Structure of RhlG, an essential beta-ketoacyl reductase in the rhamnolipid biosynthetic pathway of Pseudomonas aeruginosa

Rhamnolipids are extracellular biosurfactants and virulence factors secreted by the opportunistic human pathogen Pseudomonas aeruginosa that are required for swarming motility. The rhlG gene is essential for rhamnolipid formation, and the RhlG enzyme is thought to divert fatty acid synthesis intermediates into the rhamnolipid biosynthetic pathway based on its similarity to FabG, the beta-ketoacyl-acyl carrier protein (ACP) reductase of type II fatty acid synthesis. Crystallographic analysis reveals that the overall structures of the RhlG.NADP+ and FabG.NADP+ complexes are indeed similar, but there are key differences related to function. RhlG does not undergo the conformational changes upon NADP(H) binding at the active site that in FabG are the structural basis of negative allostery. Also, the acyl chain-binding pocket of RhlG is narrow and rigid compared with the larger, flexible substrate-binding subdomain in FabG. Finally, RhlG lacks a positively charged/hydrophobic surface feature adjacent to the active site that is found on enzymes like FabG that recognize the ACP of fatty acid synthesis. RhlG catalyzed the NADPH-dependent reduction of beta-ketodecanoyl-ACP to beta-d-hydroxydecanoyl-ACP. However, the enzyme was 2000-fold less active than FabG in carrying out the same reaction. These structural and biochemical studies establish RhlG as a NADPH-dependent beta-ketoacyl reductase of the SDR protein superfamily and further suggest that the ACP of fatty acid synthesis does not carry the substrates for RhlG.     

A new mechanism for anaerobic unsaturated fatty acid formation in Streptococcus pneumoniae

The anaerobic pathway for unsaturated fatty acid synthesis was established in the 1960s in Escherichia coli. The double bond is introduced into the growing acyl chain by FabA, an enzyme capable of both the dehydration of beta-hydroxydecanoyl-acyl carrier protein (ACP) to trans-2-decenoyl-ACP, and the isomerization of trans-2 to cis-3-decenoyl-ACP. However, there are a number of anaerobic bacteria whose genomes do not contain a fabA homolog, although these organisms nonetheless produce unsaturated fatty acids. We cloned and biochemically characterized a new enzyme in type II fatty acid synthesis from Streptococcus pneumoniae that carries out the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP, but is not capable of catalyzing the dehydration of beta-hydroxy intermediates. This tetrameric enzyme, designated FabM, has no similarity to FabA, but rather is a member of the hydratase/isomerase superfamily. Thus, the branch point in the biosynthesis of unsaturated fatty acids in S. pneumoniae occurs following the formation of trans-2-decenoyl-ACP, in contrast to E. coli where the branch point takes place after the formation of beta-hydroxydecanoyl-ACP.     

金黄色葡萄球菌细胞膜磷脂合成的研究进展

金黄色葡萄球菌引起的危害是目前我国微生物安全的重要问题之一。金黄色葡萄球菌通过脂肪酸生物合成磷脂酸(磷脂合成必需中间体)合成细胞膜磷脂以完成自身繁殖。因此,抑制菌体磷脂酸合成可有效防控金黄色葡萄球菌对环境及生物体造成危害。然而,金黄色葡萄球菌可经II型脂肪酸合成(type II fatty acid synthesis, FASII)通路和旁路两条途径合成磷脂酸,常规抑菌剂仅靶向抑制FASII通路,可能导致菌体在富含外源脂肪酸条件下出现“旁路逃逸”,形成防控漏洞。为此,本文系统总结金黄色葡萄球菌基于FASII通路和旁路合成细胞磷脂酸及磷脂酸向其他磷脂类物质转化的信号传导过程,讨论抑菌物质靶向抑制上述信号传导过程中可能的关键靶点,为新型抑菌剂开发提供理论指导。     

