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• W2061859547 abstract "SoxB is an essential component of the bacterial Sox sulfur oxidation pathway. SoxB contains a di-manganese(II) site and is proposed to catalyze the release of sulfate from a protein-bound cysteine S-thiosulfonate. A direct assay for SoxB activity is described. The structure of recombinant Thermus thermophilus SoxB was determined by x-ray crystallography to a resolution of 1.5 Å. Structures were also determined for SoxB in complex with the substrate analogue thiosulfate and in complex with the product sulfate. A mechanistic model for SoxB is proposed based on these structures. SoxB is an essential component of the bacterial Sox sulfur oxidation pathway. SoxB contains a di-manganese(II) site and is proposed to catalyze the release of sulfate from a protein-bound cysteine S-thiosulfonate. A direct assay for SoxB activity is described. The structure of recombinant Thermus thermophilus SoxB was determined by x-ray crystallography to a resolution of 1.5 Å. Structures were also determined for SoxB in complex with the substrate analogue thiosulfate and in complex with the product sulfate. A mechanistic model for SoxB is proposed based on these structures. The oxidation of reduced inorganic sulfur species by sulfur bacteria is an important component of the biogeochemical sulfur cycle and has practical applications in biomining, agriculture, biocorrosion, fuel desulfuration, and waste treatment (1Dahl C. Friedrich C.G. Microbial Sulfur Metabolism. Springer-Verlag, 2007Google Scholar, 2Lengeler J.W. Drews G. Schlegel H.G. Biology of the Prokaryotes. Blackwell Science, Oxford, UK1999Google Scholar). Sulfur bacteria use the electrons liberated in sulfur oxidation reactions as the reductant for carbon dioxide fixation and/or as donors to respiratory electron transport chains. The Sox (sulfur oxidizing) system is one of the most widely distributed sulfur oxidation pathways and is found in both photosynthetic and nonphotosynthetic sulfur-oxidizing eubacteria (3Friedrich C.G. Bardischewsky F. Rother D. Quentmeier A. Fischer J. Curr. Opin. Microbiol. 2005; 8: 253-259Crossref PubMed Scopus (364) Google Scholar). Substrates of the Sox system are reported to include thiosulfate, sulfide, elemental sulfur, sulfite, and tetrathionate (4Wodara C. Bardischewsky F. Friedrich C.G. J. Bacteriol. 1997; 179: 5014-5023Crossref PubMed Google Scholar, 5Appia-Ayme C. Little P.J. Matsumoto Y. Leech A.P. Berks B.C. J. Bacteriol. 2001; 183: 6107-6118Crossref PubMed Scopus (82) Google Scholar, 6Mukhopadhyaya P.N. Deb C. Lahiri C. Roy P. J. Bacteriol. 2000; 182: 4278-4287Crossref PubMed Scopus (65) Google Scholar). The Sox pathway has been best characterized in the α-Proteobacterium Paracoccus pantotrophus. In this bacterium thiosulfate is oxidized to sulfate by the four periplasmic protein complexes SoxYZ, SoxAX, SoxB, and SoxCD (3Friedrich C.G. Bardischewsky F. Rother D. Quentmeier A. Fischer J. Curr. Opin. Microbiol. 2005; 8: 253-259Crossref PubMed Scopus (364) Google Scholar, 7Rother D. Henrich H.J. Quentmeier A. Bardischewsky F. Friedrich C.G. J. Bacteriol. 2001; 183: 4499-4508Crossref PubMed Scopus (116) Google Scholar, 8Friedrich C.G. Rother D. Bardischewsky F. Quentmeier A. Fischer J. Appl. Environ. Microbiol. 