Cloning and Expression of a cDNA Encoding Bovine Lipoyltransferase

Fujiwara, Kazuko; Okamura‐Ikeda, Kazuko; Motokawa, Yutaro

doi:10.1074/jbc.272.51.31974

Cited by 40 publications

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“…The N-terminal domain comprises two ␤-sheets, a large mixed ␤-sheet consisting of six strands (␤ 1 , ␤ 2 , ␤ 6 , ␤ 8 , ␤ 9 , and ␤ 10 ) and a small mixed ␤-sheet consisting of three strands (␤ 3 -␤ 5 ), with seven ␣-helices (␣ 1 -␣ 7 ) and a 3 10 -helix (3 10 1) surrounding the ␤-sheets. The C-terminal domain consists of three ␣-helices (␣ 8 -␣ 10 ), two 3 10 -helices (3 10 2 and 3 10 3), and a ␤-sheet comprising three strands (␤ 12 -␤ 14 ). An open and solvent-exposed cleft is formed between the two domains.…”

Section: Resultsmentioning

confidence: 99%

“…The secondary structure elements of E. coli the LplA are assigned using the program DSSP (37). ␣-Helices, 3 10 -helices, and ␤-strands are represented with magenta rectangles, orange rectangles, and blue arrows, respectively. Amino acid numbering of E. coli LplA starts at the serine residue following the initiation methionine (5), that of S. pneumoniae LplA starts at the initiation methionine residue (30), that of human LT starts at the N-terminal amino acid of the mature proteins (11), and that of LipB starts at the methionine residue added to the N-terminal extended active LipB (9).…”

Section: Methodsmentioning

confidence: 99%

“…1). However, the mammalian lipoyltransferases have no ability to activate lipoate to lipoyl-AMP and catalyze only the second half-reaction (10,11). Strong homology in the N-terminal half region suggests that the region may be responsible for the second transfer reaction, whereas some residue(s) in the C-terminal region of the E. coli enzyme may be essential for the lipoate-activating reaction, which are lacking in the mammalian sequences (10).…”

Section: Reactionmentioning

confidence: 99%

“…Lipoic acid is a prosthetic group of acyltransferase (E2) subunit of the pyruvate, ␣-ketoglutarate, and branched-chain ␣-ketoacid dehydrogenase complexes and of H-protein of the glycine cleavage system (1)(2)(3)(4). It attaches to a specific lysine residue on the proteins via an amide linkage between the ⑀-amino group of the lysine residue and the carboxyl group of lipoic acid.…”

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Crystal Structure of Lipoate-Protein Ligase A from Escherichia coli

Fujiwara

Toma

Okamura‐Ikeda

et al. 2005

Journal of Biological Chemistry

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Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of ␣-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 Å resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.Lipoic acid is a prosthetic group of acyltransferase (E2) subunit of the pyruvate, ␣-ketoglutarate, and branched-chain ␣-ketoacid dehydrogenase complexes and of H-protein of the glycine cleavage system (1-4). It attaches to a specific lysine residue on the proteins via an amide linkage between the ⑀-amino group of the lysine residue and the carboxyl group of lipoic acid. In the reaction sequence of the complexes, the lypoyllysine arm shuttles the reaction intermediates and reducing equivalents between the active sites of the components of the complexes.The attachment of lipoic acid to the proteins occurs by two-step reactions in which a lipoyl-AMP intermediate is formed from lipoic acid and ATP, and pyrophosphate is released in the initial activation reaction (Reaction 1).The lipoyl moiety of the intermediate is then transferred to apoproteins in the second transfer reaction, yielding the lipoylated protein and AMP (Reaction 2).Lipoyl-AMP ϩ apoprotein 3 lipoylated protein ϩ AMP (5, 6). LplA has a molecular mass of 37,795 Da, consisting of 337 amino acids excluding the initiating methionine residue, which is cleaved off during the biosynthesis (5). Strains with lplA null mutations have severe defects in the incorporation of exogenously supplied lipoic acid and lipoic acid analogues into apoproteins (5, 7). In E. coli, there is another enzyme, LipB, responsible for the covalent attachment of lipoic acid. LipB transfers lipoic acid/octanoic acid endogenously synthesized on the acyl carrier protein by the function of LipA to the lipoate-dependent enzymes (7-9). LipB consists of 213 amino acids, whose amino acid sequence shares only 12.7% identity with that of LplA (Fig. 1). On the other hand, the amino acid sequence of LplA shows 31 and 35% identity with those of human and bovine lipoyltransferase, the mammalian LplA homologues, respectively (Fig. 1). However, the mammalian lipoyltransferases have no ability to activate l...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Reactionmentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal Structure of Lipoate-Protein Ligase A from Escherichia coli

