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Purification, Characterization, and cDNA Cloning of Lipoate-activating Enzyme from Bovine Liver

2001; Elsevier BV; Volume: 276; Issue: 31 Linguagem: Inglês

10.1074/jbc.m101748200

ISSN

1083-351X

Autores

Kazuko Fujiwara, S. Takeuchi, Kazuko Okamura‐Ikeda, Yutaro Motokawa,

Tópico(s)

Alcoholism and Thiamine Deficiency

Resumo

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 Mg2+ 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. 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 Mg2+ 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. lipoate-activating enzyme (p-amidinophenyl)methanesulfonyl fluoride hydrochloride dithiothreitol high performance liquid chromatography polyacrylamide gel electrophoresis Lipoic acid is a prosthetic group of H-protein of the glycine cleavage system and the acyltransferase components (E2s) of the pyruvate, α-ketoglutarate, and branched-chain α-ketoacid dehydrogenase complexes (1Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1986; 261: 8836-8841Abstract Full Text PDF PubMed Google Scholar, 2Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1992; 267: 20011-20016Abstract Full Text PDF PubMed Google Scholar, 3Perham R.N. Biochemistry. 1991; 30: 8501-8512Crossref PubMed Scopus (362) Google Scholar, 4Reed L.J. Hackert M.L. J. Biol. Chem. 1990; 265: 8971-8974Abstract Full Text PDF PubMed Google Scholar). The lipoyl moiety is attached to the specific lysine residue of the proteins via an amide linkage. The lipoyllysine arm is responsible for the shuttling of the reaction intermediate and reducing equivalents between the active sites of the complexes.The covalent attachment of lipoic acid occurs in two steps as follows.Lipoic acid+ATP→lipoyl­AMP+PPiREACTION1Lipoyl­AMP+apoprotein→holoprotein+AMPREACTION2Lipoate-protein ligase A of Escherichia coli catalyzes both Reactions 1 and 2 (5Morris T.W. Reed K.E. Cronan Jr., J.E. J. Biol. Chem. 1994; 269: 16091-16100Abstract Full Text PDF PubMed Google Scholar). In contrast, the two reactions are catalyzed by separate enzymes in mammals; lipoate-activating enzyme (LAE)1 catalyzes Reaction 1 (6Tsunoda J.N. Yasunobu K.T. Arch. Biochem. Biophys. 1967; 118: 395-401Crossref PubMed Scopus (27) Google Scholar) and lipoyltransferase catalyzes Reaction 2 (7Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1994; 269: 16605-16609Abstract Full Text PDF PubMed Google Scholar, 8Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1996; 271: 12932-12936Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We demonstrated the intramitochondrial lipoylation of in vitro translated apoH-protein precursor (9Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1990; 265: 17463-17467Abstract Full Text PDF PubMed Google Scholar) and purified lipoyltransferase from bovine liver mitochondria (7Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1994; 269: 16605-16609Abstract Full Text PDF PubMed Google Scholar). The purified enzyme efficiently transferred the lipoyl moiety from lipoyl-AMP to H-protein and the lipoyl domains of E2s of pyruvate, α-ketoglutarate, and branched-chain α-ketoacid dehydrogenase complexes but had no ability to activate lipoate (8Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1996; 271: 12932-12936Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Despite an early finding of the presence of LAE in bovine liver, less is known about the properties of the enzyme.To better understand a mechanism of the protein lipoylation, we tried to isolate an enzyme that catalyzes the activation of lipoic acid employing a naturally occurring enantiomer of lipoic acid, (R)-(+)-lipoic acid, as a substrate. Studies of the intracellular distribution indicated that LAE activity was exclusively localized in mitochondria when GTP was employed as a high energy compound. The purified LAE utilized GTP preferentially for the activation of lipoic acid. Nucleotide sequencing analysis of cDNAs encoding the purified LAE revealed that the enzyme is identical with xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III (10Vessey D.A. Lau E. Kelley M. J. Biochem. Mol. Toxicol. 2000; 14: 11-19Crossref PubMed Google Scholar). The results indicate a novel function of the medium-chain acyl-CoA synthetase employing GTP. Lipoic acid is a prosthetic group of H-protein of the glycine cleavage system and the acyltransferase components (E2s) of the pyruvate, α-ketoglutarate, and branched-chain α-ketoacid dehydrogenase complexes (1Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1986; 261: 8836-8841Abstract Full Text PDF PubMed Google Scholar, 2Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1992; 267: 20011-20016Abstract Full Text PDF PubMed Google Scholar, 3Perham R.N. Biochemistry. 1991; 30: 8501-8512Crossref PubMed Scopus (362) Google Scholar, 4Reed L.J. Hackert M.L. J. Biol. Chem. 1990; 265: 8971-8974Abstract Full Text PDF PubMed Google Scholar). The lipoyl moiety is attached to the specific lysine residue of the proteins via an amide linkage. The lipoyllysine arm is responsible for the shuttling of the reaction intermediate and reducing equivalents between the active sites of the complexes. The covalent attachment of lipoic acid occurs in two steps as follows.Lipoic acid+ATP→lipoyl­AMP+PPiREACTION1Lipoyl­AMP+apoprotein→holoprotein+AMPREACTION2Lipoate-protein ligase A of Escherichia coli catalyzes both Reactions 1 and 2 (5Morris T.W. Reed K.E. Cronan Jr., J.E. J. Biol. Chem. 1994; 269: 16091-16100Abstract Full Text PDF PubMed Google Scholar). In contrast, the two reactions are catalyzed by separate enzymes in mammals; lipoate-activating enzyme (LAE)1 catalyzes Reaction 1 (6Tsunoda J.N. Yasunobu K.T. Arch. Biochem. Biophys. 1967; 118: 395-401Crossref PubMed Scopus (27) Google Scholar) and lipoyltransferase catalyzes Reaction 2 (7Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1994; 269: 16605-16609Abstract Full Text PDF PubMed Google Scholar, 8Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1996; 271: 12932-12936Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We demonstrated the intramitochondrial lipoylation of in vitro translated apoH-protein precursor (9Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1990; 265: 17463-17467Abstract Full Text PDF PubMed Google Scholar) and purified lipoyltransferase from bovine liver mitochondria (7Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1994; 269: 16605-16609Abstract Full Text PDF PubMed Google Scholar). The purified enzyme efficiently transferred the lipoyl moiety from lipoyl-AMP to H-protein and the lipoyl domains of E2s of pyruvate, α-ketoglutarate, and branched-chain α-ketoacid dehydrogenase complexes but had no ability to activate lipoate (8Fujiwara K. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 1996; 271: 12932-12936Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Despite an early finding of the presence of LAE in bovine liver, less is known about the properties of the enzyme. To better understand a mechanism of the protein lipoylation, we tried to isolate an enzyme that catalyzes the activation of lipoic acid employing a naturally occurring enantiomer of lipoic acid, (R)-(+)-lipoic acid, as a substrate. Studies of the intracellular distribution indicated that LAE activity was exclusively localized in mitochondria when GTP was employed as a high energy compound. The purified LAE utilized GTP preferentially for the activation of lipoic acid. Nucleotide sequencing analysis of cDNAs encoding the purified LAE revealed that the enzyme is identical with xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III (10Vessey D.A. Lau E. Kelley M. J. Biochem. Mol. Toxicol. 2000; 14: 11-19Crossref PubMed Google Scholar). The results indicate a novel function of the medium-chain acyl-CoA synthetase employing GTP. We thank ASTA-Medica for the generous gift of (R)-(+)- and (S)-(−)-lipoate and Ryoichi Kunai (University of Tokushima) for amino acid sequencing.

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