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• W1974804581 abstract "The nitrilase superfamily proteins are involved in a wide variety of nonpeptide carbon–nitrogen hydrolysis reactions, characterized by a thioacyl-enzyme intermediate formed through the attack of a cyano or carbonyl carbon by a novel conserved catalytic triad of Glu-Lys-Cys,1 to produce important natural products such as auxin, biotin, precursors of antibiotics, etc. On the basis of extensive sequence analysis, members of the nitrilase superfamily were classified into six2 or 131 functionally district groups. However, although sequence searching can identify polypeptides as members of the nitrilase superfamily, their annotations have been perplexing. For example, nitrile hydratases that convert a nitrile to the corresponding amide3, 4 were not classified as members of the nitrilase superfamily while most enzymes assigned to the nitrilase superfamily are actually amidases. It is therefore necessary to reclassify these protein members preferably by structure-based methods. Currently only a few protein structures are available in this superfamily, including a NitFhit protein from Caenorhabditis elegans,5 two N-carbamyl-D-amino acid amidohydrolases (DCases) from Agrobacterium sp. Strain KNK7126 and Agrobacterium radiobacter,7 and a putative CN hydrolase from Sacchromyces cerevisiae strain S288C.8 They are either dimeric or tetrameric α-β-β-α sandwich proteins. We now report the crystal structure of XC1258, a putative nitrilase superfamily protein, from the plant pathogen Xanthomonas campestris pv. campestris str. 17 (Xcc). On the basis of a multiple sequence alignment, XC1258 was found to exhibit only low sequence identities (23%, 17%, 14.2%, and 16.1%, respectively) with other nitrilase superfamily proteins for which the tertiary structures have been determined (1F89, 1EMS, 1ERZ, and 1FO6).9 However, conserved catalytic triad residues of Glu-Lys-Cys in the active site region were observed.1 Extensive consensus of the characteristic signature sequence surround these catalytic triad residues further establishes XC1258 as a Nit protein.1 We have determined the crystal structure of XC1258 to a resolution of 1.73 Å using the two-wavelength MAD approach. Interestingly, a cacodylate or dimethylarsinic acid compound was found to situate perfectly in the active region, forming a strong arsenic adduct with the active cysteine residue. This observation confirms the previously proposed reaction mechanism for the nitrilase superfamily proteins and suggests that their activity could be inhibited by the dimethylarsinic compound through a sulfur-arsinic covalent bond. The cloning of XC1258 gene, along with its expression and protein purification, have been described in a previous communication.9 Se-Met labeled XC1258 was produced using a nonauxotroph E. coli strain BL21(DE3) as host in the absence of methionine but with ample amounts of Se-met (100 mg/L). The induction was conducted at 37°C for 4 h by the addition of 0.5 mM IPTG in M9 medium consisting of 1 g of NH4Cl, 3 g of KH2PO4, and 6 g of Na2HPO4 supplemented with 20% (W/V) of glucose, 0.3% (W/V) of MgSO4, and10 mg of FeSO4. Purification and crystallization of the Se-Met labeled XC1258 were performed using the protocols as established for the native protein.9 Cubic-like crystals appeared in 3 days from a reservoir solution comprising 100 mM sodium cacodylate pH 6.5 and 1M sodium citrate in the absence of any reducing agent. The data collection statistics has been published before.9 Crystals were flash-cooled at 100 K under a stream of cold nitrogen. X-ray diffraction data were collected using the National Synchrotron Radiation Research Center (NSRRC) beamline 13B1 in Taiwan. A two-wavelength 1.73 Å resolution of MAD data set was obtained. The data were indexed and integrated using HKL2000 processing software,10 giving a data set that was 98.5% complete with an overall Rmerge of 4.3% on intensities. The crystals belong to the orthorhombic space group P21212. The data collection statistics are summarized in Table 1. The refinement of selenium atom positions, phase calculation, density modification, and building of initial model were performed using the program SOLVE/RESOLVE.11 The program O12 (or XtalView13) and REFMAC514 were then used for rounds of manual model rebuilding and refinement. The final statistics of structural refinement of XC1258 was listed in Table 1. The atomic coordinates of XC1258 have been deposited in the PDB (2E11). The structure of XC1258 consisting of 265 amino acid residues has been determined to a resolution of 1.73 Å using