Ducasse-Cabanot Stéphanie, Cohen-Gonsaud Martin, Marrakchi Hedia, Nguyen Michel, Zerbib Didier, Bernadou Jean, Daffé Mamadou, Labesse Gilles, Quémard Annaïk
Institut de Pharmacologie et de Biologie Structurale, Département des Mécanismes Moléculaires des Infections Mycobactériennes, CNRS, Toulouse, France.
Antimicrob Agents Chemother. 2004 Jan;48(1):242-9. doi: 10.1128/AAC.48.1.242-249.2004.
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 beta-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 Mn(III)-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 beta-ketoacyl reduction step of fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrug-resistant tubercle bacilli.
一线抗结核特效药异烟肼可抑制参与分枝菌酸生物合成的脂肪酸延伸系统（FAS）FAS-II，分枝菌酸是结核分枝杆菌包膜的主要脂质。催化FAS-II延伸循环第二步的MabA蛋白在结构和功能上与异烟肼的体内靶点InhA相关，InhA是一种依赖NADH的烯酰-酰基载体蛋白还原酶。目前的研究表明，异烟肼在体外可通过与抑制InhA活性类似的机制有效抑制MabA的NADPH依赖性β-酮酰基还原活性。这涉及到Mn(III)激活的异烟肼与MabA辅因子之间形成共价加合物。液相色谱-质谱分析表明，异烟酰-NADP加合物具有多种处于动态平衡的化学形式。对分离形式的动力学实验以及酶-配体复合物的纯化均有力地表明，对MabA活性有活性的分子是氧化衍生物和一种主要的环状形式。荧光光谱法表明，该加合物与MabA活性位点结合。MabA-加合物复合物的建模预测了抑制剂的异烟酰部分与Tyr185之间的相互作用。这一假设得到以下事实的支持：与野生型酶相比，MabA Y185L对加合物的50%抑制浓度更高，而两种蛋白对NADP(+)的亲和力相似。解析得到的MabA Y185L晶体结构表明，Tyr185的取代未引起显著的构象变化。对脂肪酸生物合成β-酮酰基还原步骤的首个抑制剂的描述应有助于设计对耐多药结核杆菌有效的新型抗结核药物。
