Lipoic acid metabolism in the rat

dl-[1,6-¹⁴C]Lipoic acid was administered by intraperitoneal injection to rats at the level of 0.5 mg/100 g body weight. Approximately 56% of the radioactivity was recovered in the urine. When acidified and extracted with benzene, 92% of the radioactivity remained in the aqueous phase. Gel-filtration and paper chromatography were used to identify three of the compounds in the benzene extract as lipoic, bisnorlipoic and tetranorlipoic acids. In addition, a keto compound appears to be present. The aqueous phase contained several radioactive components separable by ion-exchange and paper chromatographies. Two of these compounds were identified as lipoate and β-hydroxybisnorlipoate. No evidence for oxidation of the dithiolane ring of lipoic acid was observed. dl-[7,8-¹⁴C]Lipoic acid was administered to rats under the same conditions. The urine contained 81% of the radioactivity, 72% of which remained in the aqueous phase and 28% was extracted into benzene. In contrast to over 30% of the label from dl-(1,6-¹⁴C] lipoate being expired as ¹⁴CO₂, a negligible amount of ¹⁴CO₂ was produced by rats injected with dl-[7,8-¹⁴C]lipoate. The catabolites identified were the same as those found using the 1,6-labeled lipoate. Another dithiolane-intact compound was also isolated. It appears that the rat, similar to Pseudomonas putida LP, metabolizes lipoate mainly via β-oxidation of the valeric acid side chain.     

The metabolism of dl-(1,6-14C)lipoic acid in the rat

dl-[1,6-¹⁴C]Lipoic acid was synthesized and administered to rats or incubated in vitro with rat liver systems. The urinary excretion of radioactivity after labeled lipoate was administered intraperitoneally at a level of 0.5 mg/100 g body weight was maximal at 3–6 hr, with 60% of the injected radioactivity recovered within 24 hr. Respiratory ¹⁴CO₂ from the same animals is maximal at 3 hr, after which it falls off markedly. Approximately 30% of the injected radioactivity was recovered as ¹⁴CO₂ within 24 hr. The excretion of radioactivity after lipoate was administered by stomach tube was similar to that after intraperitoneal injection. Localization of radioactivity in the body was greatest in liver, intestinal contents, and muscle in all cases. Ionexchange and paper chromatographies of 24-hr pooled urine revealed several watersoluble radioactive metabolites. Incubation of [¹⁴C]lipoate with homogenates or mitochondrial preparations in vitro resulted in the production of ¹⁴CO₂, which was decreased by incubation with unlabeled fatty acids and unaffected by the addition of carnitine or (+)-decanoylcarnitine. The rat, like certain bacteria, metabolizes lipoate via β-oxidation of the valeric acid side chain and by other metabolic reactions on the dithiolane ring, which render the molecule more water soluble.     

Regulation of Cellular Thiols in Human Lymphocytes by α-Lipoic Acid: A Flow Cytometric Analysis

Modulation of cellular thiols is an effective therapeutic strategy, particularly in the treatment of AIDS. Lipoic acid, a metabolic antioxidant, functions as a redox modulator and has proven clinically beneficial effects. It is also used as a dietary supplement. We utilized the specific capabilities of N-ethylmaleimide to block total cellular thiols, phenylarsine oxide to block vicinal dithiols, and buthionine sulfoximine to deplete cellular GSH to flow cytometrically investigate how these thiol pools are influenced by exogenous lipoate treatment. Low concentrations of lipoate and its analogue lipoamide increased Jurkat cell GSH in a dose-dependent manner between 10 (25 μM for lipoamide) to 100 μM. This was also observed in mitogenically stimulated peripheral blood lymphocytes (PBL). Studies with Jurkat cells and its Wurzburg subclone showed that lipoate dependent increase in cellular GSH was similar in CD4+ and − cells. Chronic (16 week) exposure of cells to lipoate resulted in further increase of total cellular thiols, vicinal dithiols, and GSH. High concentration (2 and 5 mM) of lipoate exhibited cell shrinkage, thiol depletion, and DNA fragmentation effects. Based on similar effects of octanoic acid, the cytotoxic effects of lipoate at high concentration could be attributed to its fatty acid structure. In certain diseases such as AIDS and cancer, elevated plasma glutamate lowers cellular GSH by inhibiting cystine uptake. Low concentrations of lipoate and lipoamide were able to bypass the adverse effect of elevated extracellular glutamate. A heterogeneity in the thiol status of PBL was observed. Lipoate, lipoamide, or N-acetylcysteine corrected the deficient thiol status of cell subpopulations. Hence, the favorable effects of low concentrations of lipoate treatment appears clinically relevant. © 1997 Elsevier Science Inc.     