the MAD method, with the refinement statistics listed in Table 1. The final model (Fig. 1) comprises four protein molecules from residue 1 to residue 265 and 940 water molecules. The Ramachandran plot calculated from the PdbViewer15 shows that 91.7% of the residues are in the most favored regions, with 7.5% of the residues in the additional allowed regions. Only two residues were found to deviate from the allowed torsional angles. The first residue C143 is located in a turn connecting β7 strand with helix α5, forming a nucleophile elbow in the active site similar to other α/β hydrolases.16, 17 Because of the strategic position of the active site residue, its backbone torsion angles fall in the disallowed region (Φ = 72°, Ψ = −52°), which may be a unique feature of this active site.8 The second residue E232 is also located in a sharp turn connecting β11 with β12 with torsion angles (Φ = 30°, Ψ = −102°) in the disallowed region. Two specific H-bonds were found in this region; one is E232 HN – A214 CO, and another one is R231 CO – Q233 HN. (a) The XC1258 monomer tertiary structure color-coded from blue (N-terminal) to red (C-terminal). (b) The primary sequence of XC1258. The 12 β-strands were marked in red arrows and the six α-helices in green tubes. The conserved catalytic triad residues E43, K109, and C143 were shown in red capitals. (c) The stereo ribbon picture of the XC1258 tetramer. The α-helices were shown in red, the β-strands in yellow, and the random coil in green. The twofold axes were also shown and annotated in arrows. (d) The stereo view of the active site complexed with dimethyl arsenic acid. The H-bonds were drawn in dotted green lines, while unconventional CH…π bonds in dotted gray lines. The 2F0-Fc electron density maps of the dimethyl arsenic adduct and the surrounding aromatic acid residues were shown in red and blue, respectively, in the background. The arsenic atom is obviously coordinated to two methyl groups and one oxygen atom. Unlike the catalytic cavity in DCase,6, 7 that of XC1258 is filled mainly with hydrophobic residues. Figure 1(a, c, d) were drawn by PyMol program (DeLano Scientific LLC, 2004) and Figure 2 by PdbViewer.15 XC1258 monomer is a globular α/β protein comprising mainly six α helices and two six-stranded β sheets [Fig. 1(a,b)]. Helices α1 and α3 form one layer, interacting with the β12 β1 β2 β3 β4 β5 β-sheet layer, and α4 and α5 form the other layer, interacting with the β6 β7 β8 β9 β10 β11 β-sheet layer. The axis of helix α6 is approximately perpendicular to other helices and does not form part of the αββα four-layer sandwich. As in DCase6, 7 and NitFhit5 proteins, XC1258 monomers assemble into a tetramer that holds two types of interfaces (A/C or B/D and A/B or C/D) via three diad axes. The A/C interface is formed by a helix bundle (α4/α5) and an antiparallel β-sheet mediated by the C-terminal S261-G265 residues [bottom of Fig. 1(c)]. Considerable numbers of salt-bridges and H-bonds (16 in total) were detected in the A/C interface, turning a four-layered αββα sandwich into an eight-layered αββααββα sandwich. On the other hand, the A/B interface is formed mainly by two antiparallel β-strand interactions involving strands 11-11 and 12-12, respectively. However it is interesting to note that these two antiparallel β-strands pair in different ways; strand 11 of subunit A interacts with strand 11 of subunit B through backbone-backbone H-bonds (R231 HN – Q227 CO and E229 HN – E229 CO and vice versa, 4 in total), while those between strands 12 go through backbone/side-chain or side-chain/side-chain salt-bridges (Q235Oϵ – T239 Oγ, Q235 Nϵ – T238 CO and vice versa, four in total). Therefore, the Cα-Cα distances increase from ∼4.1 Å between strands 11 to ∼7.4Å between strands 12. Including the four extra salt-bridges between the E232Oζ – P226 CO and E2332Oζ – D221 Oδ atoms, there are 12 H-bonds/salt bridges in total to stabilize the A/B subunit interfaces. Altogether, there are two 12 β-strand sheets in each A/B and C/D dimers, forming a 48-stranded beta box in the XC1258 tetramer. This situation is similar to the Nit tetramer in the NitFhit protein, which forms a 52-stranded beta box in the protein core.5 The XC1258 crystals were grown in 100 mM cacodylate buffer (dimethyl arsenate), and found to yield a tetrahedral covalent adduct between the C143 Sγ and arsenate, which was unequivocally identified from the X-ray fluorescence scan (data not shown). In the final refined complex, the bond lengths from As to divalent oxygen is 1.77 Å, from As to C143 Sγ is 2.21 Å, and from As to the methyl groups are 1.97 Å. These bond lengths