Branched-chain amino acid metabolism controls membrane phospholipid structure in Staphylococcus aureus

Branched-chain amino acids (primarily isoleucine) are important regulators of virulence and are converted to precursor molecules used to initiate fatty acid synthesis in Staphylococcus aureus. Defining how bacteria control their membrane phospholipid composition is key to understanding their adaptation to different environments. Here, we used mass tracing experiments to show that extracellular isoleucine is preferentially metabolized by the branched-chain ketoacid dehydrogenase complex, in contrast to valine, which is not efficiently converted to isobutyryl-CoA. This selectivity creates a ratio of anteiso:iso C₅-CoAs that matches the anteiso:iso ratio in membrane phospholipids, indicating indiscriminate utilization of these precursors by the initiation condensing enzyme FabH. Lipidomics analysis showed that removal of isoleucine and leucine from the medium led to the replacement of phospholipid molecular species containing anteiso/iso 17- and 19-carbon fatty acids with 18- and 20-carbon straight-chain fatty acids. This compositional change is driven by an increase in the acetyl-CoA:C₅-CoA ratio, enhancing the utilization of acetyl-CoA by FabH. The acyl carrier protein (ACP) pool normally consists of odd carbon acyl-ACP intermediates, but when branched-chain amino acids are absent from the environment, there was a large increase in even carbon acyl-ACP pathway intermediates. The high substrate selectivity of PlsC ensures that, in the presence or the absence of extracellular Ile/Leu, the 2-position is occupied by a branched-chain 15-carbon fatty acid. These metabolomic measurements show how the metabolism of isoleucine and leucine, rather than the selectivity of FabH, control the structure of membrane phospholipids.     

Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action.

The effects of inhibition of Escherichia coli phospholipid synthesis on the accumulation of intermediates of the fatty acid synthetic pathway have been previously investigated with conflicting results. We report construction of an E. coli strain that allows valid [14C]acetate labeling of fatty acids under these conditions. In this strain, acetate is a specific precursor of fatty acid synthesis and the intracellular acetate pools are not altered by blockage of phospholipid synthesis. By use of this strain, we show that significant pools of fatty acid synthetic intermediates and free fatty acids accumulate during inhibition of phospholipid synthesis and that the rate of synthesis of these intermediates is 10 to 20% of the rate at which fatty acids are synthesized during normal growth. Free fatty acids of abnormal chain length (e.g., cis-13-eicosenoic acid) were found to accumulate in glycerol-starved cultures. Analysis of extracts of [35S]methionine-labeled cells showed that glycerol starvation resulted in the accumulation of several long-chain acyl-acyl carrier protein (ACP) species, with the major species being ACP acylated with cis-13-eicosenoic acid. Upon the restoration of phospholipid biosynthesis, the abnormally long-chain acyl-ACPs decreased, consistent with transfer of the acyl groups to phospholipid. The introduction of multicopy plasmids that greatly overproduced either E. coli thioesterase I or E. coli thioesterase II fully relieved the inhibition of fatty acid synthesis seen upon glycerol starvation, whereas overexpression of ACP had no effect. Thioesterase I overproduction also resulted in disappearance of the long-chain acyl-ACP species. The release of inhibition by thiosterase overproduction, together with the correlation between the inhibition of fatty acid synthesis and the presence of abnormally long-chain acyl-ACPs, suggests with that these acyl-ACP species may act as feedback inhibitors of a key fatty acid synthetic enzyme(s).     

Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli.

Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the beta-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.     