2001; 67: 2873-2882Crossref PubMed Scopus (474) Google Scholar). Intermediates in the pathway are covalently bound to a cysteine residue located in a conserved Gly-Gly-Cys-Gly-Gly sequence at the C terminus of the SoxY protein (9Quentmeier A. Friedrich C.G. FEBS Lett. 2001; 503: 168-172Crossref PubMed Scopus (52) Google Scholar). This C-terminal peptide acts as a swinging arm enabling the cysteine and its bound adducts to enter the active sites of the other pathway components (10Sauvé V. Bruno S. Berks B.C. Hemmings A.M. J. Biol. Chem. 2007; 282: 23194-23204Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In the current pathway model the heme protein SoxAX (11Bamford V.A. Bruno S. Rasmussen T. Appia-Ayme C. Cheesman M.R. Berks B.C. Hemmings A.M. EMBO J. 2002; 21: 5599-5610Crossref PubMed Scopus (123) Google Scholar) oxidatively conjugates thiosulfate to the SoxY swinging arm to form a cysteine S-thiosulfonate, which is then degraded by a combination of SoxB and SoxCD. View Large Image Figure ViewerDownload Hi-res image Download (PPT) View Large Image Figure ViewerDownload Hi-res image Download (PPT) View Large Image Figure ViewerDownload Hi-res image Download (PPT) REACTIONS 1–4 The electrons produced in the two oxidative steps are fed into the electron transfer chain via a small c-type cytochrome. Many bacteria with a Sox system lack the SoxCD complex found in P. pantotrophus and are instead thought to feed the sulfane group of thiosulfate into other sulfur oxidation pathways (12Hensen D. Sperling D. Trüper H.G. Brune D.C. Dahl C. Mol. Microbiol. 2006; 62: 794-810Crossref PubMed Scopus (127) Google Scholar, 13Beller H.R. Chain P.S. Letain T.E. Chakicherla A. Larimer F.W. Richardson P.M. Coleman M.A. Wood A.P. Kelly D.P. J. Bacteriol. 2006; 188: 1473-1488Crossref PubMed Scopus (286) Google Scholar, 14Ogawa T. Furusawa T. Nomura R. Seo D. Hosoya-Matsuda N. Sakurai H. Inoue K. J. Bacteriol. 2008; 190: 6097-6110Crossref PubMed Scopus (31) Google Scholar). The reaction assigned to SoxB in the Sox pathway model is the hydrolysis of a sulfur-sulfur bond. This is an unusual enzymatic reaction that has only otherwise been suggested for enzymes designated as trithionate or tetrathionate hydrolases (15Meulenberg R. Pronk J.T. Frank J. Hazeu W. Bos P. Kuenen J.G. Eur. J. Biochem. 1992; 209: 367-374Crossref PubMed Scopus (22) Google Scholar, 16De Jong G.A. Hazeu W. Bos P. Kuenen J.G. Eur. J. Biochem. 1997; 243: 678-683Crossref PubMed Scopus (45) Google Scholar, 17Rzhepishevska O.I. Valdés J. Marcinkeviciene L. Gallardo C.A. Meskys R. Bonnefoy V. Holmes D.S. Dopson M. Appl. Environ. Microbiol. 2007; 73: 7367-7372Crossref PubMed Scopus (46) Google Scholar, 18Kanao T. Kamimura K. Sugio T. J. Biotechnol. 2007; 132: 16-22Crossref PubMed Scopus (57) Google Scholar). The thiosulfohydrolase activity proposed for SoxB has yet to be directly demonstrated. It is, instead, inferred from two key observations. First, in vitro pathway reconstitution experiments show that SoxB catalyzes a nonoxidative reaction (7Rother D. Henrich H.J. Quentmeier A. Bardischewsky F. Friedrich C.G. J. Bacteriol. 2001; 183: 4499-4508Crossref PubMed Scopus (116) Google Scholar). Second, SoxB has sequence similarity to the 5′-nucleotidase family of enzymes (19Knöfel T. Sträter N. Nat. Struct. Biol. 1999; 6: 448-453Crossref PubMed Scopus (103) Google Scholar). Because 5′-nucleotidases catalyze the hydrolytic cleavage of phosphate groups from nucleotides, this sequence similarity suggests that SoxB also carries out a hydrolytic reaction. Catalytically active SoxB purified from P. pantotrophus or the closely related bacterium Paracoccus versutus contains up to two atoms of manganese but only traces of other metal ions (20Cammack R. Chapman A. Lu W.P. Karagouni A. Kelly D.P. FEBS Lett. 1989; 253: 239-243Crossref Scopus (40) Google Scholar, 21Friedrich C.G. Quentmeier A. Bardischewsky F. Rother D. Kraft R. Kostka S. Prinz H. J. Bacteriol. 