Isioniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: Catalase, peroxidase, and INH-NADH adduct formation activities
Isioniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: Catalase, peroxidase, and INH-NADH adduct formation activities
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DOI：
10.1002/pro.324
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发表时间：
2010-03-01
期刊：
影响因子：
8
通讯作者：
Ghiladi, Reza A.
中科院分区：
文献类型：
--
作者：
Cade, Christine E.;Dlouhy, Adrienne C.;Ghiladi, Reza A.
结核分枝杆菌过氧化氢酶-过氧化物酶(KatG)是一种双功能的血红素蛋白，已被证明可以激活异烟肼(INH)，异烟肼是一线抗结核治疗不可或缺的前体药物。被激活的物种被认为是异烟酰基，与NAD(+)/NADH偶联，形成异烟肼-NADH加合物，最终具有抗结核活性。为了更好地了解异烟肼激活的机制以及KatG衍生的异烟肼耐药的起源，我们比较了野生型酶的催化特性(包括形成INH-NADH加合物的能力)和23个与结核分枝杆菌临床分离株的异烟肼耐药相关的KatG突变体。没有发现过氧化氢酶和过氧化物酶活性，这两种固有的酶功能与异烟肼耐药性有关。此外，在缺乏Met-Tyr-Trp交联物的突变体中，过氧化氢酶功能丧失。Met-Tyr-Trp交联物是KatG中的生物辅因子，以前已被证明是该活性所必需的。然而，存在或不存在交联物本身也被发现与异烟肼抵抗无关。然后，对与KatG抗性相关的突变体在有过氧化氢(t-BuOOH和过氧化氢超氧化物，没有外源氧化剂)的情况下(仅空气背景对照)产生INH-NADH加合物的能力进行了检测。结果表明，残基位置在确定与异烟肼激活相关的异烟肼抗性机制中起着关键作用；然而，同一位置上的不同突变可以产生非常不同的氧化剂特异性反应活性。此外，这些数据可以解释为存在与异烟肼-NADH加合物的形成无关的第二种异烟肼抵抗机制。
Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH) a pro-drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD(+)/NADH forming an isoniazid-NADH adduct that ultimately confers anti-tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG-derived INH-resistance, we have compared the catalytic properties (including the ability to form the INH-NADH adduct) of the wild-type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis, isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met-Tyr-Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance-conferring mutants were then assayed for their ability to generate the INH-NADH adduct in the presence of peroxide (t-BuOOH and H2O2 superoxide, and no exogenous oxidant (air-only background control). The results demonstrate that residue location plays a critical role in determining INH-resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant-specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH-resistance that is not correlated with the formation of the INH-NADH adduct.