Interplay between Yersinia pestis and its flea vector in lipoate metabolism

To thrive, vector-borne pathogens must survive in the vector’s gut. How these pathogens successfully exploit this environment in time and space has not been extensively characterized. Using Yersinia pestis (the plague bacillus) and its flea vector, we developed a bioluminescence-based approach and employed it to investigate the mechanisms of pathogenesis at an unprecedented level of detail. Remarkably, lipoylation of metabolic enzymes, via the biosynthesis and salvage of lipoate, increases the Y. pestis transmission rate by fleas. Interestingly, the salvage pathway’s lipoate/octanoate ligase LplA enhances the first step in lipoate biosynthesis during foregut colonization but not during midgut colonization. Lastly, Y. pestis primarily uses lipoate provided by digestive proteolysis (presumably as lipoyl peptides) rather than free lipoate in blood, which is quickly depleted by the vector. Thus, spatial and temporal factors dictate the bacterium’s lipoylation strategies during an infection, and replenishment of lipoate by digestive proteolysis in the vector might constitute an Achilles’ heel that is exploited by pathogens.Subject terms: Bacterial pathogenesis, Metabolism     

The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains

Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-amino group of a specific lysine present on a highly conserved acceptor protein domain, resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains were rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP.     

Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.     

Effect of DL alpha-lipoic acid on some carbohydrate metabolising enzymes in stone forming rats.

DL alpha-lipoic acid has been shown to prevent the induced precipitation of calcium oxalate crystals in the renal tissues of laboratory animals. The acid seems to have a profound influence on carbohydrate metabolism in diabetic rats. Here the effect of alpha-lipoic acid was studied on certain key carbohydrate metabolising enzymes in the tissues of calcium oxalate stone forming rats administered with glycollate as oxalate precursor. There was augmentation of glycolysis in the renal tissues of stone forming as well as lipoate administered rats. The two major gluconeogenic enzymes, glucose-6-phosphatase (G6P) and fructose-1, 6 diphosphatase (FDP) were significantly inhibited in tissues of calculogenic rats. Lipoic acid also reduced the enzyme activities significantly. The citric acid cycle enzymes were not influenced to an appreciable extent. The observed alterations are likely to be due to the regulatory effects of oxalate and lipoate on the enzyme systems.     

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C₈-acyl carrier protein (C₈-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4′-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C₈-CoA. Furthermore, our ¹H saturation transfer difference and ³¹P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved ⁷¹RGG⁷³ loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.     

Fatty Acid Binding Proteins FABP9 and FABP10 Participate in Antibacterial Responses in Chinese Mitten Crab,Eriocheir sinensis

Invertebrates rely solely on the innate immune system for defense against pathogens and other stimuli. Fatty acid binding proteins (FABP), members of the lipid binding proteins superfamily, play a crucial role in fatty acid transport and lipid metabolism and are also involved in gene expression induced by fatty acids. In the vertebrate immune system, FABP is involved in inflammation regulated by fatty acids through its interaction with peroxidase proliferator activate receptors (PPARs). However, the immune functions of FABP in invertebrates are not well characterized. For this reason, we investigated the immune functionality of two fatty acid binding proteins, Es-FABP9 and Es-FABP10, following lipopolysaccharide (LPS) challenge in the Chinese mitten crab (Eriocheir sinensis). An obvious variation in the expression of Es-FABP9 and Es-FABP10 mRNA in E. sinensis was observed in hepatopancreas, gills, and hemocytes post-LPS challenge. Recombinant proteins rEs-FABP9 and rEs-FABP10 exhibited distinct bacterial binding activity and bacterial agglutination activity against Escherichia coli and Staphylococcus aureus. Furthermore, bacterial growth inhibition assays demonstrated that rEs-FABP9 responds positively to the growth inhibition of Vibrio parahaemolyticuss and S. aureus, while rEs-FABP10 responds positively to the growth inhibition of Aeromonas hydrophila and Bacillus subtilis. Coating of agarose beads with recombinant rEs-FABP9 and rEs-FABP10 dramatically enhanced encapsulation of the beads by crab hemocytes in vitro. In conclusion, the data presented here demonstrate the participation of these two lipid metabolism-related proteins in the innate immune system of E. sinensis.     

A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis

In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl‐GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester‐linked acyl‐enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys‐Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.     

Global Conformational Change Associated with the Two-step Reaction Catalyzed by Escherichia coli Lipoate-Protein Ligase A

Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645–33651). Here, we report two new LplA structures, LplA·lipoyl-5′-AMP and LplA·octyl-5′-AMP·apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5′-AMP reaction intermediate and rotation of the C-terminal domain by about 180°. These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys¹³³ residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.     