2000; 182: 4677-4687Crossref PubMed Scopus (116) Google Scholar). EPR studies suggest that the manganese ions are present in the form of a dinuclear Mn(II) cluster with bis(μ-hydroxo) (μ-carboxylato) bridging ligands (20Cammack R. Chapman A. Lu W.P. Karagouni A. Kelly D.P. FEBS Lett. 1989; 253: 239-243Crossref Scopus (40) Google Scholar, 22Epel B. Schäfer K.O. Quentmeier A. Friedrich C. Lubitz W. J. Biol. Inorg. Chem. 2005; 10: 636-642Crossref PubMed Scopus (37) Google Scholar). In phylogenetic and environmental studies the presence of a soxB gene has been used as a marker for the presence of the Sox pathway and as an indicator of the ability of the organism to oxidize thiosulfate (23Petri R. Podgorsek L. Imhoff J.F. FEMS Microbiol. Lett. 2001; 197: 171-178Crossref PubMed Google Scholar, 24Meyer B. Imhoff J.F. Kuever J. Environ. Microbiol. 2007; 9: 2957-2977Crossref PubMed Scopus (163) Google Scholar). Here we report experiments aimed at establishing a direct assay of SoxB activity. We have used x-ray crystallography to determine the structure of recombinant SoxB from the thermophilic bacterium Thermus thermophilus. This is the first structure of an enzyme catalyzing the hydrolysis of a sulfur-sulfur bond. We have also obtained structures of T. thermophilus SoxB in complex with mechanistically relevant ligands. Based on these structures, we propose a model for the SoxB mechanism. T. thermophilus HB27 SoxB was expressed in the cytoplasm of Escherichia coli with a Strep II tag replacing the N-terminal Tat signal peptide. The soxB gene was amplified from genomic DNA using the forward primer 5′-GCTAGCTGGAGCCACCCGCAGTTCGAAAAAGGCGCCCTGGAGGACCCCAGGTCC-3′ and the reverse primer 5′-ACGCCTGGTACCTCATC CCGTCACCTCCGG-3′. The amplicon was used as the target in a second round of PCR using the same reverse primer and 5′-CAGGACGAATTCATTAAAGAGGAGAAATTAACTATGGCTAGCTGGAGCCACCCG-3′ as the forward primer. This second amplicon was digested with EcoRI and KpnI and ligated into the same sites in pQE80L (Qiagen) to produce the expression plasmid pVS048. E. coli strain Rosetta 2 (DE3) (Novagen) containing pVS048 was cultured aerobically at 30 °C in 2 liters of LB medium (25Sambrook J. Russell D. Molecular Cloning: A Laboratory Manual. Vol. 3. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: A2.2Google Scholar) that had been supplemented with 1 mm MnCl2, 100 μg ml−1 ampicillin, and 25 μg ml−1 chloramphenicol. When the culture reached an A600 nm of 0.6, soxB expression was induced with 100 μm isopropyl Β-d-thiogalactopyranoside, and growth was continued for a further 17 h. The bacteria were harvested by centrifugation and resuspended in 100 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm MnCl2, with EDTA-free Complete protease inhibitors (Roche Applied Science) and a few crystals each of lysozyme and DNase I (both Sigma-Aldrich). The resuspended cells were broken by three passages through a French Press at 8000 p.s.i. The lysate was centrifuged at 155,000 × g for 30 min at 4 °C. The supernatant was collected and heat-treated at 65 °C for 20 min. Denatured material was removed by centrifugation at 10,000 × g at 4 °C for 15 min, and the supernatant was loaded onto a 2.5-ml Strep-Tactin Superflow column (IBA) previously equilibrated with 100 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm MnCl2. The column was washed with 60 ml of the equilibration buffer. SoxB was then eluted with 10 ml of 100 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm MnCl2, 2.5 mm dethiobiotin. SoxB fractions were concentrated and further