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

The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in Escherichia coli, transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid dehydrogenases of aerobic metabolism. Strains containing null mutations in lipB are auxotrophic for either lipoic acid or octanoic acid. We report the isolation of two spontaneously arising mutant strains that allow growth of lipB strains on glucose minimal medium; we determined that suppression was caused by single missense mutations within the coding sequence of the gene (lplA) that encodes lipoate-protein ligase. The LplA proteins encoded by the mutant genes have reduced K m values for free octanoic acid and thus are able to scavenge cytosolic octanoic acid for octanoylation of lipoyl domains.Escherichia coli has three lipoic acid-dependent enzyme systems: pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (OGDH), and the glycine cleavage system (GCV) (8). PDH catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A (CoA), the tricarboxylic acid (TCA) cycle substrate and fatty acid building block. OGDH functions in the TCA cycle, where it catalyzes the decarboxylation of 2-oxoglutarate to succinyl-CoA, the precursor of several amino acids. GCV is involved in the breakdown of glycine into ammonia and C 1 units. Whereas GCV is expressed only in the presence of glycine, PDH and OGDH are required for aerobic growth. (During anaerobic growth, acetyl-CoA is synthesized by other enzymes and an OGDH-independent branched form of the TCA cycle forms succinyl-CoA from succinate.) The three enzyme systems contain subunits (the E2 subunits of PDH and OGDH and the H protein of GCV) which contain at least one lipoyl domain, a conserved structure of ca. 80 residues (8). Lipoic acid is attached in an amide bond to a specific lysine residue of these domains, where it functions as a classical "swinging arm," carrying reaction intermediates between the active sites of the lipoate-dependent systems (27).Lipoic acid [R-5-(1,2-dithiolan-3-yl)pentanoic acid, also called 6,8-dithiooctanoic acid and thioctic acid] is composed of an eight-carbon fatty acid backbone to which two sulfur atoms are attached at carbons 6 and 8 (Fig. 1). In the oxidized state, the sulfur atoms are in a disulfide linkage forming a fivemembered ring with three backbone carbons. The disulfide bond is reduced upon binding of the intermediates (an acetyl moiety in the case of PDH, a succinyl moiety in the case of OGDH, and an aminomethyl moiety in the case of GCV). Following release of the intermediates to form the products of the enzyme complexes, the reduced lipoyl moiety must be reoxidized before entering another catalytic cycle. Oxidation is catalyzed by lipoamide dehydrogenase, a subunit component of the three lipoic acid-dependent enzyme systems (8). E. coli strains defective in lipoic acid biosynthesis are unable to grow on aerobic glucose minimal media unless the media are supplemented with acetate and succinat...

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“…Moreover, not all of the available lipoyl domains need be lipoylated for optimal PDH and OGDH complex activities (13,19).…”

Section: Discussionmentioning

confidence: 99%

Scavenging of Cytosolic Octanoic Acid by Mutant LplA Lipoate Ligases Allows Growth of Escherichia coli Strains Lacking the LipB Octanoyltransferase of Lipoic Acid Synthesis

Hermes¹,

Cronan

2009

J Bacteriol

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“…An analogue-resistant mutant that did not map at the lplA locus (209) was shown to be a chromosomal amplification of the lipA lipB region of the chromosome (224). The increased lipB dosage resulted in greater LipB activity that resulted in increased levels of lipoylation by endogenously synthesized lipoic acid that competed with the utilization of exogenous selenolipoic acid via the LplA-dependent pathway.…”

Section: Protein Lipoylation Pathwaysmentioning

confidence: 99%

“…The increased lipB dosage resulted in greater LipB activity that resulted in increased levels of lipoylation by endogenously synthesized lipoic acid that competed with the utilization of exogenous selenolipoic acid via the LplA-dependent pathway. A very modest (two- to threefold) increase in lipB dosage was sufficient to give resistance which was explained by the known highly nonlinear relationship between the degree of protein lipoylation and the activity of the 2-oxoacid dehydrogenase complexes plus the fact that E. coli does not require full activity of the 2-oxoacid dehydrogenases for growth on minimal medium containing glucose (224). Thus, synthesis of sufficient lipoic acid to modify a few percent of the 2-oxo acid dehydrogenase complexes allowed growth in presence of selenolipoic acid.…”

Section: Protein Lipoylation Pathwaysmentioning

confidence: 99%

Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation

Cronan

2014

EcoSal Plus

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Summary Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well-described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized and thus there is no transcriptional control of the synthetic genes. In contrast transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA a dual function protein that both represses biotin operon transcription and ligates biotin to its cognate protein.

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“…The Δ lipB deletion phenotype is known to be suppressed by overexpression of lplA (Morris et al ., ; Hermes and Cronan, ). Therefore, Southern blot analyses were performed using probes specific to the lipB and lplA genes in order to eliminate the possibility that gene duplication caused the suppression [prior work from this laboratory had demonstrated duplication of the lipB locus (Jordan and Cronan, )]. The genomic DNA blot patterns of strains FH6 and FH34 were identical (data not shown).…”

Section: Resultsmentioning

confidence: 98%

An NAD synthetic reaction bypasses the lipoate requirement for aerobic growth of Escherichia coli strains blocked in succinate catabolism

Hermes

Cronan

2014

Molecular Microbiology

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SUMMARY The lipoate coenzyme is essential for function of the pyruvate (PDH) and 2-oxoglutarate (OGDH) dehydrogenases and thus for aerobic growth of Escherichia coli. LipB catalyzes the first step in lipoate synthesis, transfer of an octanoyl moiety from the fatty acid synthetic intermediate, octanoyl-ACP, to PDH and OGDH. E. coli also encodes LplA, a ligase that in presence of exogenous octanoate (or lipoate) can bypass loss of LipB. LplA imparts ΔlipB strains with a “leaky” growth phenotype on aerobic glucose minimal medium supplemented with succinate (which bypasses the OGDH-catalyzed reaction), because it scavenges an endogenous octanoate pool to activate PDH. Here we characterize a ΔlipB suppressor strain that did not require succinate supplementation, but did require succinyl-CoA ligase, confirming the presence of alternative source(s) of cytosolic succinate. We report that suppression requires inactivation of succinate dehydrogenase (SDH), which greatly reduces the cellular requirement for succinate. In the suppressor strain succinate is produced by three enzymes, any one of which will suffice in the absence of SDH. These three enzymes are: trace levels of OGDH, the isocitrate lyase of the glyoxylate shunt and an unanticipated source, aspartate oxidase, the enzyme catalyzing the first step of nicotinamide biosynthesis.

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

Cited by 11 publications

References 35 publications

Scavenging of Cytosolic Octanoic Acid by Mutant LplA Lipoate Ligases Allows Growth of Escherichia coli Strains Lacking the LipB Octanoyltransferase of Lipoic Acid Synthesis

Scavenging of Cytosolic Octanoic Acid by Mutant LplA Lipoate Ligases Allows Growth of Escherichia coli Strains Lacking the LipB Octanoyltransferase of Lipoic Acid Synthesis

Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation

An NAD synthetic reaction bypasses the lipoate requirement for aerobic growth of Escherichia coli strains blocked in succinate catabolism

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