Escherichia coli LipA is a lipoyl synthase: In vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein

The Escherichia coli lipA gene product has been genetically linked to carbo n-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Re ed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], alt hough in vitro lipoate biosynthesis with LipA has never been observed. In t his study, the lipA gene and a hexahistidine tagged lipA construct (LipA-Hi s) were overexpressed in E. coli as soluble proteins. The proteins were pur ified as a mixture of monomeric and dimeric species that contain approximat ely four iron atoms per LipA polypeptide and a similar amount of acid-labil e sulfide. Electron paramagnetic resonance and electronic absorbance spectr oscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S ] cluster states. Reduction with sodium dithionite results in small quantit ies of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein con taining a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was a ssayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein li gase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo- PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183 ]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, Lip B, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirma tion that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was o btained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding doma in that had been lipoylated in a LipA reaction. These results provide infor mation about the mechanism of LipA catalysis and place LipA within the fami ly of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.