purified on a Superdex 200 10/300 size exclusion column (GE Healthcare) that had been equilibrated in 30 mm Tris-HCl, pH 8.0, 150 mm NaCl. The T. thermophilus HB27 SoxYZ complex was expressed in the cytoplasm of E. coli with a hexahistidine tag replacing the N-terminal signal peptide of SoxY. The soxY gene was amplified from genomic DNA using the primers 5′-CCTGCGGGATCCCAGGGCCTCGAGGGCGAG-3′ and 5′-GCCGCAGGTACCTTAACCGCAGCCCCCCACG-3′. The resulting amplicon was digested with BamHI and KpnI and cloned into the same sites in pQE80L to produce plasmid pVS054. The soxZ gene was amplified from genomic DNA using the primers 5′-ACCGTGGGTACCATTAAAGAGGAGAAATTAACTATGCCTTTTAGGACCATCGCG-3′ and 5′-CGGGCAAAGCTTTTAGGCCAGCTCCAGCTTGA-3′. The resulting amplicon was digested with KpnI and HindIII and cloned into the same sites in pVSO54 to produce plasmid pVS056. E. coli strain Rosetta 2 (DE3) containing pVS056 was cultured, and a heat-treated soluble extract was prepared using the same protocol that was employed in the purification of recombinant SoxB except that the growth medium was not supplemented with MnCl2. The heat-treated extract was supplemented with 1 mm DTT 4The abbreviations used are: DTTdithiothreitolPEGpolyethylene glycol. and loaded onto a 5-ml His-Trap column (GE Healthcare) previously equilibrated in 50 mm Tris-HCl, pH 8.0, 0.5 m NaCl, 10 mm imidazole, 2 mm DTT. The column was washed with equilibration buffer containing 25 mm imidazole and then developed with a linear 25–210 mm imidazole gradient. SoxYZ fractions were concentrated by ultrafiltration and further purified on a Superdex 75 10/300 size exclusion column (GE Healthcare) in 30 mm Tris-HCl, pH 8.0, 0.2 m NaCl, 2 mm DTT. dithiothreitol polyethylene glycol. N-terminal sequencing and mass spectrometry of the purified SoxYZ complex showed that the N-terminal methionine residue of the recombinant SoxZ protein had been removed. The N-terminal amino acid of SoxY released by the Edman reaction ran at the same chromatographic position as phenylalanine. In addition, the recombinant SoxY protein had a mass 14 Da greater than the expected mass value of 14,668 Da. These observations are consistent with the N-terminal methionine of SoxY being methylated, as has been reported previously for proteins that have a positively charged residue as the second amino acid (this residue is arginine in T. thermophilus SoxY) (26Apostol I. Aitken J. Levine J. Lippincott J. Davidson J.S. Abbott-Brown D. Protein Sci. 1995; 4: 2616-2618Crossref PubMed Scopus (19) Google Scholar). To produce DTT-free reduced SoxYZ, the purified protein was supplemented with a further 5 mm DTT to ensure complete reduction of the active site cysteine. Excess DTT was then removed by gel filtration on a Superdex 75 10/300 column in 10 mm Tris-HCl, pH 8.0. To produce the S-thiosulfonate derivative of SoxYZ, the purified protein was dialyzed against 10 mm Tris-HCl, pH 8.0, 50 mm potassium tetrathionate for 16 h at 22 °C. Excess tetrathionate together with the thiosulfate reaction product were removed from the protein by gel filtration as described above. The samples were desalted by a 30-min dialysis against 0.1% formic acid using a 0.025-μm pore floating membrane (Millipore). After dialysis the samples were diluted with acetonitrile to a SoxYZ concentration of 10 μm. Electrospray mass spectrometry data were obtained using a QTof1 (Micromass) operated using MassLynx v4.0. The samples were sprayed from borosilicate emitters (Proxeon Biosystems) using a capillary voltage tuned between 1.5 and 2.1 kV to obtain a stable spray. Cone voltage was maintained