The Mycobacterium tuberculosis FAS-II dehydratases and methyltransferases define the specificity of the mycolic acid elongation complexes

Background

The human pathogen Mycobacterium tuberculosis (Mtb) has the originality of possessing a multifunctional mega-enzyme FAS-I (Fatty Acid Synthase-I), together with a multi-protein FAS-II system, to carry out the biosynthesis of common and of specific long chain fatty acids: the mycolic acids (MA). MA are the main constituents of the external mycomembrane that represents a tight permeability barrier involved in the pathogenicity of Mtb. The MA biosynthesis pathway is essential and contains targets for efficient antibiotics. We have demonstrated previously that proteins of FAS-II interact specifically to form specialized and interconnected complexes. This finding suggested that the organization of FAS-II resemble to the architecture of multifunctional mega-enzyme like the mammalian mFAS-I, which is devoted to the fatty acid biosynthesis.

Principal Findings

Based on conventional and reliable studies using yeast-two hybrid, yeast-three-hybrid and in vitro Co-immunoprecipitation, we completed here the analysis of the composition and architecture of the interactome between the known components of the Mtb FAS-II complexes. We showed that the recently identified dehydratases HadAB and HadBC are part of the FAS-II elongation complexes and may represent a specific link between the core of FAS-II and the condensing enzymes of the system. By testing four additional methyltransferases involved in the biosynthesis of mycolic acids, we demonstrated that they display specific interactions with each type of complexes suggesting their coordinated action during MA elongation.

Significance

These results provide a global update of the architecture and organization of a FAS-II system. The FAS-II system of Mtb is organized in specialized interconnected complexes and the specificity of each elongation complex is given by preferential interactions between condensing enzymes and dehydratase heterodimers. This study will probably allow defining essential and specific interactions that correspond to promising targets for Mtb FAS-II inhibitors.     

X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase II (mtKasB)

Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.     

A novel interaction linking the FAS-II and phthiocerol dimycocerosate (PDIM) biosynthetic pathways

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.     

The biosynthesis of mycolic acids in Mycobacterium tuberculosis relies on multiple specialized elongation complexes interconnected by specific protein-protein interactions

Tuberculosis kills about two million people every year and remains one of the leading causes of mortality worldwide. As a result of the increasing antibiotic resistance of Mycobacterium tuberculosis (Mtb) strains, there is an urgent need for new antitubercular drugs. Several efficient antibiotics, including isoniazid, specifically target the fatty acid synthase-II (FAS-II) complex of mycolic acid biosynthesis. We have previously shown that there are protein-protein interactions between the components of FAS-II that are essential for mycobacterial survival. We have now looked at the potential partners of FAS-II, mtFabD, the methyltransferases MmaAs, and Pks13. A combination of yeast two-hybrid and co-immunoprecipitation experiments showed that mtFabD interacts with each beta-ketoacyl-synthase (KasA, KasB and mtFabH) and with the core of FAS-II (InhA and MabA). The methyltransferases have a greater affinity for KasA and KasB than for mtFabH, suggesting that modifications on the meromycolic chains may occur during their elongation. Finally, Pks13, which catalyzes the final Claisen condensation of mycolic acids, interacts specifically with KasB. These data allowed us to determine the architecture of the multiple specialized FAS-II complexes, giving us insights into the organization of the complete mycolic acids biosynthesis. Our studies suggest a new and crucial interaction (KasB-Pks13) as a putative target for peptidomimetic antibiotics.     

Crystal structure of MabA from Mycobacterium tuberculosis,a reductase involved in long-chain fatty acid biosynthesis

The fatty acid elongation system FAS-II is involved in the biosynthesis of mycolic acids, which are major and specific long-chain fatty acids of the cell envelope of Mycobacterium tuberculosis and other mycobacteria, including Mycobacterium smegmatis. The protein MabA, also named FabG1, has been shown recently to be part of FAS-II and to catalyse the NADPH-specific reduction of long chain beta-ketoacyl derivatives. This activity corresponds to the second step of an FAS-II elongation round. FAS-II is inhibited by the antituberculous drug isoniazid through the inhibition of the 2-trans-enoyl-acyl carrier protein reductase InhA. Thus, the other enzymes making up this enzymatic complex represent potential targets for designing new antituberculous drugs. The crystal structure of the apo-form MabA was solved to 2.03 A resolution by molecular replacement. MabA is tetrameric and shares the conserved fold of the short-chain dehydrogenases/reductases (SDRs). However, it exhibits some significant local rearrangements of the active-site loops in the absence of a cofactor, particularly the beta5-alpha5 region carrying the unique tryptophan residue, in agreement with previous fluorescence spectroscopy data. A similar conformation has been observed in the beta-ketoacyl reductase from Escherichia coli and the distantly related dehydratase. The distinctive enzymatic and structural properties of MabA are discussed in view of its crystal structure and that of related enzymes.     

Mycobacterium tuberculosis Proteins Involved in Mycolic Acid Synthesis and Transport Localize Dynamically to the Old Growing Pole and Septum

Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles'' heel of the bacillus at all its growing stages.     

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.     

The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation

Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the beta-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages.     

Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability

Despite the existence of efficient chemotherapy, tuberculosis remains a leading cause of mortality worldwide. New drugs are urgently needed to reduce the potential impact of the emergence of multidrug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb). The front-line antibiotic isoniazid (INH), and several other drugs, target the biosynthesis of mycolic acids and especially the Fatty Acid Synthase-II (FAS-II) elongation system. This biosynthetic pathway is essential and specific for mycobacteria and still represents a valuable system for the search of new anti-tuberculous agents. Several data, in the literature, suggest the existence of protein-protein interactions within the FAS-II system. These interactions themselves might serve as targets for a new generation of drugs directed against Mtb. By using an extensive in vivo yeast two-hybrid approach and in vitro co-immunoprecipitation, we have demonstrated the existence of both homotypic and heterotypic interactions between the known components of FAS-II. The condensing enzymes KasA, KasB and mtFabH interact with each other and with the reductases MabA and InhA. Furthermore, we have designed and constructed point mutations of the FAS-II reductase MabA, able to disrupt its homotypic interactions and perturb the interaction pattern of this protein within FAS-II. Finally, we showed by a transdominant genetic approach that these mutants are dominant negative in both non-pathogenic and pathogenic mycobacteria. These data allowed us to draw a dynamic model of the organization of FAS-II. They also represent an important step towards the design of a new generation of anti-tuberculous agents, as being inhibitors of essential protein-protein interactions.     

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA,a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60–90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C_38–42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors.     

Mutations in the essential FAS II β‐hydroxyacyl ACP dehydratase complex confer resistance to thiacetazone in Mycobacterium tuberculosis and Mycobacterium kansasii

It has recently been shown that the anti‐mycobacterial pro‐drug thiacetazone (TAC) inhibits the conversion of double bonds of mycolic acid precursors into cyclopropyl rings in Mycobacterium bovis var BCG, M. marimum and M. chelonae by affecting the cyclopropyl mycolic acid synthases (CMASs) as judged by the build‐up of unsaturated mycolate precursors. In our hands, TAC inhibits mycolic acid biosynthesis in Mycobacterium tuberculosis and M. kansasii with almost negligible accumulation of those precursors. Our observations that ‘de novo’ biosynthesis of all the mycolic acid families decreased upon TAC treatment prompted us to analyse the role of each one of the Type II Fatty Acid Synthase (FASII) enzymes. Overexpression of the hadABC operon, encoding the essential FASII dehydratase complex, but not of any of the remaining FASII genes acting on the elongation of fatty acyl chains leading to the synthesis of meromycolic acids, resulted in high level of resistance to TAC in M. tuberculosis. Spontaneous M. tuberculosis and M. kansasii TAC‐resistant mutants isolated during this work revealed mutations in the hadABC genes strongly supporting our proposal that these enzymes are new players in the resistance to this anti‐mycobacterial compound.     

Inhibition of mycolic acid biosynthesis in a cell-wall preparation from Mycobacterium smegmatis by methyl 4-(2-octadecylcyclopropen-1-y1) butanoate, a structural analogue of a key precursor

[¹⁴C]acetate was incorporated into mycolic acids by a cell-free, cell-wall fraction from Mycobacterium smegmatis . This activity was inhibited by methyl 4-(2-octadecylcyclopropen-1-y1) butanoate which was designed as a structural analogue of cis -tetracos-5-enoate, a precursor of mycolic acid biosynthesis. Other fatty acids and their methyl esters failed to inhibit mycolic acid biosynthesis at the concentration 1–2 mg ml^-1, at which methyl 4-(2-octadecylcyclopropen-1-y1) butanoate was effective. Thus a novel agent was shown to act against an enzyme activity or target involved specifically in biosynthesis of a characteristic, mycobacterial, cell-wall component.     

Interpreting Expression Data with Metabolic Flux Models: Predicting Mycobacterium tuberculosis Mycolic Acid Production

Metabolism is central to cell physiology, and metabolic disturbances play a role in numerous disease states. Despite its importance, the ability to study metabolism at a global scale using genomic technologies is limited. In principle, complete genome sequences describe the range of metabolic reactions that are possible for an organism, but cannot quantitatively describe the behaviour of these reactions. We present a novel method for modeling metabolic states using whole cell measurements of gene expression. Our method, which we call E-Flux (as a combination of flux and expression), extends the technique of Flux Balance Analysis by modeling maximum flux constraints as a function of measured gene expression. In contrast to previous methods for metabolically interpreting gene expression data, E-Flux utilizes a model of the underlying metabolic network to directly predict changes in metabolic flux capacity. We applied E-Flux to Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB). Key components of mycobacterial cell walls are mycolic acids which are targets for several first-line TB drugs. We used E-Flux to predict the impact of 75 different drugs, drug combinations, and nutrient conditions on mycolic acid biosynthesis capacity in M. tuberculosis, using a public compendium of over 400 expression arrays. We tested our method using a model of mycolic acid biosynthesis as well as on a genome-scale model of M. tuberculosis metabolism. Our method correctly predicts seven of the eight known fatty acid inhibitors in this compendium and makes accurate predictions regarding the specificity of these compounds for fatty acid biosynthesis. Our method also predicts a number of additional potential modulators of TB mycolic acid biosynthesis. E-Flux thus provides a promising new approach for algorithmically predicting metabolic state from gene expression data.     

Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria

Background

Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.

Methodology/Principle Findings

We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the α- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs.

Conclusions/Significance

This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.     

Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II

Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA:AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.     

Discovery of novel bacterial elongation condensing enzyme inhibitors by virtual screening

The elongation condensing enzymes in the bacterial fatty acid biosynthesis pathway represent desirable targets for the design of novel, broad-spectrum antimicrobial agents. A series of substituted benzoxazolinones was identified in this study as a novel class of elongation condensing enzyme (FabB and FabF) inhibitors using a two-step virtual screening approach. Structure activity relationships were developed around the benzoxazolinone scaffold showing that N-substituted benzoxazolinones were most active. The benzoxazolinone scaffold has high chemical tractability making this chemotype suitable for further development of bacterial fatty acid synthesis inhibitors.     

Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate.

Enoyl-ACP reductases participate in fatty acid biosynthesis by utilizing NADH to reduce the trans double bond between positions C2 and C3 of a fatty acyl chain linked to the acyl carrier protein. The enoyl-ACP reductase from Mycobacterium tuberculosis, known as InhA, is a member of an unusual FAS-II system that prefers longer chain fatty acyl substrates for the purpose of synthesizing mycolic acids, a major component of mycobacterial cell walls. The crystal structure of InhA in complex with NAD+ and a C16 fatty acyl substrate, trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester, reveals that the substrate binds in a general "U-shaped" conformation, with the trans double bond positioned directly adjacent to the nicotinamide ring of NAD+. The side chain of Tyr158 directly interacts with the thioester carbonyl oxygen of the C16 fatty acyl substrate and therefore could help stabilize the enolate intermediate, proposed to form during substrate catalysis. Hydrophobic residues, primarily from the substrate binding loop (residues 196-219), engulf the fatty acyl chain portion of the substrate. The substrate binding loop of InhA is longer than that of other enoyl-ACP reductases and creates a deeper substrate binding crevice, consistent with the ability of InhA to recognize longer chain fatty acyl substrates.     

Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis

Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs.     

Further insight into S-adenosylmethionine-dependent methyltransferases: structural characterization of Hma, an enzyme essential for the biosynthesis of oxygenated mycolic acids in Mycobacterium tuberculosis

Mycolic acids are major and specific components of the cell envelope of Mycobacteria that include Mycobacterium tuberculosis, the causative agent of tuberculosis. Their metabolism is the target of the most efficient antitubercular drug currently used in therapy, and the enzymes that are involved in the production of mycolic acids represent important targets for the development of new drugs effective against multidrug-resistant strains. Among these are the S-adenosylmethionine-dependent methyltransferases (SAM-MTs) that catalyze the introduction of key chemical modifications in defined positions of mycolic acids. Some of these subtle structural variations are known to be crucial for both the virulence of the tubercle bacillus and the permeability of the mycobacterial cell envelope. We report here the structural characterization of the enzyme Hma (MmaA4), a SAM-MT that is unique in catalyzing the introduction of a methyl branch together with an adjacent hydroxyl group essential for the formation of both keto- and methoxymycolates in M. tuberculosis. Despite the high propensity of Hma to proteolytic degradation, the enzyme was produced and crystallized, and its three-dimensional structure in the apoform and in complex with S-adenosylmethionine was solved to about 2 A. Thestructuresshowtheimportantroleplayedbythemodificationsfound within mycolic acid SAM-MTs, especially thealpha2-alpha3 motif and the chemical environment of the active site. Essential information with respect to cofactor and substrate binding, selectivity and specificity, and about the mechanism of catalytic reaction were derived.     

Trafficking pathways of mycolic acids: structures, origin, mechanism of formation, and storage form of mycobacteric acids

Mycolic acids, the hallmark of mycobacteria and related bacteria, are major and specific components of their cell envelope and essential for the mycobacterial survival. Mycobacteria contain structurally related long-chain lipids, but the metabolic relationships between these various classes of compounds remain obscure. To address this question a series of C(35) to C(54) nonhydroxylated fatty acids (mycobacteric acids), ketones, and alcohols structurally related to the C(70-80) dicyclopropanated or diethylenic mycolic acids were characterized in three mycobacterial strains and their structures compared. The relationships between these long-chain acids and mycolic acids were established by following the in vivo traffic of (14)C labeled alpha-mycolic acids purified from the same mycobacterial species. The labeling was exclusively found in mycobacteric acids. The mechanism of this degradation was established by incorporation of (18)O(2) into long-chain lipids and shown to consist in the rupture of mycolic acids between carbon 3 and 4 by a Baeyer-Villiger-like reaction. We also demonstrated that mycobacteric acids occur exclusively in the triacylglycerol (TAG) fraction where one molecule of these acids esterifies one of the three hydroxyl groups of glycerol. Altogether, these data suggest that these compounds represent a pathway of metabolic energy that would be used by mycobacteria in particular circumstances.