Thematic review series: Glycerolipids. Acyltransferases in bacterial glycerophospholipid synthesis

Phospholipid biosynthesis is a vital facet of bacterial physiology that begins with the synthesis of the fatty acids by a soluble type II fatty acid synthase. The bacterial glycerol-phosphate acyltransferases utilize the completed fatty acid chains to form the first membrane phospholipid and thus play a critical role in the regulation of membrane biogenesis. The first bacterial acyltransferase described was PlsB, a glycerol-phosphate acyltransferase. PlsB is a key regulatory point that coordinates membrane phospholipid formation with cell growth and macromolecular synthesis. Phosphatidic acid is then produced by PlsC, a 1-acylglycerol-phosphate acyltransferase. These two acyltransferases use thioesters of either CoA or acyl carrier protein (ACP) as the acyl donors and have homologs that perform the same reactions in higher organisms. However, the most prevalent glycerol-phosphate acyltransferase in the bacterial world is PlsY, which uses a recently discovered acyl-phosphate fatty acid intermediate as an acyl donor. This unique activated fatty acid is formed from the acyl-ACP end products of the fatty acid biosynthetic pathway by PlsX, an acyl-ACP:phosphate transacylase.     

Regulation of fatty acid biosynthesis in Escherichia coli.

Our understanding of fatty acid biosynthesis in Escherichia coli has increased greatly in recent years. Since the discovery that the intermediates of fatty acid biosynthesis are bound to the heat-stable protein cofactor termed acyl carrier protein, the fatty acid synthesis pathway of E. coli has been studied in some detail. Interestingly, many advances in the field have aided in the discovery of analogous systems in other organisms. In fact, E. coli has provided a paradigm of predictive value for the synthesis of fatty acids in bacteria and plants and the synthesis of bacterial polyketide antibiotics. In this review, we concentrate on four major areas of research. First, the reactions in fatty acid biosynthesis and the proteins catalyzing these reactions are discussed in detail. The genes encoding many of these proteins have been cloned, and characterization of these genes has led to a better understanding of the pathway. Second, the function and role of the two essential cofactors in fatty acid synthesis, coenzyme A and acyl carrier protein, are addressed. Finally, the steps governing the spectrum of products produced in synthesis and alternative destinations, other than membrane phospholipids, for fatty acids in E. coli are described. Throughout the review, the contribution of each portion of the pathway to the global regulation of synthesis is examined. In no other organism is the bulk of knowledge regarding fatty acid metabolism so great; however, questions still remain to be answered. Pursuing such questions should reveal additional regulatory mechanisms of fatty acid synthesis and, hopefully, the role of fatty acid synthesis and other cellular processes in the global control of cellular growth.     

A mutant of Arabidopsis deficient in desaturation of palmitic Acid in leaf lipids

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by elevated amounts of palmitic acid and a decreased amount of unsaturated 16-carbon fatty acids as a consequence of a single nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids suggested that the mutant is deficient in the activity of a chloroplast ω9 fatty acid desaturase which normally introduces a double bond in 16-carbon acyl chains esterified to monogalactosyldiacylglycerol (MGD). The mutant exhibited an increased ratio of 18- to 16-carbon fatty acids in MGD due to a change in the relative contribution of the prokaryotic and eukaryotic pathways of lipid biosynthesis. This appears to be a regulated response to the loss of chloroplast ω9 desaturase and presumably reflects a requirement for polyunsaturated fatty acids for the normal assembly of chloroplast membranes. The reduction in mass of prokaryotic MGD species involved both a reduction in synthesis of MGD by the prokaryotic pathway and increased turnover of MGD molecular species which contain 16:0.     

Sphingolipid Metabolic Disorders in Trembler Mouse Peripheral Nerves In Vivo Result from an Abnormal Substrate Supply

Abstract: Sphingolipid metabolic pathways in the peripheral nerves of dysmyelinating Trembler mice were studied in vivo, using intraneurally injected [³H]palmitate as the exogenous substrate. The kinetic analysis of the experimental data obtained for the mutant revealed that, as in normal nerves, two metabolically and kinetically independent pathways are implicated in the biosynthesis of the major peripheral nerve sphingolipids: the ceramide pathway and another pathway in which there is no detectable labeled intermediate ("direct amidification"). The results also show that, in the Trembler mouse sciatic nerves: (a) The severely deficient sphingolipid biosynthesis results from the constitution of a qualitatively and quantitatively abnormal fatty acid substrate pool destined for metabolism via the ceramide pathway, which ensures the totality of the galactocerebroside labeling and two-thirds of that of sphingomyelin. The ceramide intermediates of this pathway are labeled only on their fatty acyl moiety, which contains only 16-carbon atom chains. (b) "Direct amidification" events implicated in sphingolipid labeling are decreased compared with normal and account for the remaining sphingomyelin formation.     

