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W2080851578 abstract "The human MOCS3 gene encodes a protein involved in activation and sulfuration of the C terminus of MOCS2A, the smaller subunit of the molybdopterin (MPT) synthase. MPT synthase catalyzes the formation of the dithiolene group of MPT that is required for the coordination of the molybdenum atom in the last step of molybdenum cofactor (Moco) biosynthesis. The two-domain protein MOCS3 catalyzes both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A by its C-terminal rhodanese-like domain (RLD). The low activity of MOCS3-RLD with thiosulfate as sulfur donor and detailed mutagenesis studies showed that thiosulfate is most likely not the physiological sulfur source for Moco biosynthesis in eukaryotes. It was suggested that an l-cysteine desulfurase might be involved in the sulfuration of MOCS3 in vivo. In this report, we investigated the involvement of the human l-cysteine desulfurase Nfs1 in sulfur transfer to MOCS3-RLD. A variant of Nfs1 was purified in conjunction with Isd11 in a heterologous expression system in Escherichia coli, and the kinetic parameters of the purified protein were determined. By studying direct protein-protein interactions, we were able to show that Nfs1 interacted specifically with MOCS3-RLD and that sulfur is transferred from l-cysteine to MOCS3-RLD via an Nfs1-bound persulfide intermediate. Because MOCS3 was shown to be located in the cytosol, our results suggest that cytosolic Nfs1 has an important role in sulfur transfer for the biosynthesis of Moco. The human MOCS3 gene encodes a protein involved in activation and sulfuration of the C terminus of MOCS2A, the smaller subunit of the molybdopterin (MPT) synthase. MPT synthase catalyzes the formation of the dithiolene group of MPT that is required for the coordination of the molybdenum atom in the last step of molybdenum cofactor (Moco) biosynthesis. The two-domain protein MOCS3 catalyzes both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A by its C-terminal rhodanese-like domain (RLD). The low activity of MOCS3-RLD with thiosulfate as sulfur donor and detailed mutagenesis studies showed that thiosulfate is most likely not the physiological sulfur source for Moco biosynthesis in eukaryotes. It was suggested that an l-cysteine desulfurase might be involved in the sulfuration of MOCS3 in vivo. In this report, we investigated the involvement of the human l-cysteine desulfurase Nfs1 in sulfur transfer to MOCS3-RLD. A variant of Nfs1 was purified in conjunction with Isd11 in a heterologous expression system in Escherichia coli, and the kinetic parameters of the purified protein were determined. By studying direct protein-protein interactions, we were able to show that Nfs1 interacted specifically with MOCS3-RLD and that sulfur is transferred from l-cysteine to MOCS3-RLD via an Nfs1-bound persulfide intermediate. Because MOCS3 was shown to be located in the cytosol, our results suggest that cytosolic Nfs1 has an important role in sulfur transfer for the biosynthesis of Moco. Among the metabolic pathways requiring sulfur transfer are those leading to the formation of iron-sulfur (FeS) 2The abbreviations used are: FeSiron-sulfurMocomolybdenum cofactorMPTmolybdopterinNi-NTAnickel-nitrilotriacetic acidIPTGisopropyl thio-β-d-galactosideRLDrhodanese-like domainSPRsurface plasmon resonancePLPpyridoxal phosphateRUresonance units. 