Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes

Abstract

Two genes, lipA and lipB, involved in lipoic acid biosynthesis or metabolism were characterized by DNA sequence analysis. The translational initiation site of the lipA gene was established, and the lipB gene product was identified as a 25-kDa protein. Overproduction of LipA resulted in the formation of inclusion bodies, from which the protein was readily purified. Cells grown under strictly anaerobic conditions required the lipA and lipB gene products for the synthesis of a functional glycine cleavage system. Mutants carrying a null mutation in the lipB gene retained a partial ability to synthesize lipoic acid and produced low levels of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase activities. The lipA gene product failed to convert protein-bound octanoic acid moieties to lipoic acid moieties in vivo; however, the growth of both lipA and lipB mutants was supported by either 6-thiooctanoic acid or 8-thiooctanoic acid in place of lipoic acid. These data suggest that LipA is required for the insertion of the first sulfur into the octanoic acid backbone. LipB functions downstream of LipA, but its role in lipoic acid metabolism remains unclear.