We have developed a radiochemical method for the measurement of biotin synthase activity in vitro. A cell-free extract from an Escherichia coli strain containing a cloned bioB (biotin synthase) gene was incubated with [C]dethiobiotin, which was converted to [C]biotin. The assay was used to identify the low molecular weight compounds and two of the proteins that, in addition to the bioB gene product, are required for biotin synthase activity in vitro. The low molecular weight compounds are cysteine; S-adenosylmethionine; thiamine pyrophosphate; Fe; a pyridine nucleotide (the most effective being NADPH); and one of the amino acids asparagine, aspartate, glutamine, or serine. The proteins are flavodoxin and ferredoxin (flavodoxin)-NADP reductase (EC 1.18.1.2). A third thiamine pyrophosphate-dependent protein is also required for activity. When the cell-free extract was incubated with nonlabeled dethiobiotin and either [S]cysteine or [S]cystine, S was incorporated into biotin, and we present further evidence that cysteine, and not S-adenosylmethionine or methionine, is the sulfur donor for the biotin synthase reaction.

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