2The abbreviations used are: FeSiron-sulfurMocomolybdenum cofactorMPTmolybdopterinNi-NTAnickel-nitrilotriacetic acidIPTGisopropyl thio-β-d-galactosideRLDrhodanese-like domainSPRsurface plasmon resonancePLPpyridoxal phosphateRUresonance units. clusters, biotin, thiamine, lipoic acid, molybdopterin (MPT), and sulfur-containing bases in tRNA (1Marquet A. Curr. Opin. Chem. Biol. 2001; 5: 541-549Crossref PubMed Scopus (73) Google Scholar). MPT, the basic component of the molybdenum cofactor (Moco), is a tricyclic pterin derivative that bears the cis-dithiolene group essential for molybdenum ligation (2Rajagopalan K.V. Johnson J.L. Hainline B.E. Fed. Proc. 1982; 41: 2608-2612PubMed Google Scholar). Moco is essential for the activity of sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase in humans (3Johnson J.L. Duran M. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 3163-3177Google Scholar). The biosynthetic pathway of Moco can be divided into three steps: (i) conversion of GTP to precursor Z; (ii) transformation of precursor Z to MPT; and (iii) insertion of molybdenum onto MPT to form Moco (4Rajagopalan K.V. Neidhardt F.C. Escherichia coli and Salmonella. Cellular and Molecular Biology. ASM Press, Washington, DC1996: 674-679Google Scholar).Recent studies have identified the human genes involved in the biosynthesis of Moco (5Reiss J. Hum. Genet. 2000; 106: 157-163Crossref PubMed Scopus (96) Google Scholar). Human MPT synthase, like the Escherichia coli (MoaD-MoaE)2 counterpart, is a heterotetramer, which is composed of two so-called MOCS2A (∼9,700 Da) and MOCS2B (∼20,800 Da) subunits (6Leimkühler S. Freuer A. Santamaria Araujo J.A. Rajagopalan K.V. Mendel R.R. J. Biol. Chem. 2003; 278: 26127-26134Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The sulfur used to generate the dithiolene moiety of MPT is carried on the MOCS2A subunit in the form of a C-terminal thiocarboxylate that must be regenerated during each catalytic cycle. The reaction mechanism of resulfuration of E. coli MPT synthase has been described in detail (7Lake M.W. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nature. 2001; 414: 325-329Crossref PubMed Scopus (203) Google Scholar, 8Leimkühler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 22024-22031Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 9Leimkühler S. Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 34695-34701Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 10Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Similar to ubiquitin-activating enzymes (E1), the MOCS3 protein activates the C terminus of MOCS2A to form an acyl adenylate (11Schmitz J. Mullich Cowdhury M. Hänzelmann P. Lee E.-Y. Schindelin H. Leimkühler S. Biochemistry. 2008; 47: 4679-4689Crossref Scopus (75) Google Scholar). Subsequently, the MOCS2A acyl adenylate is converted to a thiocarboxylate by action of the C-terminal rhodanese-like domain (RLD) of MOCS3 (8Leimkühler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 22024-22031Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This RLD is present in all eukaryotic homologues, including the Saccharomyces cerevisiae Uba4 protein, and several bacterial homologues, but not in the E. coli MoeB protein (12Krepinsky K. Leimkühler S. FEBS J. 2007; 274: 2778-2787Crossref PubMed Scopus (27) Google Scholar). MOCS3 was shown to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A during Moco biosynthesis (13Matthies A. Nimtz M. Leimkühler S. Biochemistry. 2005; 44: 7912-7920Crossref PubMed Scopus (64) Google Scholar). All three proteins were shown to be localized in the cytosol in humans (14Matthies A. Rajagopalan K.V. Mendel R.R. Leimkühler S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5946-5951Crossref PubMed Scopus (105) Google Scholar). In addition, a recent report showed that MOCS3 also catalyzes the adenylation and sulfuration of hUrm1, a protein suggested to be involved in protein conjugation in humans (11Schmitz J. Mullich Cowdhury M. Hänzelmann P. Lee E.