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• W2023707186 abstract "The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2′-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8Å or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. In the substrate complexes, biphenyl rings of substrates are recognized by extensive hydrophobic interactions with the active site residues. Binding of substrates accompanies structural changes of the active site loops and recruits His60 to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser27, His60, and Arg70, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys27 functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His60 and Arg70 orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys27 and stabilize the negatively charged reaction intermediate. Cys, His, and Arg residues are conserved in putative proteins homologous to DszB, which are presumed to constitute a new family of desulfinases. The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2′-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8Å or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. In the substrate complexes, biphenyl rings of substrates are recognized by extensive hydrophobic interactions with the active site residues. Binding of substrates accompanies structural changes of the active site loops and recruits His60 to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser27, His60, and Arg70, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys27 functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His60 and Arg70 orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys27 and stabilize the negatively charged reaction intermediate. Cys, His, and Arg residues are conserved in putative proteins homologous to DszB, which are presumed to constitute a new family of desulfinases. Sulfur oxides released into the atmosphere by combustion of fossil fuel cause serious air pollution, which leads to acid rain and destroys forests and soils. Sulfur oxides also raise public health concerns associated with cardiopulmonary diseases (1Pope III, C.A. Burnett R.T. Thun M.J. Calle E.E. Krewski D. Ito K. Thurston G.D. J. Am. Med. Assoc. 2002; 287: 1132-1141Crossref PubMed Scopus (6551) Google Scholar). To alleviate these problems, efforts are being made to limit the sulfur content in diesel and gasoline fuel. Inorganic or nonaromatic organic sulfur compounds can easily be removed from fossil fuels by the conventional metal-catalyzed hydrodesulfurization method (2Houalla M. Broderick D.H. Sapre A.V. Nag N.K. Beer V. Gates B.C. Kwart H. J. Catal. 1980; 61: 523-527Crossref Scopus (336) Google Scholar), but it is difficult to remove polycyclic aromatic sulfur compounds such as benzothiophene and dibenzothiophene (DBT) 2The abbreviations used are: DBT, dibenzothiophene; HBP, 2-hydroxybiphenyl; HBPS, 2′-hydroxybiphenyl-2-sulfinic acid; BPS, biphenyl-2-sulfinic acid; PDB, Protein Data Bank; r.m.s.d., root mean square deviation. (3Kabe T. Akamatsu K. Ishihara A. Otsuki S. Godo M. Zhang Q. Qian W.H. Ind. Eng. Chem. Res. 1997; 36: 5146-5152Crossref Scopus (82) Google Scholar). In particular, DBT is considered as a model compound of polycyclic organic sulfur compounds in fossil fuels, and degradation of DBT by microbial activities has been studied with a keen interest in biodesulfurization (4Monticello D.J. Finnerty W.R. Annu. Rev. Microbiol. 1985; 39: 371-389Crossref PubMed Scopus (173) Google Scholar, 5Van Hamme J.D. Singh A. Ward O.P. Microbiol. Mol. Biol. Rev. 2003; 67: 503-549Crossref PubMed Scopus (1008) Google Scholar). Sulfur-specific DBT degradation pathway, which is capable of transforming DBT to sulfite and 2-hydroxybiphenyl (HBP), was identified in the soil bacterium Rhodococcus erythropolis (6Gallagher J.R. Olson E.S. Stanley D.C. FEMS Microbiol. Lett. 1993; 107: 31-35Crossref PubMed Scopus (213) Google Scholar, 7Izumi Y. Ohshiro T. Ogino H. Hine Y. Shimao M. Appl. Environ. Microbiol. 1994; 60: 223-226Crossref PubMed Google Scholar) and found to be catalyzed by the following three enzymes: DszA, DszB, and DszC (8Denome S.A. Oldfield C. Nash L.J. Young K.D. J. Bacteriol. 1994; 176: 6707-6716Crossref PubMed Google Scholar, 9Piddington C.S. Kovacevich B.R. Rambosek J. Appl. Environ. Microbiol. 