Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, USA.
Protein Sci. 2010 Mar;19(3):458-74. doi: 10.1002/pro.324.
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 H(2)O(2)), 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.
结核分枝杆菌过氧化氢酶-过氧化物酶（KatG）是一种双功能血红素蛋白，已被证明能激活异烟肼（INH），这是一线抗结核治疗的关键药物。该激活物质，被认为是异烟酸自由基，与 NAD（+）/NADH 偶联形成异烟肼-NADH 加合物，最终赋予抗结核活性。为了更好地理解异烟肼的激活机制以及 KatG 衍生的 INH 耐药性的起源，我们比较了野生型酶与 23 种与临床结核分枝杆菌分离株中异烟肼耐药相关的 KatG 突变体的催化特性（包括形成 INH-NADH 加合物的能力）。既没有发现过氧化氢酶或过氧化物酶活性（KatG 的两种固有酶功能）与异烟肼耐药性相关。此外，在缺乏 Met-Tyr-Trp 交联的突变体中失去了过氧化氢酶功能，该交联是 KatG 的生物生成辅因子，先前已证明该交联对于该活性是必不可少的。然而，交联本身的存在与否也被发现与 INH 耐药性无关。然后，在存在过氧化物（叔丁基过氧化物和 H2O2）、超氧化物和没有外源氧化剂（仅空气背景对照）的情况下，对 KatG 耐药性赋予突变体进行了生成 INH-NADH 加合物的能力测定。结果表明，残基位置在决定与 INH 激活相关的 INH 耐药机制方面起着关键作用；然而，同一位置的不同突变可以产生截然不同的反应性，这些反应性是特定于氧化剂的。此外，数据可以解释为存在第二种与 INH-NADH 加合物形成无关的 INH 耐药机制。