-Y. Schindelin H. Leimkühler S. Biochemistry. 2008; 47: 4679-4689Crossref Scopus (75) Google Scholar).Rhodaneses (thiosulfate:cyanide sulfurtransferases, EC 2.8.1.1) are widespread enzymes that in vitro catalyze the transfer of a sulfane sulfur atom from thiosulfate to cyanide (15Bordo D. Bork P. Embo. Reports. 2002; 3: 741-746Crossref PubMed Scopus (261) Google Scholar). They are not only found in combination with other proteins but also as single domain proteins or as tandem repeats serving as versatile sulfur carriers (15Bordo D. Bork P. Embo. Reports. 2002; 3: 741-746Crossref PubMed Scopus (261) Google Scholar, 16Mueller E.G. Nat. Chem. Biol. 2006; 2: 185-194Crossref PubMed Scopus (272) Google Scholar). A cysteine is the first residue of a six amino acid active site loop defining the ridge of the catalytic pocket that is expected to play a key role in substrate recognition and catalytic activity (15Bordo D. Bork P. Embo. Reports. 2002; 3: 741-746Crossref PubMed Scopus (261) Google Scholar).Detailed studies of the MOCS3-RLD sulfur transferase activity showed that MOCS3 acts in vitro as a thiosulfate:cyanide sulfur transferase; however, the activity was determined to be more than 1000 times lower compared with the activity of bovine rhodanese (14Matthies A. Rajagopalan K.V. Mendel R.R. Leimkühler S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5946-5951Crossref PubMed Scopus (105) Google Scholar). A detailed mutagenesis study of the 6-amino acid active site loop of MOCS3-RLD suggested that thiosulfate is most likely not the physiological sulfur source for MOCS3 in humans. It has been proposed that an l-cysteine desulfurase might act as a direct sulfur donor for cytosolic MOCS3 in humans (14Matthies A. Rajagopalan K.V. Mendel R.R. Leimkühler S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5946-5951Crossref PubMed Scopus (105) Google Scholar).l-Cysteine desulfurases are pyridoxal phosphate (PLP)-containing enzymes that catalyze the formation of l-alanine and a protein-bound persulfide group by using l-cysteine as substrate (17Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (496) Google Scholar, 18Mihara H. Esaki N. Appl. Microbiol. Biotechnol. 2002; 60: 12-23Crossref PubMed Scopus (218) Google Scholar).A single l-cysteine desulfurase homologue, named Nfs1, was identified in humans (19Land T. Rouault T.A. Mol. Cell. 1998; 2: 807-815Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 20Biederbick A. Stehling O. Rosser R. Niggemeyer B. Nakai Y. Elsasser H.P. Lill R. Mol. Cell Biol. 2006; 26: 5675-5687Crossref PubMed Scopus (143) Google Scholar). However, it was shown that two distinct Nfs1 isoforms are produced through alternative utilization of in-frame AUGs (19Land T. Rouault T.A. Mol. Cell. 1998; 2: 807-815Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 21Li K. Tong W.H. Hughes R.M. Rouault T.A. J. Biol. Chem. 2006; 281: 12344-12351Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 22Tong W.H. Rouault T. EMBO J. 2000; 19: 5692-5700Crossref PubMed Scopus (168) Google Scholar). The major form is generated by initiation of the first AUG of the Nfs1 transcript and contains a mitochondrial targeting signal at the N terminus that undergoes cleavage to yield a mature mitochondrial protein of 47 kDa in size (19Land T. Rouault T.A. Mol. Cell. 1998; 2: 807-815Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In mitochondria, Nfs1 is involved in FeS cluster biosynthesis (23Tong W.H. Rouault T.A. Cell Metab. 