1995; 61: 468-475Crossref PubMed Google Scholar). DszA and DszC are flavin-dependent monooxygenases and responsible for oxidation of DBT to 2′-hydroxybiphenyl-2-sulfinic acid (HBPS). A 39-kDa protein DszB (EC 3.13.1.3) participates in the last desulfurization step and hydrolyzes the sulfinate group of HBPS (Scheme 1). The reaction catalyzed by DszB is the slowest in the pathway and thus was proposed as the rate-limiting step in desulfurization (10Gray K.A. Pogrebinsky O.S. Mrachko G.T. Xi L. Monticello D.J. Squires C.H. Nat. Biotechnol. 1996; 14: 1705-1709Crossref PubMed Scopus (307) Google Scholar). DszB is inhibited by the reaction product of HBP but not by biphenyl, which indicates that the hydroxyl group is required for the inhibition. However, DszB can accept biphenyl-2-sulfinic acid (BPS) as a substrate, and the hydroxyl group of HBPS does not seem to be essential for the activity (11Nakayama N. Matsubara T. Ohshiro T. Moroto Y. Kawata Y. Koizumi K. Hirakawa Y. Suzuki M. Maruhashi K. Izumi Y. Kurane R. Biochim. Biophys. Acta. 2002; 1598: 122-130Crossref PubMed Google Scholar). DszB is also strongly inhibited by cysteine-modifying reagents, and mutation of the unique cysteine residue, Cys27, into serine abolished the activity (11Nakayama N. Matsubara T. Ohshiro T. Moroto Y. Kawata Y. Koizumi K. Hirakawa Y. Suzuki M. Maruhashi K. Izumi Y. Kurane R. Biochim. Biophys. Acta. 2002; 1598: 122-130Crossref PubMed Google Scholar). Besides biodesulfurization, knowledge of sulfur metabolism by soil bacteria is particularly interesting because of their competition for sulfur nutrient with plants. Although biochemical studies have shown that DszB is a unique desulfinase, its detailed molecular reaction mechanism remains largely unknown. In an attempt to expand our understanding of bacterial desulfurization in molecular detail, we determined crystal structures of DszB in complex with substrates HBPS and BPS. Materials—BPS was synthesized as reported previously (12Hanson G. Kemp D.S. J. Org. Chem. 1981; 46: 5441-5443Crossref Scopus (40) Google Scholar). Sodium salt of HBPS was a gift from Petroleum Energy Center, Shimizu, Japan. Reagents for crystallization buffer were purchased from Hampton Research. Other chemicals used were of the finest grade commercially available. Crystallization and Diffraction Data Collection—Protein purification and crystallization were performed as described previously (11Nakayama N. Matsubara T. Ohshiro T. Moroto Y. Kawata Y. Koizumi K. Hirakawa Y. Suzuki M. Maruhashi K. Izumi Y. Kurane R. Biochim. Biophys. Acta. 2002; 1598: 122-130Crossref PubMed Google Scholar, 13Lee W.C. Ohshiro T. Matsubara T. Izumi Y. Tanokura M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1636-1638Crossref PubMed Scopus (9) Google Scholar). For phasing, native crystals of DszB were soaked in 10 mm KAu(CN)2 for 2 days before the diffraction data collection. Single-wavelength anomalous diffraction data set was collected on beamline BL-6A at the Photon Factory (Tsukuba, Japan), at the wavelength of 0.978 Å. Diffraction images were integrated and scaled using MOSFLM and SCALA programs from the CCP4 program suite (14Collaborative Computational Project, No. 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1995; 50: 760-763Crossref Scopus (19797) Google Scholar). Native data sets of DszB and substrate complexes were collected on beamlines BL-18B and BL-6A at the Photon Factory, respectively (Table 1).TABLE 1Selected crystallographic data and statisticsDszBGold derivativeDszB-HBPSDszB-BPSData collectionSpace groupP212121P212121C2C2Wavelength, Å1.0000.9780.9780.978Resolution, Å28.8-1.834.9-2.054.2-1.631.0-1.8No. of measured reflections139,971189,431353,812236,875No. of unique reflections39,99129,35995,47565,706Completeness, % (last shell)99.4 (98.6)98.8 (94.1)100 (99.8)99.4 (96.3)I/σ (last shell)6.8 (3.2)11.3 (6.4)7.1 (3.7)9.2 (4.8)Rmerge (last shell)aRmerge = ∑hkl∑i|Ii(hkl) - 〈I(hkl)) 〉|/∑hkl∑i(hkl), where Ii(hkl) is the i-th intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl) 〉 is its average.0.072 (0.214)0.042 (0.106)0.058 (0.199)0.054 (0.138)SAD phasingResolution, Å34.9-2.2RcullisbRcullis is as defined in CNS.0.847Phasing powercPhasing power is as defined in CNS.0.962Figure of merit0.216RefinementResolution28.8-1.854.2-1.631.0-1.8R-factor (Rfree)0.188 (0.201)0.180 (0.196)0.169 (0.198)Average B-factor, protein, Å216.53513.46313.452Average B-factor, water, Å231.50723.57226.770Average B-factor, ligands,dAverage B-factors of a glycerol molecule, HBPS, and BPS are bound to the active