Annaïk Quémard

The missing piece of the type II fatty acid synthase system from Mycobacterium tuberculosis

The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3R)-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis, as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (≥C12) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3R)-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae, and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3R)-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs.

Isoniazid inhibition of mycolic acid synthesis by cell extracts of sensitive and resistant strains of Mycobacterium aurum.

Isonicotinic acid hydrazide (isoniazid; INH) inhibition of mycolic acid synthesis was studied by using cell extracts from both INH-sensitive and -resistant strains of Mycobacterium aurum. The cell extract of the INH-sensitive strain was inhibited by INH, while the preparation from the INH-resistant strain was not. This showed that the INH resistance of mycolic acid synthesis was not due to a difference in drug uptake or the level of peroxidase activity (similar in both extracts). As INH did not induce accumulation of any labeled intermediates, it is postulated that the drug acts either on production of labeled chain elongation precursors of mycolic acids or an early step of this elongation. The level of inhibition was not changed by addition of NAD or nicotinamide; thus, INH does not act on mycolic acid synthesis as an NAD antimetabolite. Benzoic or acetic acid hydrazides and known or postulated metabolites of INH (i.e., the corresponding acid, aldehyde, or alcohol) were not inhibitors of cell-free mycolic acid synthesis; the complete structure of INH was required, as already known for inhibition of mycobacterial culture growth. Extracts prepared from INH-treated cells showed reduced mycolic acid synthesis, and the inhibition level was not modified by either extensive dialysis or pyridoxal phosphate. This latter molecule efficiently antagonized INH action by reacting rapidly with INH, as shown by differential spectroscopy. Moreover, pyridoxal phosphate did not release inhibition of INH-treated extracts. It is proposed that INH may covalently react with an essential component of the mycolic acid synthesis system.

The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACPPks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACPPks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce α-alkyl β-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.

Mn(III) Pyrophosphate as an Efficient Tool for Studying the Mode of Action of Isoniazid on the InhA Protein of Mycobacterium tuberculosis

ABSTRACT The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III) pyrophosphate. In the presence of nicotinamide coenzymes (NAD+, NADH, nicotinamide mononucleotide [NMN+]) and nicotinic acid adenine dinucleotide (DNAD+), INH oxidation produced the formation of INH-coenzyme adducts in addition to known biologically inactive products (isonicotinic acid, isonicotinamide, and isonicotinaldehyde). A pool of INH-NAD(H) adducts preformed in solution allowed the rapid and strong inhibition of in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids: the inhibition was 90 or 60% when the adducts were formed in the presence of NAD+ or NADH, respectively. Under similar conditions, no inhibitory activity of INH-NMN(H) and INH-DNAD(H) adducts was detected. When an isolated pool of 100 nM INH-NAD(H) adducts was first incubated with InhA, the enzyme activity was inhibited by 80%; when present in excess, both NADH and decenoyl-coenzyme A are able to prevent this phenomenon. InhA inhibition by several types of INH-coenzyme adducts coexisting in solution is discussed in relation with the structure of the coenzyme, the stereochemistry of the adducts, and their existence as both open and cyclic forms. Thus, manganese(III) pyrophosphate appears to be an efficient and convenient alternative oxidant to mimic the activity of the Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations of reactive INH species in order to promote the design of new inhibitors of InhA as potential antituberculous 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 Å. Thestructuresshowtheimportantroleplayedbythemodificationsfound within mycolic acid SAM-MTs, especially theα2-α3 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.

A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone

Background: The anti-TB prodrugs isoxyl (ISO) and thiacetazone (TAC) inhibit mycolic acid biosynthesis. Results: We show that ISO and TAC both target the dehydration step of the FAS-II elongation system. Conclusion: ISO and TAC share the same mode of action. Significance: ISO and TAC are the first antibiotics reported to target the FAS-II dehydratase(s) of Mycobacterium tuberculosis. Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C18, C20, and C22 fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.

The Dual Function of the Mycobacterium tuberculosis FadD32 Required for Mycolic Acid Biosynthesis

Mycolic acids are major and specific lipids of Mycobacterium tuberculosis cell envelope. Their synthesis requires the condensation by Pks13 of a C(22)-C(26) fatty acid with the C(50)-C(60) meromycolic acid activated by FadD32, a fatty acyl-AMP ligase essential for mycobacterial growth. A combination of biochemical and enzymatic approaches demonstrated that FadD32 exhibits substrate specificity for relatively long-chain fatty acids. More importantly, FadD32 catalyzes the transfer of the synthesized acyl-adenylate onto specific thioester acceptors, thus revealing the protein acyl-ACP ligase function. Therefore, FadD32 might be the prototype of a group of M. tuberculosis polyketide-synthase-associated adenylation enzymes possessing such activity. A substrate analog of FadD32 inhibited not only the enzyme activity but also mycolic acid synthesis and mycobacterial growth, opening an avenue for the development of novel antimycobacterial agents.

Mycolic acid synthesis: a target for ethionamide in mycobacteria?

Striking structural analogies exist between the two specific antimycobacterial drugs ethionamide (ETH) and isoniazid (INH), and they share several inhibitory properties in susceptible species of mycobacteria. The effect of ETH on mycolic acid synthesis was studied in whole cells and in cell extracts of various species, since this synthesis is one direct target for INH, as we recently demonstrated in cell extracts of Mycobacterium aurum. It was shown in the present study that there is not a direct relationship between ETH susceptibility and mycolic acid inhibition. This observation could explain the lack of cross-resistance between the two drugs. The presence of ETH disturbed mycolic acid synthesis in both resistant and susceptible mycobacteria. Synthesis of oxygenated species of mycolic acid was inhibited, while that of diunsaturated acids was either slightly altered or even increased. In contrast, INH inhibited the synthesis of all kinds of mycolic acids in the same way in all susceptible strains and had no effect on mycolic acid synthesis in resistant strains. In the presence of ETH, the unsaturated mycolic acid molecules presented a methyl end different from the usual one. These data strongly suggest that the normal unsaturated mycolic acid species are not the precursors of the oxygenated types. Moreover, they show that ETH probably acts early in the pathway leading to oxygenated mycolic acid.

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition.

Development of isoniazid-NAD truncated adducts embedding a lipophilic fragment as potential bi-substrate InhA inhibitors and antimycobacterial agents.

Isoniazid-NAD truncated adducts embedding a lipophilic fragment were designed, synthesized and evaluated as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (InhA) of Mycobacterium tuberculosis and as antimycobacterial agents. These compounds, planned as bi-substrate inhibitors and inspired from the active metabolite of isoniazid, combine both the nicotinamide moiety of the cofactor NAD and a lipophilic hydrocarbon chain mimic of the InhA substrate. The lipophilic fragment was introduced using either Suzuki-Miyaura cross-coupling or a classical nucleophilic substitution reaction. Several compounds developed in this work were indeed able to inhibit the InhA activity and showed promising antimycobacterial activities. However a direct correlation between the expressed activity and the bi-substrate mode of action could not yet be unambiguously demonstrated.