Didier Zerbib

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.

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.

In Vitro Inhibition of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA by Isoniazid

ABSTRACT The first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present work shows that the NADPH-dependent β-ketoacyl reduction activity of MabA is efficiently inhibited by isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the formation of a covalent adduct between MnIII-activated isoniazid and the MabA cofactor. Liquid chromatography-mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand complex strongly suggested that the molecules active against MabA activity are the oxidized derivative and a major cyclic form. Spectrofluorimetry showed that the adduct binds to the MabA active site. Modeling of the MabA-adduct complex predicted an interaction between the isonicotinoyl moiety of the inhibitor and Tyr185. This hypothesis was supported by the fact that a higher 50% inhibitory concentration of the adduct was measured for MabA Y185L than for the wild-type enzyme, while both proteins presented similar affinities for NADP+. The crystal structure of MabA Y185L that was solved showed that the substitution of Tyr185 induced no significant conformational change. The description of the first inhibitor of the β-ketoacyl reduction step of fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrug-resistant tubercle bacilli.

The regulatory role of the IS 1‐encoded InsA protein in transposition

We show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1‐mediated cointegrate formation and transposition of a synthetic IS 1‐based transposon (‘omegon’Ω‐on). These results also indicate that the Ω‐on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA‐lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1‐encoded genes both in cis or in trans. The experiments involving Ω‐on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.

Functional organization of the ends of IS 1: specific binding site for an IS1‐encoded protein

The IS 1‐encoded protein InsA binds specifically to both ends of IS 1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel ‘foot‐printing’ and gel retardation, that the InsA binding sites are located within the 24/25bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS 1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This ‘architecture’ of the ends of IS 1 is remarkably similar to that of IS elements IS10, IS50 and IS903.

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.

Knr4: a disordered hub protein at the heart of fungal cell wall signalling

The most highly connected proteins in protein–protein interactions networks are called hubs; they generally connect signalling pathways. In Saccharomyces cerevisiae, Knr4 constitutes a connecting node between the two main signal transmission pathways involved in cell wall maintenance upon stress: the cell wall integrity and the calcium–calcineurin pathway. Knr4 is required to enable the cells to resist many cell wall‐affecting stresses, and KNR4 gene deletion is synthetic lethal with the simultaneous deletion of numerous other genes involved in morphogenesis and cell wall biogenesis. Knr4 has been shown to engage in multiple physical interactions, an ability conferred by the intrinsic structural adaptability of major disordered regions present in the N‐terminal and C‐terminal parts of the protein. Taking all together, Knr4 is an intrinsically disordered hub protein. Available data from other fungi indicate the conservation of Knr4 homologs cellular function and localization at sites of polarized growth among fungal species, including pathogenic species. Because of their particular role in morphogenesis control and of their fungal specificity, these proteins could constitute interesting new pharmaceutical drug targets for antifungal combination therapy.