Purification, characterization, and cDNA cloning of lipoate-activating enzyme from bovine liver.
In mammals, lipoate-activating enzyme (LAE) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. The lipoyl moiety is then transferred to the specific lysine residue of lipoate-dependent enzymes by the action of lipoyltransferase. We purified LAE from bovine liver mitochondria to apparent homogeneity. LAE activated lipoate with GTP at a 1000-fold higher rate than with ATP. The reaction absolutely required lipoate, GTP, and Mg(2+) ion, and the reaction product was lipoyl-GMP. LAE activated both (R)- and (S)-lipoate to the respective lipoyl-GMP, although a preference for (R)-lipoate was observed. Similarly, lipoyltransferase equally transferred both the (R)- and (S)-lipoyl moieties from the respectively activated lipoates to apoH-protein. Interestingly, however, only H-protein carrying (R)-lipoate was active in the glycine cleavage reaction. cDNA clones encoding a precursor LAE with a mitochondrial presequence were isolated. The predicted amino acid sequence of LAE is identical with that of xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III, but an amino acid substitution due to a single nucleotide polymorphism was found. These results indicate that the medium-chain acyl-CoA synthetase in mitochondria has a novel function, the activation of lipoate with GTP.
Fujiwara
K
K
Institute for Enzyme Research, the University of Tokushima, Tokushima 770-8503, Japan.
Takeuchi
S
S
Okamura-Ikeda
K
K
Motokawa
Y
Y
D000217:Acyltransferases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary; D017886:Durapatite; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008322:Mammals; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D008063:Thioctic Acid / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D008063:Thioctic Acid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017886:Durapatite; D000217:Acyltransferases; C109735:lipoyltransferase I; C109736:lipoyltransferase II; D009713:Nucleotidyltransferases; C434474:lipoate activating enzyme; D008274:Magnesium