Biotin synthase: purification, characterization as a [2Fe-2S]cluster protein, and in vitro activity of the Escherichia coli bioB gene product.
We report here the first purification of the protein encoded by the Escherichia coli bioB gene. One species of this protein runs on native gels with an electrophoretic mobility typical of a protein with m = 82 kDa, suggesting the protein is a dimer (gene sequence predicts m = 38.7 kDa). There are two iron- and two acid-labile sulfur atoms per protein monomer. Solutions containing the protein are red and have an absorbance spectrum characteristic of proteins with [2Fe-2S] clusters. In its oxidized native state, the protein is EPR-silent. Upon addition of dithionite, the protein's UV-visible absorbance spectrum is very slowly bleached, and an EPR active species is produced that displays a signal at gavg = 1.95. All these results are consistent with this protein containing one [2Fe-2S] cluster per monomer. The EPR spin quantitation is only 5-10% of expected. Since this protein loses iron upon reduction with dithionite, the low-spin quantitation is probably due to cluster instability in the reduced state. Another species of the bioB gene products has also been purified which runs on native gels with an electrophoretic mobility typical of a protein with m = 104 kDa. This species appears to be a dimer with one [2Fe-2S] cluster per dimer. The 104-kDa protein can be converted to the 82-kDa protein upon incubation with Fe3+ and S2-. The bioB gene product we have isolated is active in the conversion of dethiobiotin to biotin in vitro in the presence of NADPH, AdoMet, Fe3+ or Fe2+, and additional unidentified factors from the crude extracts of E. coli. The Km for dethiobiotin in this reaction has been found to be 2 microM.
Sanyal
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E. I. du Pont de Nemours and Company, Wilmington, Delaware 19880-0328.
Cohen
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D000595:Amino Acid Sequence; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D011189:Potassium Chloride / Q000494:pharmacology; D013053:Spectrophotometry; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D004227:Dithionite; D009249:NADP; D011189:Potassium Chloride; D001710:Biotin; C004749:desthiobiotin; D013466:Sulfurtransferases; C057985:biotin synthetase