site, respectively. Å232.59512.38811.609r.m.s.d. bonds, Å0.0050.0050.004r.m.s.d. angles, °1.6941.2071.208a Rmerge = ∑hkl∑i|Ii(hkl) - 〈I(hkl)) 〉|/∑hkl∑i(hkl), where Ii(hkl) is the i-th intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl) 〉 is its average.b Rcullis is as defined in CNS.c Phasing power is as defined in CNS.d Average B-factors of a glycerol molecule, HBPS, and BPS are bound to the active site, respectively. Open table in a new tab Structure Determination, Model Building, and Refinement—The x-ray crystal structure of DszB was determined by the single wavelength anomalous dispersion method (Table 1). The single gold site was located, and the program CNS was used to refine the heavy atom parameters (15Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). After phase improvement by density modification, the single wavelength data produced an interpretable electron density map. The programs ARP/wARP and XtalView were used to build the model (16Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar, 17McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar). The structures of DszB-HBPS and DszB-BPS were solved by the molecular replacement method using the program MolRep (18Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar). Topology files of ligands were prepared using PRODRG server, and models were refined by the program CNS (15Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar, 19Schuttelkopf A.W. van Aalten D.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4360) Google Scholar) (Table 1). The stereochemistry of the final models was assessed with the program PROCHECK (20Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The coordinates and structure factors of DszB, DszB-HBPS, and DszB-BPS structures have been deposited in the Protein Data Bank (PDB) as 2DE2, 2DE3, and 2DE4, respectively. Secondary structures were designated with the program DSSP (21Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12421) Google Scholar). Structure figures were prepared with the programs Molscript and Raster3D (22Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar, 23Merritt E.A. Murphy M.E. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar). Site-directed Mutagenesis—Site-directed mutagenesis study was conducted as described previously (11Nakayama N. Matsubara T. Ohshiro T. Moroto Y. Kawata Y. Koizumi K. Hirakawa Y. Suzuki M. Maruhashi K. Izumi Y. Kurane R. Biochim. Biophys. Acta. 2002; 1598: 122-130Crossref PubMed Google Scholar). pKK223-3 expression vectors (Amersham Biosciences) coding for wild-type DszB or mutants were transformed into Escherichia coli BL21 strain harboring pKY206 plasmid (BL21/pKY206), which contains E. coli chaperone genes (24Mizobata T. Akiyama Y. Ito K. Yumoto N. Kawata Y. J. Biol. Chem. 1992; 267: 17773-17779Abstract Full Text PDF PubMed Google Scholar). Cultured cells were resuspended in 50 mm potassium phosphate buffer (10% glycerol (v/v) and 1 mm dithiothreitol) and lysed by sonication (Branson Instruments). The cell debris was removed by centrifugation at 12,000 × g for 30 min, and relative protein concentration was verified by SDS-PAGE. Thus prepared soluble fractions of cell crude extracts were used for activity assay. Activity of DszB and its mutants was assayed at 28 °C by measuring the amount of HBP produced from HBPS with high pressure liquid chromatography system. 1 unit of activity was defined as the amount of DszB necessary to produce 1 nmol of 2-HBP or sulfite per min. Sequence Alignment—Multiple alignment of amino acid sequences was carried out using ClustalX program (25Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35620) Google Scholar). Fig. 5 was prepared using ESPript program (26Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics. 1999; 15: 305-308Crossref PubMed Scopus (2540) Google Scholar). The Overall Structure of DszB—The crystals of DszB are in space group P212121 with a = 36.7, b = 82.6, and c = 139.6 Å. The asymmetric unit consists of a single molecule of DszB. In addition to 330 water molecules, 7 glycerol molecules and 1 acetate ion were modeled and included in the structure. The structure of DszB is monomeric and oval-shaped with approximate dimensions of 60 × 50 × 40 Å (Fig. 1A). In the final model of DszB, more than 91% of residues are located in the most favored regions in the Ramachandran plot, and only Leu226 is located in generously allowed regions. However, this residue