title: Molybdenum cofactor biosynthesis in humans: identification of a persulfide group in the rhodanese-like domain of MOCS3 by mass spectrometry.
abstract: The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese. MOCS3 is proposed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C-terminus of the smaller subunit of molybdopterin (MPT) synthase during Moco biosynthesis in humans. Recent studies have shown that the MOCS3 rhodanese-like domain (MOCS3-RLD) catalyzes the transfer of sulfur from thiosulfate to cyanide and is also able to provide the sulfur for the thiocarboxylation of MOCS2A in a defined in vitro system for the generation of MPT from precursor Z. MOCS3-RLD contains four cysteine residues of which only C412 in the six amino acid active loop is conserved in homologous proteins from other organisms. ESI-MS/MS studies gave direct evidence for the formation of a persulfide group that is exclusively formed on C412. Simultaneous mutagenesis of the remaining three cysteine residues showed that none of them is involved in the sulfur transfer reaction in vitro. A disulfide bridge was identified to be formed between C316 and C324, and possible roles of the three noncatalytic cysteine residues are discussed. By ESI-MS/MS a partially gluconoylated N-terminus of the His6-tagged MOCS3-RLD was identified (mass increment of 178 Da) which resulted in a heterogeneity of the protein but did not influence sulfurtransferase activity.
authors.affiliation: Institut für Pflanzenbiologie, Technische Universität Braunschweig, 38023 Braunschweig, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Matthies
authors.forename: Manfred
authors.initials: M
authors.lastname: Nimtz
authors.forename: Silke
authors.initials: S
authors.lastname: Leimkühler
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003067:Coenzymes / Q000096:biosynthesis*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D006639:Histidine / Q000235:genetics; D006801:Humans; D008667:Metalloproteins / Q000096:biosynthesis*; D051379:Mice; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D011621:Pteridines; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013440:Sulfides / Q000737:chemistry*; D013466:Sulfurtransferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000235:genetics
chemical_list: D003067:Coenzymes; D003545:Cysteine; D004220:Disulfides; D006639:Histidine; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009713:Nucleotidyltransferases; D011621:Pteridines; D013438:Sulfhydryl Compounds; D013440:Sulfides; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; C051552:persulfides; C022261:molybdopterin; C502790:MOCS3 protein, human