at 40 V. Raw spectra were combined and processed using MaxEnt1 (Micromass). Crystals were grown at 20 °C by the sitting drop vapor diffusion method by mixing 1 μl of SoxB at 6.5 mg/ml in 10 mm Tris-HCl, pH 8.0, 0.2 m NaCl, and 1 μl of 24–26% (v/v) tert-butanol, and 0.1 m Tris acetate, pH 8.5. To ensure that the two metal sites were well occupied with manganese, 2 mm MnCl2 was added to the mother liquor for some structures. For crystallization in the presence of sulfur compounds, 2 mm disodium thiosulfate or diammonium sulfate was added directly to the protein prior to setting up the drops. The crystal used for the phasing was cryoprotected in the crystallization solution by the addition of 25% (v/v) glycerol to the mother liquor. The structure derived from this crystal showed glycerol molecules occupying the active site. To avoid interference of cryoprotectant with the active site, 25% (v/v) PEG 400 replaced glycerol for the cryoprotection of all other crystals analyzed. 100 mm disodium thiosulfate or diammonium sulfate were added to the cryosolutions for crystals grown in the presence of these chemicals. All of the data sets were collected at 100 K either in-house using a Bruker MicroStar microfocus x-ray generator with Montel optics and a Bruker platinum 135 CCD detector or at the European Synchrotron Radiation Facility (ID14-1, Grenoble, France) (Table 1).TABLE 1Data collection, phasing, and refinement statistics for T. thermophilus SoxBSoxB-glycerolSoxB-Mn2+SoxB-Mn2+-thiosulfateSoxB-Mn2+-sulfateAdditive(s)Mn2+Sodium thiosulfate and Mn2+Ammonium sulfate and Mn2+Cryoprotectant agentGlycerolPEG 400PEG 400PEG s400X-ray source (wavelength)In-house (1.54 Å)In-house (1.54 Å)In-house (1.54 Å)ESRF ID14-1 (0.93 Å)Space groupP212121P212121P212121P212121Cell dimensions (Å)a = 71.3, b = 86.5, c = 96.0a = 70.4, b = 86.7, c = 95.1a = 70.8, b = 86.7, c = 95.4a = 70.8, b = 86.6, c = 96.1Resolution (Å)57.3-1.5 (1.54-1.50)64.1-2.1 (2.16-2.10)64.2-1.85 (1.89-1.85)39.5-1.50 (1.54-1.50)Number of unique reflections90,71932,81748,24486,525Redundancy9.8 (1.2)13.0 (10.2)7.6 (3.0)6.0 (3.0)Completeness (%)100 (100)99.9 (99.4)100 (100)95.8 (87.1)Rmerge (%)4.4 (35.3)8.4 (39.5)6.0 (37.9)5.8 (23.0)Average I/σI26 (1.6)25.6 (6.4)21.5 (2.9)8.7 (3.3)Refinement statistics R (%)17.1 (22.7)18.6 (21.9)20.0 (27.0)18.2 (23.2) Rfree (%)19.1 (26.1)23.1 (26.8)23.4 (29.0)20.7 (28.3)Root mean square deviation from idealized covalent geometry Bonds (Å)0.0080.0100.0090.009 Angles (Å)1.211.231.131.23Protein residues in model32–57330–57329–57331–573Number of atoms in model5079477848415222Number of protein atoms4437444043754599Number of nonprotein atoms2 Mn2+ (1 at 100% and 1 at 75% occupancy), 564 H2O, 30 glycerol (5), 30 tert-butanol (6), 16 acetate (4)2 Mn2+, 336 H2O2 Mn2+, 439 H2O, 20 tert-butanol (4),5 S2O32− (1)2 Mn2+, 592 H2O, 10 sulfate (2 molecules at 25% occupancy each), 15 tert-butanol (3), 4 acetate (1)Mean <B> protein (Å2)13.920.718.214.8Mean <B> nonprotein (Å2)30.928.728.929.9Residues in most favorite Ramachandran regions (%)98.097.696.597.41Ramachandran outliers (%)0.00.00.920.0 Open table in a new tab The images were processed using SAINT (27Bruker SMART, SAINT, SADABS and XPREP Software Reference Manual. Bruker AXS Inc., Madison, WI2000Google Scholar) for the in-house data and MOSFLM (28Leslie A.G. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography. 