are within the range expected for AsO (1.67 Å) and AsS (2.24 Å) bonds.18 However, the AsO bond is somewhat longer (by 0.1 Å), and AsS bond somewhat shorter (by 0.03 Å). It is possible that some resonance occurs between the CH2SAsO and CH2S+AsO− forms to decrease the AsS bond length but increase the AsO bond length.19 In the arsenic adduct, the divalent oxygen forms H-bonds with the Y144 amide proton and K109 side chain amino proton. Similar arsenate adduct with the active Ser-Oγ residue in an acetyl esterase has also been reported.20 The AsO bond length in that adduct complex is only 1.59 Å. This can be explained by the harder nature of oxygen atom compared with the sulfur atom, and hence the less contribution of the CH2O+AsO− resonance form, since most AsO bond lengths of the arsenate adducts binding with the softer sulfur atom of Cys-Sγ in a number of protein structures deposited in PDB (1K2O, 1P13, 1TAD, 1VHD, and 1VHO) are longer, ranging from 1.75 to 1.87 Å. Interestingly, the two methyl groups of cacodylate in the adduct form good CH…π interactions with the surrounding aromatic groups; that is, one with the F49, and the other with the F113 and W175. Such interactions have been proposed to contribute substantially to the stability of a protein or a protein–ligand complex.21-23 Such CH…π interactions are not simply caused through hydrophobic clustering, as they exhibit “directionality,” that is, the carbon atom of CH bond is situated directly above the aromatic groups of Phe, Tyr, or Trp, with the CH bond pointing directly toward the center of the aromatic group.21-23 The active site of the XC1258/arsenic adduct is especially rich in such interactions, with the two methyl groups of the active site cacodylate exhibiting three such interactions, two stronger (to F49 and F113) and one weaker (to W175), as revealed by the distances of the methyl Cβ atoms to the six carbon atoms in the phenyl ring (3.48–3.85 Å for F49, 3.72–4.02 Å for F113, and 4.24–5.64 Å for W175). We rationalize that the smaller and less varied of these distances (indicating that the Cβ atoms are pointing more toward the center of the aromatic ring), the stronger of such interaction. We have checked the possible formation of such CH…π bond in a number of crystal structures containing the cacodylate adduct with the Cys-Sγ atom (1VHO, 1VHD, 1TAD, 1P13, 1K2O, 1B92, 1B9F, 1B9D), and found only a few exhibit strong enough CH…π bond (one in 1VHD, and one in 1B92/1B9F/1B9D); many others have quite varied distances (3.85–5.48 Å in 1K2O, for example) or simply have the active site aromatic groups turned away from the cacodylate methyl groups. This may account for the well visibility of the electron density map containing the (CH3)2AsO/Cys-Sγ adduct in the active site of the XC1258/cacodylate complex [Fig. 1(d)]. The unexpected presence of the cacodylate adduct shows that AsV-containing organic compounds can react specifically with cysteine 143 of XC1258 to form a stable analogue of the tetrahedral transition state proposed for the deacylation of the thioacyl-enzyme intermediate formed from the starting nitrile or amide reactants (Fig. 2)1, 24; the volume occupied by the proSCH3 group on the dimethyl arsenic complex is large enough to accommodate a nucleophilic H2O molecule that can attack the carbonyl group of the thioacyl-enzyme intermediate because of the geometrically favorable position. Such H2O molecule can also be stabilized by a similar unconventional H-bond of OH…π interactions with the surrounding phenyl group of F49.25 A series of bond formation and breakage occur successively to form the free acid product to regenerate the native enzyme via a transition state complex containing partial bonds as annotated by the dotted lines in the top Fig. 2. Proposed mechanism of the hydrolysis of thioacyl-enzyme intermediates. The dimethyl arsenic acid complex resembles the transition state for hydrolysis. The electron movements were shown in curved arrows, and the partial bonds formed or broken by dotted lines. the proSCH3 group (enclosed in circle) in the dimethyl arsenic complex can be substituted by a H2O molecule to initiate the hydrolysis process. A structural fold and homolog search performed with the coordinates of XC1258 using the SSM (Secondary Structure Matching)26 and DALI27 servers have identified four similar structures that include a putative CN-hydrolase,8 a NitFhit protein in which the nitrilase domain is combined with a histidine triad (Hit) doman5 and a pair of identical N-carbamyl-D-amino acid amidohydrolases (DCases)6, 7 within the nitrilase superfamily. A multiple structural alignment