2006; 3: 199-210Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). A less abundant isoform generated by initiation of translation at the second in-frame AUG lacks the first 60 residues of the mitochondrial precursor form, and this 44-kDa protein resides both in the cytosol and in the nucleus (19Land T. Rouault T.A. Mol. Cell. 1998; 2: 807-815Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar).Recently, the Isd11 protein was identified to be essential for FeS cluster biosynthesis in mitochondria and it was shown that Isd11 forms a complex with Nfs1 (24Adam A.C. Bornhovd C. Prokisch H. Neupert W. Hell K. EMBO J. 2006; 25: 174-183Crossref PubMed Scopus (191) Google Scholar, 25Wiedemann N. Urzica E. Guiard B. Müller H. Lohaus C. Meyer H.E. Ryan M.T. Meisinger C. Mühlenhoff U. Lill R. Pfanner N. EMBO J. 2006; 25: 184-195Crossref PubMed Scopus (194) Google Scholar). Isd11 is suggested to function as an adapter and stabilizer of Nfs1 (26Shan Y. Napoli E. Cortopassi G. Hum. Mol. Genet. 2007; 16: 929-941Crossref PubMed Scopus (125) Google Scholar). Homologues of Isd11 have been identified in plant, fungi and animal genomes, which contain mitochondria, but no prokaryotic homologue has been identified (27Richards T.A. van der Giezen M. Mol. Biol. Evol. 2006; 23: 1341-1344Crossref PubMed Scopus (55) Google Scholar).To analyze whether Nfs1 acts as sulfur donor for the biosynthesis of the molybdenum cofactor, we purified an N-terminal-truncated version of Nfs1 after heterologous expression in E. coli in the presence and absence of Isd11. The l-cysteine desulfurase activity of the purified Nfs1Δ1–55/Isd11 complex was characterized. Nfs1Δ1–55 was shown to interact with both Isd11 and MOCS3-RLD, and in addition, the interaction with MOCS3-RLD was stronger when Nfs1Δ1–55 was not in a complex with Isd11. By ESI-MS analyses it was verified that the sulfur from Nfs1 is further transferred to form a persulfide on MOCS3-RLD. Our studies suggest that cytosolic Nfs1 has an additional role in the cytosol and sulfurates MOCS3 for the biosynthesis of the molybdenum cofactor.EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, Media, and Growth Conditions—E. coli BL21(DE3) cells or BL21(DE3)star cells (Novagen) were used for heterologous expression of the human Nfs1Δ1–55 and Isd11 proteins. The vectors pET15b and pACYCDuet-1 were obtained from Novagen. E. coli expression cultures were grown in LB medium under aerobic conditions at 16 °C for 16 h. Ampicillin (150 μg/ml), chloramphenicol (50 μg/ml), and isopropyl-β-d-thiogalactoside (IPTG) (100 μm) were used when required.Cloning, Expression, and Purification of Human Nfs1 and Isd11—For expression of Nfs1, primers were designed that resulted in a deletion of the first 55 amino acids of Nfs1 and that allowed cloning into the XhoI-BamHI sites of the expression vector pET15b. The resulting plasmid was designated pZM2, and expresses Nfs1Δ1–55 as an N-terminal fusion protein with a His6 tag. For coexpression with Isd11, the Isd11 cDNA fragment was cloned from a human cDNA library. Primers were designed that allowed cloning of Isd11 into the NcoI-HindIII sites of the expression vector pACYCDuet-1. The resulting plasmid was designated pZM4. For separate purification of Isd11, primers were designed that allowed cloning of Isd11 into the XhoI-BamHI sites of the expression vector pET15b. The resulting plasmid was designated pZM6, and expresses Isd11 as an N-terminal fusion protein with a His6 tag.For purification of Nfs1Δ1–55, plasmid pZM2 was cotransformed with a plasmid containing the E. coli chaperonin GroEL (6Leimkühler S. Freuer A. Santamaria Araujo J.A. Rajagopalan K.V. Mendel R.R. J. Biol. Chem. 