26. STFC Daresbury Laboratory, Warrington, England, UK1992Google Scholar) for the synchrotron data. The in-house data were processed with XPREP (27Bruker SMART, SAINT, SADABS and XPREP Software Reference Manual. Bruker AXS Inc., Madison, WI2000Google Scholar) to obtain a set of structure factors with anomalous scattering, which in turn were used in the program ShelxD (29Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1891) Google Scholar) to find the two manganese sites (f″ = 2.8 e− at λ = 1.54 Å) and five sulfur atoms. DANO/SIGMA values from SHELXD ranged from 4 to 1.5 in the resolution range 50–2.5 Å. XPREP was then rerun keeping Friedel mates intensities separate, and these intensities were processed through CCP4-combat and then CCP4-truncate (30Winn M.D. Ashton A.W. Briggs P.J. Ballard C.C. Patel P. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 1929-1936Crossref PubMed Scopus (42) Google Scholar) to produce a file of structure factor amplitudes for the phasing program SHARP (31De La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). The heavy atom model eventually comprised two manganese and six sulfur sites. Starting from the SHARP phase distribution solvent flattening in the program Solomon using a 47.5% solvent content produced a 1.8-Å map that enabled autobuilding in the program ArpWarp (32Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar). Residues before Tyr32 were not seen. All other minor gaps in the autobuilt model were easily built manually (11 residues in total). An autoBUSTER refinement 5C. Vonrhein, personal communication. was performed on the model for SoxB residues 32–573 with the two active site manganese atoms assigned a full occupancy. After 10 autoBUSTER cycles with water addition, the R factor was 23.5% and the Rfree was 25.3%. The structure was further refined with Refmac5 (33Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13911) Google Scholar), and building was completed with Coot (34Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23605) Google Scholar). Further rounds of refinement and model building used Refmac5 (33Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13911) Google Scholar) and Coot (34Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23605) Google Scholar) or X-fit (35Cheary R.W. Coelho A.A. CCP4 Powder Diffraction Library. Engeneering and Physical Sciences Research Council, Daresbury Laboratory, Warrington, UK1996Google Scholar). Pocket parameters were calculated with CASTp (36Binkowski T.A. Naghibzadeh S. Liang J. Nucleic Acids Res. 2003; 31: 3352-3355Crossref PubMed Scopus (595) Google Scholar). The coordinate root mean square deviation values were calculated with Swiss-Pdb viewer (37Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9640) Google Scholar) and Dali-Lite (38Holm L. Park J. Bioinformatics. 2000; 16: 566-567Crossref PubMed Scopus (775) Google Scholar). Sequence conservation was mapped onto SoxB using ConSurf (39Glaser F. Pupko T. Paz I. Bell R.E. Bechor-Shental D. Martz E. Ben-Tal N. Bioinformatics. 2003; 19: 163-164Crossref PubMed Scopus (951) Google Scholar, 40Landau M. Mayrose I. Rosenberg Y. Glaser F. Martz E. Pupko T. Ben-Tal N. Nucleic Acids Res. 2005; 33: W299-302Crossref PubMed Scopus (1090) Google Scholar). All of the atomic coordinates and experimental structure factors of the structures described in this paper have been deposited in the Protein Data Bank with the accession codes 2WDC for SoxB-glycerol, 2WDF for SoxB-Mn2+, 2WDE for SoxB-Mn2+-thiosulfate, and 2WDD for SoxB-Mn2+-sulfate. MicroPIXE measurements were carried out at the Ion Beam Centre (University of Surrey) on a beamline arranged as described by Grime et al. (41Grime G.W. Dawson M. Marsh M. McArthur I.C. Watt F. Nucl. Instrum. Methods Phys. Res. B. 1991; 54: 52-63Crossref Scopus (212) Google Scholar). Two SoxB crystals were dried onto 2-μm-thick mylar films in aluminum target holders (42Garman E. Structure. 