Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase (KatG) and its S315T mutant
Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase (KatG) and its S315T mutant
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DOI：
10.1021/bi051967o
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发表时间：
2006-04-04
期刊：
影响因子：
2.9
通讯作者：
Magliozzo, RS
中科院分区：
文献类型：
--
作者：
Zhao, XB;Yu, H;Magliozzo, RS
由于异烟肼 (INH) 和烟酰胺腺嘌呤二核苷酸辅因子形成异烟酰-NAD 加合物 (IN-NAD)，对结核分枝杆菌 InhA（烯酰基载体蛋白还原酶）的抑制被认为是 INH 作用模式的核心，INH 是结核感染的一线治疗方法。 INH 对分枝杆菌的作用需要过氧化氢酶-过氧化物酶 (KatG) 功能，而 IN-NAD 加合物的形成是由结核分枝杆菌 KatG 在各种条件下在体外催化的，但尚未出现与该过程相关的生理学方法，以便对最常见的携带 KatG 的耐药菌株中 INH 耐药的机制和起源进行审查[S315T]。在本报告中，我们描述了以极低浓度向 KatG 铁传递的过氧化氢如何通过形成 IN-NAD 加合物而有效抑制 InhA。在最佳条件下（H2O2 与 NAD(+) 和 INH 一起提供），野生型 KatG 介导的加合物形成速率比异烟肼抗性 KatG[S315T] 突变体介导的加合物形成速率高约 20 倍。即使在不添加过氧化物的情况下，在 KatG 存在的情况下，从 NADH 和 INH 开始也会发生缓慢的加合物形成，这是内源性过氧化物的线索。 KatG[S315T]突变体的低效率可以仅通过增加INH的浓度来增强，这与该酶对INH与静息酶和具有催化能力的酶中间体（化合物I）结合的亲和力降低一致。通过检查突变酶的三维X射线晶体结构，在结构水平上分析KatG[S315T]突变酶的耐药性起源。
Inhibition of the enzyme Mycobacterium tuberculosis InhA (enoyl-acyl carrier protein reductase) due to formation of an isonicotinoyl-NAD adduct (IN-NAD) from isoniazid (INH) and nicotinamide adenine dinucleotide cofactor is considered central to the mode of action of INH, a first-line treatment for tuberculosis infection. INH action against mycobacteria requires catalase-peroxidase (KatG) function, and IN-NAD adduct formation is catalyzed in vitro by M. tuberculosis KatG under a variety of conditions, yet a physiologically relevant approach to the process has not emerged that allows scrutiny of the mechanism and the origins of INH resistance in the most prevalent drug-resistant strain bearing KatG[S315T]. In this report, we describe how hydrogen peroxide, delivered at very low concentrations to ferric KatG, leads to efficient inhibition of InhA due to formation of the IN-NAD adduct. The rate of adduct formation mediated by wild-type KatG was about 20-fold greater than by the isoniazid-resistant KatG[S315T] mutant under optimal conditions (H2O2 Supplied along with NAD(+) and INH). Slow adduct formation also occurs starting with NADH and INH, in the presence of KatG even in the absence of added peroxide, clue to endogenous peroxide. The poor efficiency of the KatG[S315T] mutant can be enhanced merely by increasing the concentration of INH, consistent with this enzyme's reduced affinity for INH binding to the resting enzyme and the catalytically competent enzyme intermediate (Compound I). Origins of drug resistance in the KatG[S315T] mutant enzyme are analyzed at the structural level through examination of the three-dimensional X-ray crystal Structure of the mutant enzyme.