Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system

Boom, Thomas J. Vanden; Reed, Kelynne E.; Cronan, John E.

doi:10.1128/jb.173.20.6411-6420.1991

Cited by 86 publications

(84 citation statements)

References 45 publications

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“…The activity of LplA in the postulated alternate reaction would be low; otherwise, lipB-null mutant strains with a normal lplA gene would not have a lipoate auxotroph phenotype. Consistent with a low activity, lipB-null mutant strains were found to grow very slowly in the absence of lipoic acid (17,23) and to contain only low levels of lipoylated proteins (12,17). In strains with null mutations in both lipB and lplA there is no residual growth in the absence of lipoic acid and no detectable protein lipoylation (12).…”

Section: Resultsmentioning

confidence: 88%

“…Escherichia coli contains three enzyme complexes-pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and glycine cleavage enzyme-that utilize lipoic acid as a cofactor (17,23). Pyruvate dehydrogenase catalyzes a cycle of three successive reactions in the oxidative decarboxylation of pyruvate to yield acetyl coenzyme A (acetyl-CoA) plus two reducing equivalents (as NADH).…”

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confidence: 99%

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The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase

Jordan¹,

Cronan

2003

J Bacteriol

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we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that E. coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase. However, it remained possible that lipB encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that lipB encodes the lipoyltransferase. A lipB temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E. coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions.Lipoic acid (1,2-dithiolane-3-pentanoic acid) (Fig. 1) is a cofactor required for the function of key metabolic pathways in most organisms (15). The reactive sulfur moieties of lipoic acid occur at the distal ends of long flexible structures, and thus lipoic acid is able to channel reaction intermediates across remarkably long distances between the active sites of large multienzyme complexes (15). Although the general role of lipoic acid as a coenzyme has been known for decades, the mechanisms by which lipoic acid is synthesized and becomes attached to its cognate proteins continue to be elucidated.Escherichia coli contains three enzyme complexes-pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and glycine cleavage enzyme-that utilize lipoic acid as a cofactor (17, 23). Pyruvate dehydrogenase catalyzes a cycle of three successive reactions in the oxidative decarboxylation of pyruvate to yield acetyl coenzyme A (acetyl-CoA) plus two reducing equivalents (as NADH). The pyruvate and 2-oxoglutarate dehydrogenases generate energy-rich and reducing equivalents both directly and indirectly (by allowing function of the citric acid cycle), and the activity of these enzyme complexes is vita...

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Section: Resultsmentioning

confidence: 88%

mentioning

confidence: 99%

The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase

Jordan¹,

Cronan

2003

J Bacteriol

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“…The three enzymes are pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and glycine cleavage enzyme (29,37). The most-studied lipoic acid-dependent enzyme is the pyruvate dehydrogenase complex (29) (Fig.…”

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confidence: 99%

Chromosomal Amplification of the Escherichia coli lipB Region Confers High-Level Resistance to Selenolipoic Acid

Jordan¹,

Cronan

2002

J Bacteriol

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One of the mutants (slr7 mutant) of a wild-type Escherichia coli strain resistant to selenolipoic acid reported previously (K. E. Reed, T. W. Morris, and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 91:3720-3724, 1994) unexpectedly grew on minimal medium following transductional introduction of a lipA null mutation. We report that the slr7 strain carries a duplication of the lip chromosomal region that causes the phenotype of the mutant strain

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“…The first class maps at min 62.6 on the E. coli chromosome and presumably affects the gcv structural genes (12). The second class maps at min 14.8 and disrupts the lipoic acid biosynthetic pathway (18). The third class maps at min 2.7 and alters the lpd gene, encoding the L protein of the GCV enzyme complex (17).…”

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confidence: 99%

“…This strain was found to have normal transport of glycine (data not shown). Mutations in lipoic acid biosynthesis also result in a GCV-phenotype in serine auxotrophs, and growth of these mutants on glucose minimal medium (GM) containing glycine can be restored if lipoic acid is added to the medium (18). However, the addition of exogenous lipoic acid did not restore the GCV+ phenotype in strain GS786.…”

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Positive regulation of the Escherichia coli glycine cleavage enzyme system

1993

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A new mutation in Escherichia coli, designated gevAl, that results in noninducible expression of both gcv and a gcvT-kacZ gene fusion was isolated. A plasmid carrying the wild-type gcvA gene complemented the mutation and restored glycine-inducible gcv and gcvT-lacZ gene expression. These results suggest that gcvA encodes a positive-acting regulatory protein that acts in trans to increase expression of gcv.In enteric bacteria, the conversion of serine to glycine and 5,10-methylenetetrahydrofolate occurs through the action of the enzyme serine hydroxymethyltransferase, the glyA gene product (10). This reaction is an important contributor of one-carbon units in cell metabolism. The oxidative cleavage of glycine by the glycine cleavage (GCV) enzyme system provides a second pathway for one-carbon biosynthesis (14). A glycine-inducible GCV enzyme system has been demonstrated in both Eschenchia coli (8,11,12) and Salmonella typhimurium (16).E. coli mutants blocked simultaneously in the GCV enzyme system and in the serine biosynthetic pathway are unable to use glycine as a serine source and require an exogenous source of serine (the GCV-phenotype). At present, six classes of mutations have been shown to result in the GCV-phenotype under the appropriate growth conditions. The first class maps at min 62.6 on the E. coli chromosome and presumably affects the gcv structural genes (12). The second class maps at min 14.8 and disrupts the lipoic acid biosynthetic pathway (18). The third class maps at min 2.7 and alters the lpd gene, encoding the L protein of the GCV enzyme complex (17). The fourth class maps at min 54.8 and partially inactivates serine hydroxymethyltransferase, the glyA gene product (13). The fifth class maps at min 95.6 and disrupts the cycA gene involved in glycine transport (2, 4); this class results in the GCV-phenotype because of altered glycine uptake (4a). The sixth class maps at min 20 and inactivates the lIp gene (7). We report here a seventh locus that results in a GCV-phenotype because of the cell's inability to induce gcv expression.Isolation of a new class ofgcv mutations. Using a penicillin counterselection previously described (1, 11, 12), we isolated several mutants defective in the GCV enzyme pathway. One of the mutants isolated that displayed the GCVphenotype and that had a very low level of GCV enzyme activity did not map with the known structural genes encoding GCV. This strain was designated GS786 (the complete genotypes of all strains used in this study are listed in Table 1). A [2-14C]glycine uptake assay was performed to determine whether transport was altered in strain GS786. This strain was found to have normal transport of glycine (data not shown). Mutations in lipoic acid biosynthesis also result in a GCV-phenotype in serine auxotrophs, and growth of these mutants on glucose minimal medium (GM) containing glycine can be restored if lipoic acid is added to the medium (18). However, the addition of exogenous lipoic acid did not restore the GCV+ phenotype in strain GS786. These resul...

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Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system

Cited by 86 publications

References 45 publications

The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase

The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase

Chromosomal Amplification of the Escherichia coli lipB Region Confers High-Level Resistance to Selenolipoic Acid

Positive regulation of the Escherichia coli glycine cleavage enzyme system

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