1999; 7: R291-R299Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 43Garman E.F. Grime G.W. Prog. Biophys. Mol. Biol. 2005; 89: 173-205Crossref PubMed Scopus (79) Google Scholar). A 2.5-MeV proton beam 3 μm in diameter was used to induce characteristic x-ray emission from the dried crystal under vacuum. The X-rays were detected in a solid state lithium drifted silicon detector with high energy resolution. The proton beam was then scanned spatially in the x and y dimensions. Spatial maps were obtained of all elements heavier than neon that were present in the sample. Quantitative information was obtained by collecting spectra at selected points on the crystals and also at a point on the backing film. These spectra were analyzed using GUPIX (44Johansson S.A.E. Campbell J.L. Malmqvist K.G. Particle-Induced X-Ray Emission Spectrometry. John Wiley & Sons, Inc., New York1995Google Scholar) to determine the amount of each element of interest in the sample relative to the sulfur signal from the six methionine residues found in the recombinant T. thermophilus SoxB protein. The SoxB protein from the thermophilic bacterium T. thermophilus HB27 was expressed in E. coli. The identity of the recombinant protein was confirmed by N-terminal sequencing and mass spectrometry. The protein was a monomer in solution as judged by size exclusion chromatography (supplemental Fig. S1). To establish that the recombinant T. thermophilus SoxB was enzymatically active and as a means to substantiate the proposed SoxB reaction, we attempted to assay SoxB activity directly. Recombinant T. thermophilus SoxYZ was produced in E. coli, and the active site cysteine residue of the SoxY subunit was chemically modified to the S-thiosulfonate (-S-SO3−) derivative. The authenticity of this modification was confirmed by mass spectrometry showing that the derivatized SoxY protein had a molecular mass 113 Da greater than that of the native protein (the mass of thiosulfate is 112 Da) (Fig. 1, compare samples 2 and 6). The additional mass could be removed by DTT treatment. This indicated that the S-thiosulfonate group had formed a disulfide linkage with the protein and must therefore be conjugated through the sulfane sulfur to the unique active site cysteine residue in SoxY (Fig. 1, compare samples 6 and 12). The S-thiosulfonate SoxYZ protein was mixed with SoxB and incubated for 3 h at 70 °C, which is the optimum growth temperature of T. thermophilus. The reaction products were then analyzed by mass spectrometry. Following incubation, the mass peak corresponding to the S-thiosulfonate SoxY derivate had disappeared (Fig. 1, compare samples 6 and 9), whereas the SoxZ mass peak was unchanged from the start of the reaction. Identical results were obtained for a duplicate reaction that had been supplemented with Mn2+ to guard against the possibility that recombinant SoxB had not been fully loaded with metal cofactors (Fig. 1, compare samples 8 and 9). If SoxB was omitted from the reaction, no alteration of the S-thiosulfonate SoxY peak was seen even if Mn2+ was present (Fig. 1, sample 7). Taken together these observations showed that SoxB had specifically converted the SoxY derivative into a new species. The nature of the resultant species was not, however, immediately apparent because no new mass peaks were detected. Insight into the nature of the product species was instead obtained from SDS-PAGE analysis. T. thermophilus SoxYZ is sufficiently thermostable that the two subunits do not dissociate