorer match to the same hydratase. These results may indicate the presence of a DHD organization in both proteins but where the first hotdog domain is less similar to our search templates. Rv2524c is the only protein in Table 4 for which an enzymatic activity has been suggested. This is the type I fatty acid synthase (FAS-I) of M. tuberculosis, the multifunctional enzyme found in eukaryotes and advanced prokaryotes. The separate enzymatic activities have been identified and localized to functional domains. A dehydrase-related catalytic role for a histidine residue (H878) in the rat FAS-I system has been suggested (Joshi and Smith 1993). The identification of this residue was made possible by weak sequence similarity to the E. coli FabA enzyme before the crystal structure determination by Smith and coworkers (Leesong et al. 1996). In the sequence alignments of Joshi and Smith (1993) the aspartyl residue in the active site of FabA is not present. Our own alignments show the presence of an equivalent glutamyl residue in some sequences (including the mycocerosic acid synthase of M. tuberculosis, data not shown) but not in the rat and human FAS-I enzymes, for example. It should be remembered, however, that FabA is a homodimer and that the proposed catalytic residues are derived from different SHD domains. We have located a SHD in the human FAS-I sequence using PSSM, and after optimizing the sequence range to residues 820–968, we get significant scores (4.1x10 –1 to 1MKA and 1.6x10–1 to 1J1Y) with H878 at the active site. We have been unable to locate a SHD domain before or after this region of the sequence, however.
Initially, the dehydrase activity of the M. tuberculosis FAS-I was suggested to reside in the region centered on H963, and was also based on sequence analysis (Fernandes and Kolattukudy 1996). Rv2524c was, however, later identified as containing the hydratase 2 motif by Qin et al. (2000). Since the -hydroxyacyl dehydrase activity of this enzyme is the reverse of the reaction carried out by the R-specific enoyl hydratases, we can, with some confidence, assign D1229 and H1234 to be the catalytic residues for the dehydrase activity of Rv2524c. Interestingly, the type I enzyme is thought to be a homodimer (Smith et al. 2003), and although we suggest that the dehydrase activity resides in a DHD domain, an alternative hypothesis would be a dimer of SHD domains. However, the FAS-I dimer can be dissociated by lowering the ionic strength and temperature while retaining dehydrase activity on model substrates (Kumar et al. 1970). Such activity is not feasible in a solitary SHD, but would be possible in a DHD structure. Although the catalytic machinery of all members of the FAS-I family does not seem to be conserved, it is possible that an underlying DHD structure may be a common feature. However, we do not rule out the possibility of homodimer formation, despite experimental evidence to the contrary. In the M. tuberculosis enzyme, the malonyl/palmitoyl transferase catalytic domain follows the dehydrase-like domain, separated by a short linker.
M. tuberculosis also contains the FAS-II system found in bacteria and plants where the enzymatic activities exist in separate polypeptides (Bloch 1977; Smith et al. 2003; Takayama et al. 2005). The gene encoding the dehydrase activity in this system has not yet been identified. In E. coli, on the other hand, two enzymes, FabA and FabZ, have this activity with overlapping chain length specificities. In their review, Takayama et al. (2005) identify a sequence similarity between FabZ and Rv0098 from M. tuberculosis. The latter protein has been identified by Sassetti and Rubin (2003) as being essential for M. tuberculosis survival during infection in an in vivo assay, is conserved in other mycobacteria (including M. leprae), and is, therefore, a good candidate for the "missing" -hydroxyacyl-ACP dehydrase. Our PSSM analysis of the FabZ sequences from E. coli and S. pneumoniae indicates a structural relationship to the FabA of Leesong et al. (1996), with scores of 4.0x10–8 and 6.2x10–8 to 1MKA, but this relationship is not apparent in the Rv0098 sequence (no SHD or DHD hits in the top scores). Although this may merely indicate a divergence in the sequence so that the underlying structural similarity is no longer apparent, Table 4 suggests that a number of other gene products have the potential for a dehydrase active site. In particular, Rv0636 has been shown to be important in both the in vivo and in vitro studies (Sassetti and Rubin 2003; Sassetti et al. 2003) and is, therefore, an equally good candidate for the missing -hydroxyacyl-ACP dehydrase in M. tuberculosis.
To elucidate the function of the M. tuberculosis proteins in Table 4 will require detailed biochemical, genetic, and structural analyses. As a start, we have expressed and purified Rv2499c, Rv0130, and Rv0636 (A.S. Covarrubias, T. Bergfors, T. Unge, and T.A. Jones, unpubl.) and investigated their hydratase activity. Only Rv0130 has shown activity in our assays using 2-butenoyl CoA (crotonyl-CoA) as a substrate. A protein concentration of 5 ng/mL gave a steady decrease in A263 for >3 min. It must be emphasized that the known SHD and DHD hydratases/dehydrases show a variation in substrate specificity due to differences in the structure of their active sites. The precise nature of the active sites of the proposed M. tuberculosis SHD/DHD’s will require detailed crystallographic analyses and we hope that these structures will provide new insights into potential substrates.
