BIOTIN SYNTHASE FROM ESCHERICHIA-COLI, AN INVESTIGATION OF THE LOW-MOLECULAR-WEIGHT AND PROTEIN-COMPONENTS REQUIRED FOR ACTIVITY IN-VITRO

We have developed a radiochemical method for the measurement of biotin synthase activity in vitro. A cell-free extract from an Escherichia c oli strain containing a cloned bioB (biotin synthase) gene was incubat ed with [C-14]dethiobiotin, which was converted to [C-14]biotin, The a ssay was used to identify the low molecular weight compounds and two o f the proteins that, in addition to the bioB gene product, are require d for biotin synthase activity in vitro. The low molecular weight comp ounds are cysteine; S-adenosylmethionine thiamine pyrophosphate; Fe2+; a pyridine nucleotide (the most effective being NADPH); and one of th e amino acids asparagine, aspartate, glutamine, or serine. The protein s are flavodoxin and ferredoxin (flavodoxin)-NADP(+) reductase (EC 1.1 8.1.2). A third thiamine pyrophosphate-dependent protein is also requi red for activity. When the cell-free extract was incubated with nonlab eled dethiobiotin and either [S-35]cysteine or [S-35]cystine, S-35 was incorporated into biotin, and we present further evidence that cystei ne, and not S-adenosylmethionine or methionine, is the sulfur donor fo r the biotin synthase reaction.