2003; 278: 26127-26134Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) in E. coli BL21(DE3)star cells. For heterologous expression of Nfs1Δ1–55/Isd11 in E. coli, pZM2 and pZM4 were cotransformed into BL21(DE3)star cells. Cells were grown at 30 °C in 1-liter cultures of LB containing 150 μg/ml ampicillin and 50 μg/ml chloramphenicol. The expression was induced at A600 nm = 0.6 with 100 μm IPTG. Growth was continued for 16 h at 16 °C, and cells were harvested by centrifugation at 8000 × g. After cell lysis, the soluble fraction was transferred onto a column with Ni-nitrilotriacetic (Ni-NTA, Qiagen). Nfs1Δ1–55 or Nfs1Δ1–55/Isd11 were eluted with 50 mm NaH2PO4, 300 mm NaCl, 250 mm imidazole buffer, pH 8.0, containing 50 μm PLP and 10% (v/v) glycerol. Final purification of Nfs1Δ1–55 or Nfs1Δ1–55/Isd11 was achieved by chromatography on a Superose 12 size exclusion column (GE Healthcare) equilibrated in 50 mm Tris, 200 mm NaCl, 10 μm PLP, pH 8.0.For expression of Isd11, pZM6 was transformed into E. coli BL21(DE3) cells. Cells were grown at 30 °C in 1-liter cultures of LB, containing 150 μg/ml ampicillin. The expression was induced at A600 nm = 0.6 with 100 μm IPTG. Growth was continued for 16 h at 16 °C, and cells were harvested by centrifugation at 8000 × g. After cell lysis, the soluble fraction was transferred onto a column with Ni-NTA. Isd11 was eluted with 50 mm NaH2PO4, 300 mm NaCl, 250 mm imidazole buffer, pH 8.0. Final purification of Isd11 was achieved by chromatography on a Superose 12 size exclusion column equilibrated in 50 mm Tris, 200 mm NaCl, pH 9.0.Size Exclusion Chromatography—Purified Nfs1Δ1–55 or Nfs1Δ1–55/Isd11 were injected in a volume of 500 μl onto a Superdex 200 column (GE Healthcare) equilibrated in 50 mm Tris, 200 mm NaCl, 1 mm EDTA, pH 8.0.Enzyme Assays, Molar Extinction Coefficient, and Absorption Spectra of Nfs1Δ1–55—l-Cysteine desulfurase activity of Nfs1Δ1–55/Isd11 was quantified by the methylene blue method (28Fogo J.K. Popowsky M. Anal Chem. 1949; 21: 732-734Crossref Scopus (358) Google Scholar) using the parameters from Urbina et al. (29Urbina H.D. Silberg J.J. Hoff K.G. Vickery L.E. J. Biol. Chem. 2001; 276: 44521-44526Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Assay mixtures in a total volume of 0.8 ml contained 50 mm Tris, 200 mm NaCl, 10 μm PLP, 1 mm dithiothreitol, pH 8.0, and 2 μm of Nfs1Δ1–55/Isd11. The reactions were initiated by the addition of l-cysteine (0.1–1.0 mm) and incubated for 10 min at 37 °C before the reactions were stopped by the addition of 100 μl of 20 mm N,N-dimethyl-p-phenylenediamine in 7.2 m HCl and 100 μl of 30 mm FeCl3 in 1.2 m HCl. After an incubation time of 15 min, samples were centrifuged for 5 min at 12,000 × g. The supernatant was transferred to cuvettes and methylene blue was determined at 670 nm. The standard curve was recorded with sodium sulfide. Nfs1Δ1–55 concentrations were determined from the absorbance at 420 nm using the extinction coefficient of 10.9 mm–1 cm–1 for the native enzyme. The extinction coefficient was determined on the basis of the PLP content after alkaline denaturation described after the method of Peterson and Sober (30Peterson E.A. Sober H.A. J. Am. Chem. Soc. 1954; 76: 169-175Crossref Scopus (307) Google Scholar).The reduction spectrum of 9 μm Nfs1Δ1–55/Isd11 or 2 μm Nfs1Δ1–55 were recorded in the presence of 10 mm l-cysteine in 50 mm Tris, 1 mm EDTA, 200 mm NaCl, pH 8.0 at room temperature using a UV-2401PC Shimadzu spectrometer.Surface Plasmon Resonance (SPR) Measurements—All binding experiments were performed with the SPR-based instrument Biacore™ 2000 on CM5 sensor chips at a temperature