PMID:11862544

Citation	Hewitson, KS, Ollagnier-de Choudens, S, Sanakis, Y, Shaw, NM, Baldwin, JE, Münck, E, Roach, PL and Fontecave, M (2002) The iron-sulfur center of biotin synthase: site-directed mutants. J. Biol. Inorg. Chem. 7:83-93
Abstract	Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by Mössbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and Mössbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.
Links	PubMed Online version:10.1007/s007750100268
Keywords	Amino Acid Motifs/physiology; Amino Acid Substitution; Biotin/biosynthesis; Cysteine/genetics; Cysteine/metabolism; Electron Spin Resonance Spectroscopy/methods; Enzymes/chemistry; Enzymes/metabolism; Escherichia coli/enzymology; Intramolecular Transferases/chemistry; Intramolecular Transferases/metabolism; Iron/chemistry; Iron/metabolism; Iron Chelating Agents/chemistry; Iron Chelating Agents/metabolism; Iron-Sulfur Proteins/chemistry; Iron-Sulfur Proteins/isolation & purification; Iron-Sulfur Proteins/metabolism; Mutagenesis, Site-Directed; Oxidation-Reduction; Sulfurtransferases/chemistry; Sulfurtransferases/genetics; Sulfurtransferases/isolation & purification; Sulfurtransferases/metabolism
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