Fujiwara

Toma

Okamura‐Ikeda

et al. 2005

Journal of Biological Chemistry

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“…Both isoforms of lipoyltransferase catalyze the transfer of the lipoyl group from lipoyl-AMP to apoH protein but have no activity to activate lipoate to lipoyl-AMP, in contrast to the E. coli lipoyltransferase encoded by the lplA gene (Morris et al, 1995). Furthermore, these researchers cloned a cDNA for the bovine lipoyltransferase and found that both isoforms are derived from the same translated product but are processed differently (Fujiwara et al, 1997). We recently studied fatty acid synthesis in mitochondria isolated from pea and found that a major part of the de novo-synthesized fatty acids may be used for the biosynthesis of lipoic acid (Wada et al, 1997).…”

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Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase

Yasuno

Wada

1998

Plant Physiology

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Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southernhybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide.Lipoic acid (6,8-thioctic acid) is a sulfur-containing coenzyme that is required for the activity of enzyme complexes involved in the oxidative decarboxylation of ␣-ketoacids (Reed and Hackert, 1990;Perham, 1991;Mattevi et al., 1992) and in the Gly-cleavage system (Fujiwara et al., 1990;Kim and Oliver, 1990;Macherel et al., 1990). There are five lipoyl proteins: the dihydrolipoamide acyltransferase subunits of pyruvate, ␣-ketoglutarate, branched-chain ␣-ketoacid dehydrogenase complexes, protein X of the pyruvate dehydrogenase complex, and the H-protein of the Gly-cleavage system. Lipoic acid is covalently bound to these proteins via an amide linkage to the ⑀-amino group of specific Lys residues (Reed and Hackert, 1966). The lipoylLys arm functions as a carrier of reaction intermediates, and reducing equivalents interact with the active sites of the components of the complexes (Yeaman, 1989; Douce et al., 1994).Despite the importance of the lipoyl-prosthetic group in the functioning of several enzyme complexes, the biosynthesis of lipoic acid is not well understood in any organism. Molecular genetic studies (Vanden Boom et al., 1991;Reed and Cronan, 1993;Morris et al., 1994Morris et al., , 1995 of Escherichia coli have identified three genes, lipA, lipB, and lplA that are involved in the biosynthesis and transfer of lipoic acid. lipA encodes a lipoic acid synthase that is required for an insertion of the first sulfur atom into the octanoate backbone (Reed and Cronan, 1993). lplA and lipB encode lipoate ligases that are involved in the transfer of lipoic acid to cognate proteins (Morris et al., 1994(Morris et al., , 1995. LplA and LipB proteins primarily function in the utilization of lipoic acid exogenously added to the growth medium and endogenously synthesized lipoic acid in E. coli cells, respectively (Morris et al., 1994(Morris et al., , 1995. LplA protein is involved...

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Lipoic Acid

Perham

2002

Encyclopedia of Molecular Biology

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Cloning and Expression of a cDNA Encoding Bovine Lipoyltransferase

Cited by 40 publications

References 20 publications

Crystal Structure of Lipoate-Protein Ligase A from Escherichia coli

Crystal Structure of Lipoate-Protein Ligase A from Escherichia coli

Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase

Lipoic Acid

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