of these structures with XC1258 were performed using the MUSTANG program28 and are shown in Figure 3. The rmsd values between these structures are very low, ranging from 1.49 Å for 199 Cαs of 1ERZ, 1.26 Å for 224 Cαs of 1F89, to 1.38 Å for 214 Cαs of 1EMS, respectively, indicating that nitrilase fold is highly conserved. However, considerable differences exist in the lengths of hairpin loops enclosing the active site region, as clearly demonstrated in Figure 3. From the figures, one can see that for DCase (1ERZ, orange), the large Lγ and Lδ loops form a lid that encloses the catalytic triad residues in the active site cavity. For NitHis (1EMS, green), only a large Lγ loop is present. However, such large caps are absent for both CN-hydrolase (1F89, blue) and XC1258 (red). Thus CN-hydrolase and XC1258 may both exhibit broader substrate specificities. (a) Multiple sequence alignment of the XC1258 with a DCase (1ERZ), a putative yeast CN-hydrolase (1F89), and the nitrilase domain of NitFhit (1EMS) produced from MUSTANG program.28 Colors indicate the chemical nature of the amino acid (small hydrophobic including aromatic, red; acidic, blue; basic, magenta; basic amino acids with hydroxyl group and/or amino groups, black). The markup row below each stretch of the multiple alignment indicates completely conserved residue (in UPPERCASE) and partially conserved residues (3 in 4, in lowercase). Residue R155-D157 forms a small β-hairpin with residue Q163-D165 in XC1258, which was connected by a black line. (b) Multiple superimposition of these protein structures produced from the same program. The hairpin loop conformations, which differ significantly among these four structures, were drawn in thicker lines to emphasize the “lids” structures. The active site residues were drawn in ball-and-stick (oxygen, red; nitrogen, blue; sulfur, yellow). The critical loops Lγ and Lδ have different lengths, as shown in Figure 3(a). Although XC1258 is structurally similar to DCase, many critical residues necessary for the decarbamylation reaction are absent in XC1258. For example, for the three histidines identified to be important for catalysis,7 only H129 was found to be present in XC1258 (H111). The other two His residues H144 and H215 are replaced with G116 and W183 in XC1258, respectively. In fact, these three histidine residues are not even within 5 Å of the active cysteine residue. Furthermore, while the active site cavity of DCase is filled mainly with charged residues such as R174, R175, H143, and E145 excluding the three catalytic triad residues,6 in XC1258, many hydrophobic residues, such as F49, F113, V142, W175, and P176, combine with the catalytic triad residue to form the cavity. These structural information indicate that XC1258 is unlikely to be a DCase. However, from sequence comparison of the highly characteristic signature sequences surrounding the catalytic triad residues, XC1258 can be temporarily assigned to be a Nit protein belonging to the Nit and NitFhit branch due to their very high sequence identity1 (in XC1258, the signature sequences are 41LPETF45, 107YDKRHLF113, and 141QVCYDLRFP149, while those of the Nit and NitFhit branch proteins are 52LPECF56, 125YNKLHLF131, and 167SICYDVRFP175, respectively). The XC1258 structure reported here thus represents a new variant of prokaryotic Nit protein in the nitrilase superfamily without a lid. Its complex structure with the dimethylarsenic adduct in the active site confirms the common hydrolysis mechanism of the nitrilase superfamily proteins. Since the function for the Nit branch protein is currently unknown,1, 5 it would be of great help to be able to find out their ligands or substrates. More biochemical and biophysical studies are necessary to decipher its biological function. We thank the Core Facilities for Protein X-ray Crystallography in the Academia Sinica, Taiwan, for assistance of crystal screening, and the synchrotron facilities in the National Synchrotron Radiation Research Center, Taiwan, and in the SPring-8 Synchrotron facility in Japan for assistance of X-ray data collection. The National Synchrotron Radiation Research Center is a user facility supported by the National Science Council, Taiwan, ROC, and the Protein Crystallography Facility is supported by the National Research Program for Genomic Medicine, Taiwan, ROC." @default.
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• W1974804581 title "The crystal structure of XC1258 from Xanthomonas campestris: A putative procaryotic Nit protein with an arsenic adduct in the active site" @default.
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