is well defined in its electron density. The 18 N-terminal residues were not observed in the electron density map. We deleted these residues to find out whether they have relevance to function, and we found no difference in activity or stability (data not shown). DszB is composed of two α/β domains, with highly curved 5-stranded β-sheets being located between α-helix bundles. We designate the two domains as domain A (residues 19-97 and 251-365) and domain B (residues 98-250) (Fig. 1, A and B). We searched for proteins with similar structures using the DALI server (27Holm L. Sander C. Nucleic Acids Res. 1998; 26: 316-319Crossref PubMed Scopus (598) Google Scholar) and found matches, including ovotransferrin (PDB code 1NNT, Z score = 8.7, root mean square deviation (r.m.s.d.) = 4.3 Å) and substrate-binding component from a bacterial ABC transporter of sulfate (PDB code 1SBP, Z score = 7.3, r.m.s.d. = 3.8 Å) (Fig. 1C). These results show that DszB belongs to the phosphate-binding protein family as defined in the SCOP protein fold data base (28Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5610) Google Scholar). Two enzymes, porphobilinogen deaminase (29Louie G.V. Brownlie P.D. Lambert R. Cooper J.B. Blundell T.L. Wood S.P. Warren M.J. Woodcock S.C. Jordan P.M. Nature. 1992; 359: 33-39Crossref PubMed Scopus (174) Google Scholar) and thiaminase-I (30Campobasso N. Costello C.A. Kinsland C. Begley T.P. Ealick S.E. Biochemistry. 1998; 37: 15981-15989Crossref PubMed Scopus (36) Google Scholar), also belong to this family. Additionally, amino acid homology search on the NCBI protein sequence data base (www.ncbi.nlm.nih.gov/) showed that DszB has significant amino acid sequence similarity to the putative substrate-binding proteins. Specifically, the C-terminal half of DszB shares a 26% amino acid identity with AtsR, the substrate-binding protein component for the sulfate ester transport system of Pseudomonas putida S-313 (31Kahnert A. Kertesz M.A. J. Biol. Chem. 2000; 275: 31661-31667Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Based on three-dimensional structure similarity and amino acid homology, we propose that DszB has been evolved from substrate-binding proteins. The active site of DszB, in which the critical residue Cys27 is located, is in the cavity between the two domains and includes a glycerol molecule (Fig. 1D). Adding glycerol in the buffers (10% (w/v)) during the purification steps was essential in stabilizing the protein (11Nakayama N. Matsubara T. Ohshiro T. Moroto Y. Kawata Y. Koizumi K. Hirakawa Y. Suzuki M. Maruhashi K. Izumi Y. Kurane R. Biochim. Biophys. Acta. 2002; 1598: 122-130Crossref PubMed Google Scholar). The binding of a glycerol molecule in the active site may be responsible for stabilizing the protein. The domain B part of the cavity is mainly hydrophobic, whereas that of domain A contains Cys27, whose sulfhydryl group is free of interactions with nearby residues. In the course of searching for a potential general base, we found the carboxylate group of Glu192 to be adjacent to the residue, separated by about 4 Å (Fig. 1D), and we mutated it to Gln to examine its function. However, the mutation had no effect on the desulfination activity (data not shown). Structures of DszB in Complex with Substrates—To investigate the substrate-binding mode of DszB, an inactive mutant (DszB-C27S) was crystallized in complex with substrates HBPS and BPS, as attempts to soak substrates into crystals were unsuccessful (13Lee W.C. Ohshiro T. Matsubara T. Izumi Y. Tanokura M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1636-1638Crossref PubMed Scopus (9) Google Scholar). Complex crystals could be obtained by incubating the protein (∼10 mg/ml) in a buffer containing 20 mm Tris, pH 8.0, 10% (v/v) glycerol, and 20 mm HBPS or BPS prior to the crystallization setup. The structures of HBPS and BPS complexes were determined at resolutions of 1.6 and 1.8 Å, respectively, by the molecular replacement method. Crystals are in space group C2 with a = 154.7, b = 46.2, c = 114.0 Å, and β = 115.8° for DszB-HBPS and a = 153.4, b = 45.9, c = 112.9 Å, and β = 115.9° for DszB-BPS. Crystals contain two similar DszB molecules in the asymmetric unit, and each DszB molecule binds one acetate ion. One magnesium ion added in the crystallization buffer was included in each of the models. Electron densities belonging to HBPS and BPS were found in the cavity at the