LIPOIC ACID METABOLISM IN ESCHERICHIA-COLI - THE LPLA AND LIPB GENES DEFINE REDUNDANT PATHWAYS FOR LIGATION OF LIPOYL GROUPS TO APOPROTEIN

Lipoic acid is a covalently bound disulfide-containing cofactor requir ed for function of the pyruvate dehydrogenase, alpha-ketoglutarate deh ydrogenase, and glycine cleavage enzyme complexes of Escherichia coli, Recently we described the isolation of the lplA. locus, the first gen e known to encode a lipoyl-protein ligase for the attachment of lipoyl groups to lipoate-dependent apoenzymes (T.W. Morris, K.E. Reed, and J .E. Cronan, Jr,, J. Biol. Chem. 269:16091-16100, 1994), Here, we repor t an unexpected redundancy between the functions of lplA and lipB, a g ene previously identified as a putative lipoate biosynthetic locus, Fi rst, analysis of lplA null mutants revealed the existence of a second lipoyl ligase enzyme. We found that lplA null mutants displayed no gro wth defects unless combined with lipA (lipoate synthesis) or lipB muta tions and that overexpression of wild-type LplA suppressed lipB null m utations, Assays of growth, transport, lipoyl-protein content, and apo protein modification demonstrated that lplA encoded a ligase for the i ncorporation of exogenously supplied lipoate, whereas lipB was require d for function of the second lipoyl ligase, which utilizes lipoyl grou ps generated via endogenous (lipA-mediated) biosynthesis, The lipB-dep endent ligase was further shown to cause the accumulation of aberrantl y modified octanoyl-proteins in lipoate-deficient cells. Lipoate uptak e assays of strains that overproduced lipoate-accepting apoproteins al so demonstrated coupling between transport and the subsequent ligation of lipoate to apoprotein by the LplA enzyme, Although mutations in tw o genes (fadD and fadL) involved in fatty acid failed to affect lipoat e utilization, disruption of the smp gene severely decreased lipoate u tilization, DNA sequencing of the previously identified slrl selenolip oate resistance mutation (K.E. Reed, T.W. Morris, and J.E. Cronan, Jr. , Proc. Natl, Acad. Sci, USA 91:3720-3724, 1994) showed this mutation (now called lplA1) to be a G76S substitution in the LplA ligase, When compared with the wild-type allele, the cloned lplA1 allele conferred a threefold increase in the ability to discriminate against the seleni um-containing analog, These results support a two-pathway/two-ligase m odel of lipoate metabolism in E. coli.