Biotin synthase is an S-adenosyl-L-methionine (SAM) radical enzyme that inserts sulfur into dethiobiotin to produce biotin. The reaction proceeds through 5'-deoxyadenosyl radical intermediates that become reduced during the sulfur insertion step to give another product of the reaction, 5'-deoxyadenosine. We report that Escherichia coli strains lacking the 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase encoded by the pfs gene are deficient in biotin synthase activity due to accumulation of 5'-deoxyadenosine, a new substrate of the pfs-encoded nucleosidase. Physiological experiments indicate that lipoic acid synthase, another SAM radical enzyme, is also inhibited by 5'-deoxyadenosine accumulation.