in 2% (w/v) SDS at room temperature (Fig. 1, sample 1). However, if the temperature is increased to 100 °C, SoxY and SoxZ appear to become separated and migrate with identical mobility under SDS-PAGE (Fig. 1, compare samples 1 and 2). This interpretation of the electrophoretic behavior of T. thermophilus SoxYZ was confirmed by expressing and analyzing each subunit individually (data not shown). If the cysteine residue of SoxY is initially in the reduced state, it has a strong tendency to form an intermolecular disulfide bridge with the cysteine residue of another SoxY protein molecule. This disulfide-linked dimer is evident as a species of 31 kDa on SDS-PAGE, which disappears in the presence of DTT (Fig. 1, compare samples 2 and 13). Formation of this disulfide-linked SoxY dimer is enhanced by incubation at 70 °C in the presence of Mn2+ (Fig. 1, compare samples 3 and 4). Divalent metal ions are known to act as a catalyst for disulfide bond shuffling (45Fan Q.R. Long E.O. Wiley D.C. J. Biol. Chem. 2000; 275: 23700-23706Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 46Builder S. Hart R. Lester P. Reifsnyder D. U. S. Patent 5808006. 1998; (9, 15)Google Scholar). The S-thiosulfonate-SoxYZ preparation contained very little disulfide-linked SoxY dimer, and the concentration of the dimer was not altered by incubation with Mn2+ at 70 °C (Fig. 1, compare samples 6, 7, and 12). However, incubation with SoxB at 70 °C led to a significant increase in SoxY dimers with or without supplementary Mn2+ (Fig. 1, compare sample 7 with samples 8 and 9). The SoxB-dependent formation of SoxYZ complex dimers was confirmed by size exclusion chromatography (supplemental Fig. S1). The addition of DTT to the SoxB-containing reaction led to the disappearance of the SoxY dimer band on SDS-PAGE and of the SoxYZ complex dimer peak on the size exclusion column (Fig. 1, compare samples 8 and 11; supplemental Fig. S1). These changes were associated with the appearance of a mass peak corresponding to unmodified SoxY (Fig. 1, compare samples 8 and 11). Thus the SoxB reaction leads to the partial conversion of the S-thiosulfonate SoxY derivative to a species in which SoxYZ complexes form a disulfide-linked dimer through the SoxY cysteine residue. At first sight this conclusion is at odds with the current proposal that the product of the SoxB reaction should be S-sulfane SoxY rather than a disulfide-linked SoxY dimer. However, taking into account the propensity of the reduced SoxY cysteine to form disulfide links to other SoxY molecules nonenzymatically, we believe that the observed SoxB-dependent products are still consistent with SoxB catalyzing a thiosulfohydrolase reaction. Removal of the terminal sulfonate group in the SoxB reaction produces a reactive sulfane derivative that we suggest would nonenzymatically form disulfide linkages via reactions such as the following. View Large Image Figure ViewerDownload Hi-res image Download (PPT) View Large Image Figure ViewerDownload Hi-res image Download (PPT) View Large Image Figure ViewerDownload Hi-res image Download (PPT) REACTIONS 5–8 In summary, the assay described above shows that the recombinant T. thermophilus SoxB protein is" @default.
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• W2061859547 creator A5029634022 @default.
• W2061859547 creator A5034083581 @default.
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• W2061859547 title "Mechanism for the Hydrolysis of a Sulfur-Sulfur Bond Based on the Crystal Structure of the Thiosulfohydrolase SoxB" @default.
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