The Pseudomonas aeruginosa rhlG Gene Encodes an NADPH-Dependent β-Ketoacyl Reductase Which Is Specifically Involved in Rhamnolipid Synthesis

A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-β-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the rhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent β-ketoacyl reductase absolutely required for the synthesis of the β-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.     

Metabolism of saturated fatty acids by Paramecium tetraurelia

Paramecium requires oleate for growth. The phospholipids of the ciliate contain high concentrations of palmitate and 18- and 20-carbon unsaturated fatty acids. We previously showed that radiolabeled oleate is desaturated and elongated to provide these 18- and 20-carbon unsaturated acids. We now report on saturated fatty acid (SFA) metabolism in Paramecium. Radiolabeled palmitate and stearate were incorporated directly into cellular phospholipids with little or no desaturation and/or elongation. Radiolabeled acetate, malonate, pyruvate, citrate, or glucose added to cultures were not incorporated into cellular phospholipid fatty acids indicating that these exogenously supplied putative precursors were not utilized for fatty acid synthesis by Paramecium. Radiolabel from octanoate or hexanoate appeared in fatty acyl groups of phospholipids, possibly by partial beta-oxidation and reincorporation of the label. Under oleate-free conditions in which cultures do not grow, radiolabel from these shorter chain SFA were beta-oxidized and preferentially used for the formation of arachidonate, the major end-product of fatty acid synthesis in Paramecium. Cerulenin inhibited culture growth apparently by inhibiting de novo fatty acid synthesis. Cerulenin-treated cells did not incorporate radioactivity from [1-14C]octanoate into esterified palmitate. However, total saponifiable phospholipid fatty acids, including SFA, per cell increased under these conditions.     

Characterization of the Plasmodium falciparum and P. berghei glycerol 3‐phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti‐malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3‐phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N‐terminal targeting sequence to GFP and 3′ tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site‐directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3‐phosphate acyltransferase in malaria parasites.     

The Pseudomonas aeruginosa RhlA enzyme is involved in rhamnolipid and polyhydroxyalkanoate production

Pseudomonas aeruginosa produces the biosurfactant rhamnolipid, which has several potential biotechnological applications. The synthesis of this surfactant is catalyzed by rhamnosyltransferase 1, composed of the proteins RhlA and RhlB. Here we report that RhlA plays a role not only in surfactant synthesis, but also in the production of polyhydroxyalkanoates, polymers that can be used for the synthesis of biodegradable plastics.     

Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB

Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two beta-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.     

细菌3-酮脂酰ACP还原酶研究进展

3-酮脂酰ACP还原酶(FabG)在细菌中广泛存在并且十分保守,已经发现的所有FabG及其同系物都具有类似的催化活性中心序列,隶属于短链醇脱氢酶/还原酶(SDRs)超家族成员。它是Ⅱ型脂肪酸合成反应中的关键酶,将3-酮脂酰ACP还原为3-羟脂酰ACP多以NADPH作为辅酶。从搜集的文献来看,国内外针对不同细菌中3-酮脂酰ACP还原酶同系物的研究报道体现了其多样性的特点。但是,近年来,该方面的专题综述十分少见。本文主要对3-酮脂酰ACP还原酶的结构特征、在脂肪酸合成和其他方面的生物学功能,以及以该酶为作用靶点的抑菌剂等方面进行概述,以期为将来3-酮脂酰ACP还原酶的深入研究提供理论参考。     

The initial step of the glycerolipid pathway: identification of glycerol 3-phosphate/dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae.

