Stable isotope studies on the biosynthesis of lipoic acid in Escherichia coli

White, Robert H.

doi:10.1021/bi00542a003

Cited by 61 publications

(40 citation statements)

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“…Additional metabolic feeding studies demonstrated that in E. coli, lipoic acid biosynthesis does not involve either desaturation or hydroxylation of octanoic acid but does result in inversion of stereochemistry at C-6 (92-95). Sulfur is introduced at C-8 with racemization, in agreement with the formation of an intermediate carbon radical at C-8 (92,94,95). 8-Thiooctanoic and 6-thiooctanoic acids were both converted to lipoic acid, although 6-thiooctanoic acid was converted only 10 to 20% as efficiently as the other positional isomer (96).…”

Section: Biosynthesis Of Lipoic Acidmentioning

confidence: 81%

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

Cronan

2016

Microbiol Mol Biol Rev

125

137

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SUMMARYAlthough the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that ofEscherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered inBacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metabolism to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use theE. colipathway, whereas mammals and fungi probably use theB. subtilispathway. The lipoate metabolism enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enzymes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metabolism.

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Section: Biosynthesis Of Lipoic Acidmentioning

confidence: 81%

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

Cronan

2016

Microbiol Mol Biol Rev

125

137

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“…Octanoic acid has been shown to be the precursor of the carbon chain (White, 1980;Parry, 1983), but neither the origin nor the mechanism whereby the sulfur atoms are inserted into the carbon chain is known. We recently investigated the function of mitochondrial ACP, which was discovered in the mitochondria of several organisms (Shintani and Ohlrogge, 1994;Schneider et al, 1997), and found that it is involved in the biosynthesis of fatty acids in mitochondria of pea and Neurospora (Wada et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

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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“…Although the structures and reaction mechanisms of lipoic-acid-dependent proteins have been well studied, the biosynthesis of lipoic acid has been much less studied and its mechanism has not been elucidated in any organism. In bacteria, octanoic acid or its derivatives were suggested as precursors of lipoic acid (White, 1980;Parry, 1983). This was supported by several observations of octanoylated proteins upon overexpression in Escherichia coli of gene fragments encoding the lipoyl-binding domains of E2 components from pyruvate dehydrogenase (Dardel et al, 1990;Ali et al, 1990) or upon overexpression in E. coli of mature bovine H protein of the glycine decarboxylase complex (Fujiwara et al, 1992).…”

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confidence: 88%

Expression, Lipoylation and Structure Determination of Recombinant Pea H‐Protein in Escherichia coli

Macherel

Bourguignon

Forest

et al. 1996

European Journal of Biochemistry

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A synthetic gene encoding the entire mature H protein of the glycine decarboxylase complex from pea (Pisum sutivum L.) was constructed and expressed in Escherichia coli. The recombinant H protein, which after the induction period constituted more than half of the E. coli protein, was found in a soluble form. Activity measurements and mass-spectrometry analysis of the purified protein showed that, in the absence or presence of 5[3-( 1,2)-dithiolanyl]pentanoic acid (lipoic acid) in the culture medium, recombinant H protein could be produced as the unlipoylated apoform or as the lipoylated form, respectively. Addition of chloramphenicol to the culture medium after induction increased the proportion of lipoylated H protein. High rates of lipoylation of the H apoprotein were measured in vivo and in vitro, revealing that the recombinant pea H protein was an excellent substrate for the E. coli lipoyl-ligase. The threedimensional structure of the recombinant H apoprotein was determined at a 0.25-nm resolution. It was almost identical to the structure of the native pea leaf enzyme, which indicates that the recombinant protein folds properly in E. coli and that the lipoyl-ligase recognizes a three-dimensional structure in order to add lipoic acid to its specific lysine residue. It is postulated that the high level of expression and lipoylation of recombinant H protein may be due to the protein retaining the structure of the original enzyme.

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Stable isotope studies on the biosynthesis of lipoic acid in Escherichia coli

Cited by 61 publications

References 13 publications

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

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

Expression, Lipoylation and Structure Determination of Recombinant Pea H‐Protein in Escherichia coli

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