OXYGEN REGULATION OF NADH OXIDASE EXPRESSION IN STREPTOCOCCI

我们先前已经发现，在耐氧变形链球菌中，通过氧气诱导产生了两种不同的NADH氧化酶，分别对应于H_2O_2形成氧化酶(Nox-1)和H_2O形成氧化酶(Nox-2)。此外，序列搜索表明，NOx-1蛋白是鼠伤寒沙门氏菌AhpF的同源物，该蛋白是烷基过氧化氢还原酶AhpR的黄素蛋白组分，参与防御氧化应激，并提示NOx-1基因上游存在ahpC同源物，与AhpR的非黄蛋白成分鼠伤寒沙门氏菌AhpC存在同源性。证明了NOx-1和AhpC共同催化NADH依赖的有机过氧化氢或H_2O_2还原为各自的醇和/或H_2O，以及NOx-2对O_2的四电子还原，并提出变形链球菌的NOx-2含有一个半胱氨酸基的氧化还原中心；Nox-2蛋白的Cys44在O_2四电子还原为H_2O的整个过程中起着关键作用，它证明了用Ser取代Cys44提供了一种改变的O_2还原化学计量比，其中H_2O而不是2H_2O是产物。此外，通过对Nox-1、Nox-2和AhpC基因敲除突变体的分析，我们证实了Nox-1、Nox-2和AhpC蛋白是受氧诱导的，并首次提出证据表明，Nox-2通过NAD的再生在有氧能量代谢中发挥重要作用，而Nox-1的作用可以忽略不计。我们还证明了Nox-1在体内作为AhpR系统的一部分与AhpC结合的明显功能。

We have previously revealed that two distinct NADH oxidases corresponding to H_2O_2-forming oxidase (Nox-1) and H_2O-forming oxidase (Nox-2) were induced in an oxygen tolerant Streptococcus mutans by exposure to oxygen. Furthermore, sequence searches indicated that the Nox-1 protein is homologue of Salmonella typhimurium AhpF, the flavoprotein component of alkyl hydroperoxide reductase AhpR involved in defense against oxidative stress, and suggested the presence of ahpC homologue upstream of nox-1 gene with S.typhimurium AhpC, the non-flavopretein component of AhpR.In this study, we determined the complete sequence of the S.mutans ahpC comprised of 558 bp encoding AhpC of 186 amino acid residues, confirmed the identity of these proteins as an AhpR system in S.mutans, demonstrated that together the Nox-1 and AhpC catalyzes the NADH-dependent reduction of organic hydroperoxides or H_2O_2 to their respective alcohols and/or H_2O, as well as the four-electron reduction of 0_2 by Nox-2, and proposed that Nox-2 from S.mutans contains a cysteinyl redox center ; the Cys44 of Nox-2 protein plays a key role in the overall four-electron reduction of O_2 to H_2O, by demonstrating that replacement of Cys44 with Ser provides for an altered O_2 reduction stoichiometry in which H_2O_2, not 2H_2O is the product. Furthermore, we confirmed that these proteins of Nox-1, Nox-2, and AhpC are induced by oxygen, and presented the first evidence that Nox-2 plays an important role in aerobic energy metabolism through the regeneration of NAD, but Nox-1 contributes negligibly, by analyses of knockout mutants of Nox-1, Nox-2, and/or AhpC.We also demonstrated a clear function for Nox-1 as part of an AhpR system in vivo in combination with AhpC.