The initial step of phospholipid biosynthesis in yeast is carried out through the acylation of glycerol 3-phosphate (G-3-P) and dihydroxyacetone phosphate by stereospecific sn-1 acyltransferases. Here we report the identification of two key fatty acyltransferases of the glycerolipid biosynthesis pathway in Saccharomyces cerevisiae. Disruption of the open reading frame YBL011w, corresponding to a gene previously identified as a choline transporter suppressor (SCT1), resulted in a substantial decrease of total cellular G-3-P acyltransferase activity. A yeast strain disrupted at the open reading frame YKR067w, which encodes a protein closely related to Sct1p, also exhibited a dramatic reduction in G-3-P acyltransferase activity. Molecular characterizations of the genes revealed that a missense mutation in YKR067w accounted for a defect in the activities of the G-3-P acyltransferase in the yeast mutant strain TTA1. Heterologous expression of YKR067w in Escherichia coli further confirmed its enzyme activity. These results indicate that YKR067w and YBL011w, designated herein as GAT1 and GAT2(SCT1), respectively, are yeast G-3-P acyltransferase genes. Furthermore, biochemical results are presented to show that both Gat1p and Gat2p(Sct1p) are G-3-P/dihydroxyacetone phosphate dual substrate-specific sn-1 acyltransferases. The fatty acyl specificity of Gat1p is similar to that of the mammalian microsomal G-3-P acyltransferase, as it can effectively utilize a broad range of fatty acids as acyl donors. In contrast, Gat2p(Sct1p) displayed preference toward 16-carbon fatty acids. The most notable of the altered phospholipid compositions of the gat1Delta and gat2(sct1)Delta strains are a decreased phosphatidic acid pool and an increased phosphatidylserine/phosphatidylinositol ratio. This did not appear to affect the mutants as no growth defect was found. However, null mutations of both GAT1 and GAT2(SCT1) are synthetically lethal to yeast.     

Acyl-sulfamates target the essential glycerol-phosphate acyltransferase (PlsY) in Gram-positive bacteria

PlsY is the essential first step in membrane phospholipid synthesis of Gram-positive pathogens. PlsY catalyzes the transfer of the fatty acid from acyl-phosphate to the 1-position of glycerol-3-phosphate to form the first intermediate in membrane biogenesis. A series of non-metabolizable, acyl-sulfamate analogs of the acyl-phosphate PlsY substrate were prepared and evaluated as inhibitors of Staphylococcus aureus PlsY and for their Gram-positive antibacterial activities. From this series phenyl (8-phenyloctanoyl) sulfamate had the best overall profile, selectively inhibiting S. aureus phospholipid biosynthesis and causing the accumulation of both long-chain fatty acids and acyl-acyl carrier protein intermediates demonstrating that PlsY was the primary cellular target. Bacillus anthracis was unique in being more potently inhibited by long chain acyl-sulfamates than other bacterial species. However, it is shown that Bacillus anthracis PlsY is not more sensitive to the acyl-sulfamates than S. aureus PlsY. Metabolic profiling showed that B. anthracis growth inhibition by the acyl-sulfamates was not specific for lipid synthesis illustrating that the amphipathic acyl-sulfamates can also have off-target effects in Gram-positive bacteria. Nonetheless, this study further advances PlsY as a druggable target for the development of novel antibacterial therapeutics, through the discovery and validation of the probe compound phenyl (8-phenyloctanoyl) sulfamate as a S. aureus PlsY inhibitor.     

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C20 Fatty Acids to C60-to-C90 Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C₂₀ fatty acids to form C₆₀-to-C₉₀ mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Δ cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Δ cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C₄ to C₈) than was previously thought (>C₁₂). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.