A lipA (yutB) Mutant,Encoding Lipoic Acid Synthase,Provides Insight into the Interplay between Branched-Chain and Unsaturated Fatty Acid Biosynthesis in Bacillus subtilis

Lipoic acid is an essential cofactor required for the function of key metabolic pathways in most organisms. We report the characterization of a Bacillus subtilis mutant obtained by disruption of the lipA (yutB) gene, which encodes lipoyl synthase (LipA), the enzyme that catalyzes the final step in the de novo biosynthesis of this cofactor. The function of lipA was inferred from the results of genetic and physiological experiments, and this study investigated its role in B. subtilis fatty acid metabolism. Interrupting lipoate-dependent reactions strongly inhibits growth in minimal medium, impairing the generation of branched-chain fatty acids and leading to accumulation of copious amounts of straight-chain saturated fatty acids in B. subtilis membranes. Although depletion of LipA induces the expression of the Δ5 desaturase, controlled by a two-component system that senses changes in membrane properties, the synthesis of unsaturated fatty acids is insufficient to support growth in the absence of precursors for branched-chain fatty acids. However, unsaturated fatty acids generated by deregulated overexpression of the Δ5 desaturase functionally replaces lipoic acid-dependent synthesis of branched-chain fatty acids. Furthermore, we show that the cold-sensitive phenotype of a B. subtilis strain deficient in Δ5 desaturase is suppressed by isoleucine only if LipA is present.Lipoic acid (LA; 6,8-thioctic acid or 1,2-dithiolane-3-pentanoic acid) is a sulfur-containing cofactor required for the function of several key enzymes involved in oxidative and single-carbon metabolism, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, branched-chain 2-oxoacid dehydrogenase (BCKADH), acetoin dehydrogenase, and the glycine cleavage system (10). Lipoate-requiring complexes typically contain three protein subunits, E1, E2, and E3. LA is linked through an amide bond to lysine residues in the E2 subunits (42) and acts as a swinging arm, transferring covalently attached reaction intermediates among the active sites of the enzyme complexes (40).Although the general role of LA as a bound cofactor has been known for decades, the mechanisms by which LA is synthesized and becomes linked to its cognate proteins in different organisms continue to be elucidated. The reactions whereby LA-modified proteins are produced are best understood in Escherichia coli. In this organism, lipoylation is mediated by two separate enzymes, lipoyl protein ligase A (LplA) and octanoyl-acyl carrier protein-protein transferase (LipB) (30, 31). While LplA uses exogenous LA, LipB transfers endogenous octanoic acid to the target proteins (19). These octanoylated domains are then converted into lipoylated derivatives by the S-adenosyl-l-methionine-dependent enzyme lipoyl synthase (LipA), which catalyzes the insertion of sulfur atoms into the carbon-6 and -8 positions of the corresponding fatty acids (29). This process bypasses the requirement for an exogenous supply of LA.In contrast to the wealth of knowledge available on LA synthesis and utilization in E. coli, the existing information about these pathways in gram-positive bacteria is scarce. It has been found that Listeria monocytogenes mutants defective in proteins homologous to the E. coli LplA enzymes are unable to scavenge exogenous LA for modification of lipoyl domains (22, 23, 38). However, L. monocytogenes is a natural lipoate auxotroph since it does not encode the enzymes necessary for lipoate biosynthesis (15, 55). Bacillus subtilis synthesizes LA, but the biosynthesis, attachment, and function of this essential nutrient in this model gram-positive organism have not yet been studied in detail (50). Analysis of the genome sequence of B. subtilis (25) revealed that it contains an open reading frame, yutB, encoding a protein with a high degree of homology to E. coli LipA and two open reading frames encoding proteins slightly similar to LplA, while no LipB homolog was detected.LA is a critical cofactor of BCKADH, the enzyme involved in the formation of the primer carbons for the initiation of branched-chain fatty acid (BCFA) synthesis (21). Early work indicated that a bfmB mutant of B. subtilis, defective in both BCKADH and pyruvate dehydrogenase, requires short-branched-chain carboxilic acids for growth (56). However, in our hands, this mutant presented a high percentage of reversion, precluding its use in the study of lipid metabolism. Since BCFAs are the dominant acyl chains found in membrane phospholipids of B. subtilis, the goal of this study was to employ a genetic approach to investigate the role of yutB in the physiology of this organism, in particular in fatty acid metabolism. In addition, we provide compelling evidence showing that Δ5 unsaturated fatty acids (UFA), the products of the B. subtilis desaturase, can fully replace the function of BCFAs. Furthermore, we demonstrate that UFA are essential to provide cryoprotective properties in strains depleted of LipA. This work reports the first characterization of a gram-positive mutant deficient in LA synthesis and its use to study the interplay between BCFAs and UFA metabolism.     