of 25 °C and a flow rate of 10 μl/min, using the control software 2.1 and evaluation software 3.0 (Biacore AB, Uppsala, Sweden) as described previously (31Neumann M. Schulte M. Jünemann N. Stöcklein W. Leimkühler S. J. Biol. Chem. 2006; 281: 15701-15708Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The autosampler racks containing the sample vials were cooled to 4 °C. Immobilization of proteins yielded about 700–3,300 resonance units (RU) per flow cell (BSA: 1,530 RU; Isd11: 700 RU; MOCS3-RLD: 3,360 RU; Uba4: 1,870 RU).As running buffer, 20 mm phosphate, 150 mm NaCl, 0.005% Tween 20, pH 7.4 was used. Nfs1Δ1–55 and Nfs1Δ1–55/Isd11 with concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 μm were injected for 4.5 min at a flow rate of 30 μl/min followed by 15 min of dissociation using the kinject command and regeneration of the sensor surface with 50 mm HCl for 1 min. Binding curves were corrected by subtraction of buffer injection curves for all four flow cells and curves of Nfs1Δ1–55 and Nfs1Δ1–55/Isd11 for the control flow cell.Detection of Sulfur Transfer from Nfs1Δ1–55/Isd11 to MOCS3-RLD—For sulfur transfer from Nfs1Δ1–55/Isd11 to MOCS3-RLD, the proteins were incubated in a 1:1 ratio at a concentration of 2 μm in the presence of 10 mm l-cysteine and 30 mm KCN in 50 mm Tris, pH 8.0 over a period of 12 h. Produced thiocyanate was determined using the assay described by Sörbo (32Sörbo B. Biochim. Biophys. Acta. 1957; 23: 412-416Crossref PubMed Scopus (182) Google Scholar). For comparison, MOCS3-RLD was also incubated with 10 mm sodium thiosulfate. To ensure that MOCS3-RLD was added in a sulfur-free form to the assay, the protein was incubated with 5 mm KCN for 10 min at 4 °C prior to addition to the incubation mixtures, and produced thiocyanate was removed by gel filtration on a Nick column (GE Healthcare).Electrospray Ionization Mass Spectrometry (ESI-MS)—For the ESI-MS measurements, MOCS3-RLD was prepared in its persulfide-free form as described above. The persulfide-containing form of MOCS3-RLD was obtained after incubation of 50 μm MOCS3-RLD with 50 mm thiosulfate for 30 min. Persulfide-free MOCS3-RLD (50 μm) was additionally incubated with 5 mm l-cysteine for 1 h at 4 °C, and l-cysteine was subsequently removed by gel filtration.For sulfur transfer of Nfs1Δ1–55/Isd11-bound persulfide to MOCS3-RLD, 20 μm Nfs1Δ1–55/Isd11 was mixed with 50 μm persulfide-free MOCS3-RLD and 2 mm l-cysteine in 50 mm Hepes and 200 mm NaCl, pH 8.0. After incubation for 12 h at 4 °C, l-cysteine was removed by gel filtration. In addition, the MOCS3-RLD-C412A variant was used as a control, to verify the persulfide formation on Cys412 of MOCS3-RLD, which was not detected in this mutant (13Matthies A. Nimtz M. Leimkühler S. Biochemistry. 2005; 44: 7912-7920Crossref PubMed Scopus (64) Google Scholar).For ESI-MS measurements, the buffer of all proteins was changed to 3 mm NH4OAc by gel filtration, pH 8.0. 1–3 μl of the intact proteins, were diluted 1:1 with methanol followed by addition of 10% formic acid (final concentration 1–10 pmol/μl) and filled into a nanospray gold-coated glass capillary, placed orthogonally in front of the entrance hole of a QTOF-II instrument (Micromass, Manchester, UK). Approximately 1000 V was applied to the capillary, and ions were separated by the time-of-flight analyzer. Protein spectra were deconvoluted using the MaxEnt1 software package.RESULTSPurification of Nfs1Δ1–55, Isd11, and Copurification of the Nfs1Δ1–55/Isd11 Complex—For purification of Nfs1, the Nfs1 gene coding for amino acids 56–458 was cloned into the E. coli pET15b expression vector, resulting in a N-terminal