center of DszB. In the electron densities, the dihedral angle between the biphenyl rings is about 90°, and tetrahedral geometry of the sulfinate group could be recognized easily (Fig. 2A). The overall structure is similar to that of the native protein, and we can superimpose the structure of DszB with that of DszB-HBPS or DszB-BPS with an approximate r.m.s.d. of 0.5 Å (Fig. 2B) except for residues 55-62 and 187-204, which confer the most notable change as a result of substrate binding (Fig. 2C). These residues, which were loops in the native structure, form α-helices to accommodate the biphenyl rings of substrates (Fig. 2C). The structural change introduces His60 into the active site, whose side chain interacts with Ser25 and HBPS, and the conformation of loop β1-αA changes to accommodate this residue, exposing Ser27, which is Cys in the native protein, to the sulfinate group of substrates (Fig. 2D). The glycerol molecule in the active site of the native structure is replaced by phenyl sulfinate moieties of substrates in the complex structures. In the active site, Pro28, Phe61, Leu152, Trp155, Gly183, Phe203, and Leu226 mainly form hydrophobic interactions with substrates (Fig. 3A). Except for the additional hydroxyl group of HBPS, which is hydrogen-bonded to a water molecule in the active site, HBPS and BPS bind the active site in a similar manner. A sulfinate oxygen interacts with O-γ of Ser27, N-ϵ of His60, and N-η2ofArg70, all at distances of 2.6-2.7 Å. As a result, the sulfinate group, Cys27, His60, and Arg70, forms a tetrahedral hydrogen bond network, with the guanidine moiety of Arg70 forming additional hydrogen bonds with the carbonyl oxygen of Gly73 (Fig. 3B). O-γ of Ser27 also lies adjacent to the main chain nitrogen of Gly73 at a distance of ∼3.0 Å. The configuration of Gly73 is reminiscent of the oxyanion hole in serine proteases, which is usually observed in various hydrolases and is known to stabilize the negative charge of the reaction intermediate (32Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1852) Google Scholar). It is also noteworthy that a water molecule interacts with the other sulfinate oxygen at a distance of 2.8 Å (Fig. 3B). Site-directed Mutagenesis—A site-directed mutagenesis study to address the role of each residue in the active site triad demonstrates that each residue is crucial to the activity of DszB. We found that the H60Q mutation caused an ∼17-fold reduction in the specific activity (Table 2). The structural integrity of DszB may have been affected by the R70I or R70K mutations as those expressed mutants mostly existed in the insoluble fraction of cell extracts (data not shown). The highly conserved 70RXGG motif forms the structural core of domain A, and thus Arg70 may be essential in maintaining the structure of domain A. We could not detect desulfination activity from the crude cell extracts of Arg70 mutants (Table 2).TABLE 2Activity chart of DszB mutants in crude extracts of E. coli BL21 harboring pKY206 (BL21/pKY206)DszB nativeDszB H60QDszB R70IDszB R70KControl (BL21/pKY206)Protein concentration (mg/ml)39.746.326.324.136.8Activity (units/ml)99369.4NDaND, not detected.NDNDSpecific activity (units/mg)251.50NDNDNDa ND, not detected. Open table in a new tab To our knowledge, the only desulfinase documented so far is aspartate β-decarboxylase (EC 4.1.1.12), which can accept structurally similar cysteine sulfinates (33Soda K. Methods Enzymol. 1987; 143: 453-459Crossref PubMed Scopus (64) Google Scholar, 34Graber R. Kasper P. Malashkevich V.N. Strop P. Gehring H. Jansonius J.N. Christen P. J. Biol. Chem. 1999; 274: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Aspartate β-decarboxylase is a member of the pyridoxal phosphate-dependent aminotransferase family, and β-elimination of aspartate or cysteine sulfinate is considered to occur by the formation of a Schiff's base with the pyridoxal phosphate cofactor, thus stabilizing the transition state (35Eliot A.C. Kirsch J.F. Annu. Rev. Biochem. 2004; 73: 383-415Crossref PubMed Scopus (675) Google Scholar). However, DszB does not require any form of cofactor for desulfurization (11Nakayama N. Matsubara T. Ohshiro T. Moroto Y. Kawata Y. Koizumi K. Hirakawa Y. Suzuki M. Maruhashi K. Izumi Y. Kurane R. Biochim. Biophys. Acta. 2002; 1598: 122-130Crossref PubMed Google Scholar) and seems to have adopted a distinct mechanism of desulfination (36Gray K.A. Mrachko G.T. Squires C.H. Curr. Opin. Microbiol. 