Increased flexibility in the use of exogenous lipoic acid by Staphylococcus aureus

Lipoic acid is a cofactor required for intermediary metabolism that is either synthesized de novo or acquired from environmental sources. The bacterial pathogen Staphylococcus aureus encodes enzymes required for de novo biosynthesis, but also encodes two ligases, LplA1 and LplA2, that are sufficient for lipoic acid salvage during infection. S. aureus also encodes two H proteins, GcvH of the glycine cleavage system and the homologous GcvH‐L encoded in an operon with LplA2. GcvH is a recognized conduit for lipoyl transfer to α‐ketoacid dehydrogenase E2 subunits, while the function of GcvH‐L remains unclear. The potential to produce two ligases and two H proteins is an unusual characteristic of S. aureus that is unlike most other Gram positive Firmicutes and might allude to an expanded pathway of lipoic acid acquisition in this microorganism. Here, we demonstrate that LplA1 and LplA2 facilitate lipoic acid salvage by differentially targeting lipoyl domain‐containing proteins; LplA1 targets H proteins and LplA2 targets α‐ketoacid dehydrogenase E2 subunits. Furthermore, GcvH and GcvH‐L both facilitate lipoyl relay to E2 subunits. Altogether, these studies identify an expanded mode of lipoic acid salvage used by S. aureus and more broadly underscore the importance of bacterial adaptations when faced with nutritional limitation.     

Lipoate metabolism in Pseudomonas putida LP: thiolsulfinates of lipoate and bisnorlipoate.

Lipoate thiolsulfinate and two bisnorlipoate thiolsulfinates, as well as the previously identified products of β-oxidation (bisnorlipoate, tetranorlipoate, and β-hydroxybisnorlipoate), were isolated and identified as catabolites of [¹⁴C]lipoate from cultures of Pseudomonas putida LP, an organism capable of growth on lipoic acid as a sole source of carbon and sulfur. The newly identified metabolites were characterized by ion-exchange and paper chromatography and infrared, ultraviolet, and mass spectroscopies. Comparison of the isolated catabolites with synthetic standards implies that the lipoic thiolsulfinate isolated is the S-1 monoxide of 1,2-dithiolane-3-valeric acid; one bisnorlipoic thiolsulfinate isolated is the S-1 monoxide, the other apparently the S-2 monoxide. Metabolic studies with P. putida show that lipoate thiolsulfinate is taken up by this microorganism in an energy-dependent process, but less readily than lipoate; lipoate thiolsulfinate supports oxygen consumption in short-term experiments but does not support growth. These results are interpreted as meaning that the thiolsulfinates are “dead-end” metabolites, not intermediates in the sulfur metabolism of this organism. Lipoate thiolsulfinate is not detectably β-oxidized to bisnorlipoate thiolsulfinate under the usual culture conditions.     

Lipoic acid metabolism in Escherichia coli: the lplA and lipB genes define redundant pathways for ligation of lipoyl groups to apoprotein.

Lipoic acid is a covalently bound disulfide-containing cofactor required for function of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine cleavage enzyme complexes of Escherichia coli. Recently we described the isolation of the lplA locus, the first gene known to encode a lipoyl-protein ligase for the attachment of lipoyl groups to lipoate-dependent apoenzymes (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Biol. Chem. 269:16091-16100, 1994). Here, we report an unexpected redundancy between the functions of lplA and lipB, a gene previously identified as a putative lipoate biosynthetic locus. First, analysis of lplA null mutants revealed the existence of a second lipoyl ligase enzyme. We found that lplA null mutants displayed no growth defects unless combined with lipA (lipoate synthesis) or lipB mutations and that overexpression of wild-type LplA suppressed lipB null mutations. Assays of growth, transport, lipoyl-protein content, and apoprotein modification demonstrated that lplA encoded a ligase for the incorporation of exogenously supplied lipoate, whereas lipB was required for function of the second lipoyl ligase, which utilizes lipoyl groups generated via endogenous (lipA-mediated) biosynthesis. The lipB-dependent ligase was further shown to cause the accumulation of aberrantly modified octanoyl-proteins in lipoate-deficient cells. Lipoate uptake assays of strains that overproduced lipoate-accepting apoproteins also demonstrated coupling between transport and the subsequent ligation of lipoate to apoprotein by the LplA enzyme. Although mutations in two genes (fadD and fadL) involved in fatty acid failed to affect lipoate utilization, disruption of the smp gene severely decreased lipoate utilization. DNA sequencing of the previously identified slr1 selenolipoate resistance mutation (K. E. Reed, T. W. Morris, and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 91:3720-3724, 1994) showed this mutation (now called lplA1) to be a G76S substitution in the LplA ligase. When compared with the wild-type allele, the cloned lplA1 allele conferred a threefold increase in the ability to discriminate against the selenium-containing analog. These results support a two-pathway/two-ligase model of lipoate metabolism in E. coli.