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 naturally occurring form of lipoate is attached by amide linkage to the epsilon-amino group of a specific lysine residue within conserved lipoate-accepting protein domains. Lipoate-protein ligase(s) catalyze the formation of this amide bond between lipoyl groups and specific apoproteins. We report the isolation of the lplA gene which encodes an Escherichia coli lipoate-protein ligase. Strains with lplA null mutations transport lipoic acid normally but have severe defects in the incorporation and utilization of exogenously supplied lipoic acid and lipoic acid analogs. These strains are also highly resistant to selenolipoate (a growth-inhibiting lipoate analog) and contain no detectable lipoate-protein ligase activity in cell extracts. The lplA gene has been cloned, sequenced, and physically mapped to min 99.6 (4657 kilobases) of the E. coli chromosome. Upon overexpression, the 38-kDa lplA gene product was purified to homogeneity and shown to have a mass, N-terminal sequence and amino acid composition consistent with the deduced 337 residue primary sequence. Enzyme assays show that purified LplA catalyzes the ATP-dependent attachment of [35S]lipoic acid to apoprotein, thus confirming that lplA encodes lipoate-protein ligase A. Analysis of lplA null 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 ligase enzyme.
Morris
T W
TW
Department of Microbiology, University of Illinois at Urbana-Champaign 61801.
Reed
K E
KE
Cronan
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002210:Caprylates / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010453:Peptide Synthases / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D008063:Thioctic Acid / Q000378:metabolism
D002210:Caprylates; D011994:Recombinant Proteins; D008063:Thioctic Acid; D010453:Peptide Synthases; C072322:lipoate-protein ligase; C031492:octanoic acid