His6-tagged recombinant protein. To increase protein stability, E. coli groEL genes were coexpressed with Nfs1Δ1–55. The soluble fraction of Nfs1Δ1–55 was purified by Ni-NTA chromatography and size exclusion chromatography on a Superose 12 column, and after elution one major band was visible on SDS-polyacrylamide gels with a size, corresponding closely to the calculated molecular mass of 47.3 kDa for His6-tagged Nfs1Δ1–55 (Fig. 1A). Densitometric analyses showed that the protein was 65% pure. The additional band visible on the SDS-polyacrylamide gel was determined by MALDI-peptide mapping and confirmed by MS/MS analyses to be the E. coli SlyD protein, a histidine-rich FKBP-type peptidyl-prolyl cistrans isomerase, a protein that is often copurified with recombinant proteins by Ni-NTA chromatography. The purified Nfs1Δ1–55 protein was stable for about 2 days at 4 °C. In contrast, the full-length Nfs1 protein could not be purified because the majority of the protein was unstable or existed in inclusion bodies after expression in E. coli. Coexpression of GroEL resulted in a higher yield of soluble Nfs1Δ1–55, likely by preventing the formation of inclusion bodies during expression. In contrast, the stability of the purified protein was not influenced by the presence of GroEL during expression, as shown by thermal denaturation studies using circular dichroism spectroscopy (see supplemental Fig. S1, which is published as supplemental data on the JBC web site).For purification of Isd11, the Isd11 gene was cloned into the E. coli pET15b expression vector, resulting in the N-terminal His6-tagged recombinant protein. Ni-NTA affinity chromatography and size exclusion chromatography on a Superose 12 column was carried out to obtain a purified Isd11 in a soluble form. The purified Isd11 displayed a single band on Coomassie Brilliant Blue-stained SDS-polyacrylamide gels with a monomeric mass corresponding to the calculated mass of the His6-tagged Isd11 of 13.4 kDa (Fig. 1C).To increase the stability of Nfs1Δ1–55, the Isd11 gene was cloned into a vector containing the P15A origin and coexpressed with Nfs1Δ1–55 in E. coli cells. The Nfs1Δ1–55/Isd11 complex was purified by Ni-NTA and size exclusion chromatography on a Superose 12 column, and displayed two bands on Coomassie Brilliant Blue-stained SDS-polyacrylamide gels (Fig. 1B), corresponding to Nfs1Δ1–55 and Isd11, respectively. The presence of Isd11 increased the stability of Nfs1 significantly, the protein was stable for several days at 4 °C and did not lose activity after storage at –20 °C. As shown by thermal denaturation studies by circular dichroism spectroscopy, the protein also displayed a better stability at higher temperatures in comparison to Nfs1Δ1–55 expressed in the absence of Isd11 (see supplemental Fig. S1, which is published as supplemental data on the JBC web site).To identify the oligomerization state of Nfs1Δ1–55 and the Nfs1Δ1–55/Isd11 complex, the purified proteins were subjected to size exclusion chromatography. The observed elution position of Nfs1Δ1–55 from a Superdex 200 column corresponded to a mass of 90 kDa (Fig. 2A), showing that the protein existed as a dimer in solution. In comparison to proteins with a similar size, the observed elution position of the Nfs1Δ1–55/Isd11 complex revealed a mass of ∼250 kDa on the analytical size exclusion column (Fig. 2B), at least corresponding to a (Nfs1Δ1–552/Isd112)2 octamer.FIGURE 2Size exclusion chromatography of Nfs1Δ1–55 and Nfs1Δ1–55/Isd11. A, 27 μm of Nfs1Δ1–55; B,3 μm of Nfs1Δ1–55/Isd11 were analyzed by analytical size exclusion chromatography in 50 mm Tris, 1 mm EDTA, 200 mm NaCl, pH 8.0, using a Superdex 200 column. Inset, plot of the standard proteins. Size exclusion chromatography markers (Bio-Rad): gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.3 kDa).