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Our structural analysis shows that the Rv0216 gene product has a double hotdog fold, characteristic of a class of enzymes that have thiol esters as substrates. Rv0216-like sequences show conservation around a potential active site region of this class of enzyme, but this is restricted to only one of the hotdog domains. Although Rv0216 shows strong evidence for a gene duplication event, we suggest that only one functional active site has been preserved. We believe this is localized to a variation of the hydratase 2 loop (Koski et al. 2004) in the C-terminal SHD. In Rv0216-like proteins, the proposed catalytic apparatus of the hydratases is modified so that although the histidinyl residue is conserved, an asparaginyl residue replaces the catalytic aspartyl residue. Indeed, Rv0216 shows no activity on crotonyl CoA and its natural substrate remains to be identified. However, our new structure helps us evaluate seven M. tuberculosis gene products related to Rv0216. One of these is likely to be a DHD containing the dehydrase activity of the FAS-I, and five others are likely to be SHD/DHD hydratases or dehydrases. We have expressed two of these, and one shows hydratase activity on crotonyl CoA substrate. We suggest that the other, Rv0636, is a good candidate for the "missing" -hydroxyacyl-ACP dehydrase of FAS-II in M. tuberculosis. Interestingly, of the eight gene products that may contain SHD/DHD domains, four were found to be essential for survival in one of the high-density mutagenesis experiments of Rubin and coworkers (Sassetti and Rubin 2003; Sassetti et al. 2003), and six are present in M. leprae. This family may, therefore, have the potential for further structure-based drug design studies.
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SeMet was introduced into the protein by metabolic inhibition (van Duyne et al. 1993). The SeMet protein was expressed in Rosetta (DE3) pLacI cells or BL21 (DE3) cells (Invitrogen) grown in minimal media supplemented with SeMet, lysine, threonine, phenylalanine, leucine, isoleucine, and valine. Expression was done at room temperature overnight. The purification protocol for the SeMet protein was the same as for the native protein but with 10 mM -mercaptoethanol in all buffers to prevent SeMet oxidation. Mass spectrometry analyses of the native and SeMet proteins gave molecular weights of 36.69 and 36.80 kDa, respectively. The difference (0.11 kDa) indicates that 2.4, or 79%, of the three methionines have been substituted for SeMets.
Crystallization
All crystallization trials were carried out at 20°C by the vapor diffusion method (McPherson 1999). Initial crystallization conditions for the native protein were screened using Crystal Screen HT and a selection of additives from Additive Screens 1–3 (Hampton Research). Native and SeMet crystals of Rv0216 were obtained in droplets consisting of 1 µL protein (4–5 mg/mL) in 20 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, and 1 µL reservoir (100 mM MES [pH 6.5], 1.3 M ammonium sulfate, 4% PEG400, 10 mM -mercaptoethanol). The crystals grew to a size of 0.2 mmx0.2 mmx0.4 mm within 4 wk. Monomer and dimer fractions of the protein gave similar results in the crystallizations.
Data collection
Crystals of seleno-substituted Rv0216 were flash-frozen in liquid nitrogen using the mother liquor with 25% glycerol added as a cryo-protectant. Diffraction data were collected at the European Synchrotron Radiation Facility (ESRF) beamline ID29 from a single SeMet crystal at wavelengths above, below, and at the Seabsorption edge to resolutions of 2.7, 2.5, and 2.8 Å, respectively. An additional high-resolution 1.9 Å data set was collected close to the selenium peak. Indexing, integration, and scaling of the data were carried out using the HKL suite of computer programs (Otwinowski and Minor 1997). The SeMet crystal form was found to belong to space group P6322 with unit cell parameters a=77.9 Å, b=77.9 Å , and c=178.5 Å. Assuming one monomer in the asymmetric unit gives a Vm of 2.2 Å3 Da–1 (Matthews 1968). A complete native data set was later collected to a resolution of 1.9 Å. Data collection statistics are given in Table 1.
Structure determination and refinement
The structure was solved by MAD (Hendrickson 1991). The positions of two selenium atoms were found with the RSPS program (Knight 2000) using the Se-peak diffraction data set. A third selenium site was located by difference Fourier analysis after phasing with MLPHARE (Otwinowski 1991). Initial MAD phasing was carried out with SHARP (de la Fortelle and Bricogne 1997) and then extended to a resolution of 1.9 Å using SAD phasing. Electron density maps were subjected to solvent flattening and histogram matching using DM (Cowtan and Main 1998). The semi-interactive main-chain tracing and sequence decoration tools of O (Jones 2004) were used to build 330 of the 337 amino acids in the asymmetric unit. This model was improved by alternating cycles of refinement with REFMAC5 (Murshudov 1997) and interactive rebuilding with O (Jones et al. 1991). Water molecules were added with ARP/wARP (Perrakis 1997). One residue, Y94, in the putative active site shows relatively poor electron density and may interact with a sulfate group. However, since the density is not perfectly clear, it has been modeled as solvent. The final phasing and refinement statistics are given in Table 2. Coordinates and structure factors have been deposited at the PDB with access code 2BI0
[PDB]
.
Enoyl-CoA hydratase activity assay
Hydration of crotonyl-CoA (2-butenoyl-CoA) was used as an assay for enoyl-CoA hydratase activity (Moskowitz and Merrick 1969; Fukui et al. 1998). The reaction was carried out in a total volume of 1 mL. Enzyme (Rv0216, Rv0130, Rv0636, or Rv2499c) was added to 25 µM of crotonyl-CoA (Sigma) in 50 mM Tris (pH 8.5), and the decrease in absorbance at 263 nm was measured at 25°C or 30°C.
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Acknowledgments |
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References |
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