IDENTIFICATION OF THE GENE ENCODING LIPOATE-PROTEIN LIGASE-A OF ESCHERICHIA-COLI - MOLECULAR-CLONING AND CHARACTERIZATION OF THE LPLA GENE AND GENE-PRODUCT

R(+)-Lipoic acid is a cofactor required for function of the alpha-keto acid dehydrogenase and glycine cleavage enzyme complexes. The natural ly occurring form of lipoate is attached by amide linkage to the epsil on-amino group of a specific lysine residue within conserved lipoate-a ccepting protein domains. Lipoate-protein ligase(s) catalyze the forma tion of this amide bond between lipoyl groups and specific apoproteins . We report the isolation of the lplA gene which encodes an Escherichi a coli lipoate-protein ligase. Strains with lplA null mutations transp ort lipoic acid normally but have severe defects in the incorporation and utilization of exogenously supplied lipoic acid and lipoic acid an alogs. These strains are also highly resistant to selenolipoate (a gro wth-inhibiting lipoate analog) and contain no detectable lipoate-prote in Ligase activity in cell extracts. The lplA gene has been cloned, se quenced, and physically mapped to min 99.6 (4657 kilobases) of the E. coli chromosome. Upon over expression, the 38-kDa lplA gene product wa s purified to homogeneity and shown to have a mass, N-terminal sequenc e and amino acid composition consistent with the deduced 337 residue p rimary sequence. Enzyme assays show that purified LplA catalyzes the A TP-dependent attachment of [S-35]lipoic acid to apoprotein, thus confi rming that lplA encodes lipoate-protein ligase A. Analysis of lplA nul l mutants also indicates the existence of a second (lplA-independent) lipoyl-ligase enzyme in E. coli, This is the first identification of a lipoate ligase gene and the first analysis of a purified lipoate liga se enzyme.