AlipA(yutB) Mutant, Encoding Lipoic Acid Synthase, Provides Insight into the Interplay between Branched-Chain and Unsaturated Fatty Acid Biosynthesis inBacillus subtilis

Abstract

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 ⌬5 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 ⌬5 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 ⌬5 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....

Chlamydia trachomatisSerovar L2 Can Utilize Exogenous Lipoic Acid through the Action of the Lipoic Acid Ligase LplA1

“…Although LipA Ct shows a fairly high degree of homology to the E. coli LipA and has all of the canonical residues that are typical for this enzyme family, it failed to restore growth of an E. coli mutant lacking lipA. Homologs of LipA from other organisms have been shown to complement a similar E. coli lipA mutant, suggesting that heterologous enzymes are capable of functioning in vivo and complementing an E. coli mutant (26,44). Bacillus subtilis possesses two ligases and a lipoic acid synthase but no putative lipB, an arrangement similar to that encountered in chlamydiae (26).…”

“…Bacillus subtilis possesses two ligases and a lipoic acid synthase but no putative lipB, an arrangement similar to that encountered in chlamydiae (26). The B. subtilis lipA is essential for growth of the bacteria in minimal medium without exogenous lipoic acid, suggesting that it is capable of de novo lipoic acid biosynthesis, even in the absence of a putative LipB transferase (26). The existence of a similar pathway in chlamydiae cannot be ruled out since we have shown that LplA1 Ct expressed in ATM967 (⌬lplA ⌬lipB::kan) can lipoylate target proteins even in the absence of exogenous lipoic acid.…”

“…Homologs of LipA from other organisms have been shown to complement a similar E. coli lipA mutant, suggesting that heterologous enzymes are capable of functioning in vivo and complementing an E. coli mutant (26,44). Bacillus subtilis possesses two ligases and a lipoic acid synthase but no putative lipB, an arrangement similar to that encountered in chlamydiae (26). The B. subtilis lipA is essential for growth of the bacteria in minimal medium without exogenous lipoic acid, suggesting that it is capable of de novo lipoic acid biosynthesis, even in the absence of a putative LipB transferase (26).…”

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

“…The first of these more complex lipoyl synthesis pathways was found in Bacillus subtilis, a Gram-positive bacterium that is evolutionarily far removed from the Gram-negative E. coli. B. subtilis encodes a LipA that could functionally replace E. coli LipA, and disruption of the gene resulted in lipoate (or the products of the 2-oxoacid dehydrogenases) being required for growth (117). Although lipoate supplementation should require lipoate ligase activity, it was unclear why this bacterium encoded three putative lipoate ligases and no homologue of the LipB octanoyltransferase (87).…”

Dissecting Complex Metabolic Integration Provides Direct Genetic Evidence for CodY Activation by Guanine Nucleotides

“…1A). BCFAs serve as the principal fatty acids in B. subtilis membranes under standard growth conditions and also tailor membrane integrity by altering ratios of BCFAs in response to thermal perturbation, a multifaceted process known as homeoviscous adaptation (1,14,38,44,50). To synthesize BCFAs, BCKAs are converted first to their acyl-coenzyme A (acyl-CoA) derivatives and then to their carboxylic acid derivatives by functions encoded by the bkd operon (13,20).…”