Escherichia coliBiotin Synthase: An Investigation into the Factors Required for Its Activity and Its Sulfur Donor

Abstract Biotin synthase catalyzes the chemically difficult final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli. In the present work, we extend our characterization of this enzymatic reaction and the extensive set of factors required by it. A defined mixture of components that supports the biotin synthase reaction has been found. The mixture contains biotin synthase, flavodoxin, flavodoxin reductase, NADPH, Ado-Met, Fe, fructose- 1,6-bisphosphate, cysteine, and dithiothreitol. Even though this defined mixture supports the biotin synthase reaction, and in that regard is an important step forward in the study of this enzyme, it is unlikely that it contains all the physiologically significant factors involved in the biotin synthase reaction since it supports as an upper limit the synthesis of only 2 mol of biotin per mole of biotin synthase monomer. Progress in our efforts to identify additional physiologically significant factors is also reported. First, we describe evidence that the fructose 1,6-bisphosphate in the defined reaction mixture is substituting for an unknown factor of considerably higher potency present in crude extracts. Second, we have found that a labile low-molecular-weight product of the 7,8-diaminopelargonic acid aminotransferase reaction stimulates the rate of biotin formation in the defined biotin synthase reaction mixture and can increase the final amount of biotin formed by threefold. This product seems to be derived from Ado-Met, which 7,8-diaminopelargonic acid aminotransferase uses as its amino donor. However, 5′-deoxy-5′-methylthioadenosine, the postulated breakdown product from the action of 7,8-diaminopelargonic acid aminotransferase on Ado-Met, cannot be the active material since it has no stimulatory effect when added to the biotin synthase reaction mixture. Third, with a defined reaction mixture in hand, [ 35 S]cysteine and [ 35 S]Ado-Met, two potential sulfur donors present in the defined reaction mixture, were tested separately as sulfur donors. No 35 S was incorporated into newly formed biotin when either [ 35 S]cysteine or [ 35 S]Ado-Met was added to the defined biotin synthase reaction mixture.