2003; 6: 229-235Crossref PubMed Scopus (115) Google Scholar). Proposed Reaction Mechanism of DszB—In the active site of DszB-C27S in complex with substrates, Ser27 (Cys in the native protein) is positioned for nucleophilic attack on the sulfinate sulfur (Fig. 3B), and thus the thiolate ion of Cys27 and the sulfinate sulfur form a thiolsulfonate-like intermediate as a plausible first step of the reaction. It has been proposed that the Cys-His dyad in cysteine proteases forms a thiolate-imidazolium pair to activate the catalytic cysteine residue (37Polgar L. FEBS Lett. 1974; 47: 15-18Crossref PubMed Scopus (128) Google Scholar), and we find Ser27 and His60 with a similar configuration in the complex structures. However, the ion pair formation mechanism for DszB is not reasonable because S-γ of Cys27 and N-ϵ of His60 are separated by a distance of ∼17 Å in the absence of a substrate (Fig. 1D). Furthermore, the H60Q mutant retains desulfination activity, which suggests His60 does not function as a general base and may be required for the proper orientation of the sulfinate group. We could not find any other base in the vicinity of Cys27 except for the sulfinate group from the substrate. Hence, we propose the sulfinate group to be the general base. Sulfinic acids are stronger acids than carboxylic acids and consequently poor bases (38Fujihara H. Furukawa N. Patai S The Chemistry of Sulfinic Acids, Esters and Their Derivatives. John Wiley & Sons Ltd., Chichester, UK1990: 275-295Google Scholar). Nonetheless, optimal positioning and orientation of the catalytic base can increase its basicity by about several orders of magnitude (39Gandour R.D. Bioorg. Chem. 1981; 10: 169-176Crossref Scopus (209) Google Scholar). In the case of the sulfinate group, because of the lone pair electron of the sulfur atom, the syn orbital of the sulfinate oxygen and the S-C bond are predicted to be on the same side of the SO2 plane (40Basch H. Patai S The Chemistry of Sulfinic Acids, Esters and Their Derivatives. John Wiley & Sons Ltd., Chichester, UK1990: 9-34Google Scholar). Consequently, we find the sulfinate group of bound substrates in a configuration that directs its syn orbital to the hydroxyl hydrogen of Ser27 (Fig. 3B). Moreover, protonation of the sulfinate group can reduce the negative charge building on the reaction intermediate. Similar substrate-assisted catalysis has been proposed for GTPases such as p21Ras and type II restriction endonucleases (41Dall'Acqua W. Carter P. Protein Sci. 2000; 9: 1-9Crossref PubMed Scopus (111) Google Scholar). Arg70 and the main chain nitrogen of Gly73 seem to be required for the stabilization of the negatively charged reaction intermediate. A water molecule coordinated to the sulfinate oxygen could be added in a similar fashion to release a sulfite molecule in the final steps of desulfination. The proposed reaction mechanism of DszB is illustrated in Fig. 4. DszB Belongs to a New Family of Desulfinases—The structure of DszB defines a new group of hydrolases in which conserved Cys, His, and Arg participate in hydrolysis of the C-S bond in organic sulfinic acids. Based on this definition, homologs of DszB were searched for in NCBI protein sequence data base. Homologs of DszB could be found primarily in genome sequences of soil bacteria, and all include Cys, His, and Arg in their N-terminal regions (Fig. 5). A multiple alignment of the amino acid sequences also shows apparent homology (data not shown), and thus these homologs appear to have similar folds. Interestingly, some of these putative proteins are from protein clusters whose genomic contexts are different from that of dsz operon. As organic sulfur compounds of various structures exist in soil, these uncharacterized DszB homologs might function as desulfinase components of sulfur metabolic pathways yet to be discovered. We thank the beamline staff at Photon Factory for help with data collection (Proposal 2001G352) and Dr. Michael E. P. Murphy for critical reading of the manuscript." @default.
• W2023707186 created "2016-06-24" @default.
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• W2023707186 date "2006-10-01" @default.
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• W2023707186 title "Crystal Structure and Desulfurization Mechanism of 2′-Hydroxybiphenyl-2-sulfinic Acid Desulfinase" @default.
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