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Functional Complementation of the E. coli CL100(iscS–) Strain and Analysis of the Kinetic Parameters of the Nfs1Δ1–55/Isd11 Complex—Nfs1 shares an amino acid sequence identity with E. coli IscS of 60%. Thus, we analyzed whether Nfs1 was able to functionally complement the E. coli CL100(iscS–) strain (33Lauhon C.T. Kambampati R. J. Biol. Chem. 2000; 275: 20096-20103Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The E. coli CL100(iscS–) strain was either transformed with the plasmid containing Nfs1Δ1–55 or cotransformed with the Nfs1Δ1–55- and Isd11-containing plasmids, and cells were grown at 30 °C in the presence of IPTG over a period of 8 h. The corresponding growth curves showed that Nfs1Δ1–55 was able to complement the role of IscS in the E. coli CL100(iscS–) strain both in the presence or absence of Isd11, showing that the Nfs1Δ1–55 was expressed in a functional form (data not shown).The purified Nfs1Δ1–55 protein exhibited the characteristic yellow color observed for other l-cysteine desulfurases containing PLP as prosthetic group. UV-VIS absorption spectra of the purified Nfs1Δ1–55 and the Nfs1Δ1–55/Isd11 complex were similar and exhibited the absorption maximum at 420 nm (Fig. 3, A and B), characteristic for l-cysteine desulfurases (17Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (496) Google Scholar). The addition of 20 mm l-cysteine resulted in a decrease of absorbance at 420 nm and an increase of absorbance at 320 nm of both purified Nfs1Δ1–55 and Nfs1Δ1–55/Isd11 (Fig. 3), showing that both proteins were reduced by l-cysteine. However, Nfs1Δ1–55 was only able to perform one turnover and precipitated rapidly after reduction (Fig. 3A, data not shown).FIGURE 3Characterization of Nfs1Δ1–55 and Nfs1Δ1–55/Isd11 by UV-VIS absorption spectroscopy. A, spectra of 14.5 μm of air-oxidized Nfs1Δ1–55 (solid line) and of the reduced enzyme with 20 mm l-cysteine (dashed line). B, spectra of 14.5 μm of air-oxidized Nfs1Δ1–55/Isd11 (solid line) and of the reduced enzyme with 20 mm l-cysteine (dashed line). Spectra were recorded in 50 mm Tris, 1 mm EDTA, 200 mm NaCl, 10 μm PLP, pH 8.0.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the kinetic parameters of Nfs1Δ1–55, steady state kinetics were performed by varying the concentration of l-cysteine using the purified Nfs1Δ1–55 protein and the Nfs1Δ1–55/Isd11 complex. The pH optimum of Nfs1-Δ1–55/Isd11 was determined to be at 8.0 and the temperature optimum was at 46 °C (data not shown). Enzyme assays were performed at 37 °C by varying the concentrations of l-cysteine and enzyme activity was determined using the methylene blue method, detecting the release of H2S in the assay (28Fogo J.K. Popowsky M. Anal Chem. 1949; 21: 732-734Crossref Scopus (358) Google Scholar). Enzyme activity was only detectable for the Nfs1Δ1–55/Isd11 complex. For Nfs1Δ1–55, no enzyme activity could be detected even at lower temperatures (data not shown). This is consistent with the observation, that Nfs1Δ1–55 is rather unstable after reduction and precipitated under the assay conditions. Thus, the apparent kinetic con" @default.
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W2080851578 title "A Novel Role for Human Nfs1 in the Cytoplasm" @default.
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W2080851578 doi "https://doi.org/10.1074/jbc.m804064200" @default.
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