Mechanistic studies of alkyl hydroperoxide reductase

细菌的烷基过氧化氢还原酶系统用于保护免受氧化应激的毒性和诱变效应。AhpC是一种以半胱氨酸为基础的过氧化物酶，是普遍存在的“过氧化还蛋白”(PRX)家族的一员，它通过在酶上瞬时生成半胱氨酸亚磺酸并随后形成亚基间二硫键来还原过氧化氢和有机过氧化氢。AhpF是含有黄素的还原酶组分，存在于大多数细菌中，并能有效地将电子从NADH(或NADPH)传递到AhpC。哺乳动物的Prxs参与了细胞增殖和分化、免疫反应和细胞信号转导等多种过程。虽然大多数AhpC/Prx同源物都高度表达，并在氧化防御中发挥重要作用，但只有幽门螺杆菌(与胃癌有关的胃溃疡的病原体)的AhpC是已知的对该微生物的生存绝对必需的。该提案的第一个具体目标集中在(1)在过氧化氢存在下，鼠伤寒沙门氏菌AhpC和哺乳动物Prx II在周转过程中的构象、寡聚和膜结合的变化，以及(2)假定的通用碱催化剂(Arg119)参与AhpC对过氧化氢的还原。第二个具体目的是探索电子进出鼠伤寒沙门氏菌AhpF的N-端二硫中心的机制。这个中心(Cys129-Cys132)是AhpF中一个独特的氧化还原域的一部分，从我们的研究中得知，它介导了蛋白质C末端部分的氧化还原中心(FAD和Cys345-Cys348)向AhpC的电子转移。我们最近对AhpF的结晶学分析表明，N-末端结构域(NTD)和来自嗜热菌的蛋白质二硫键氧化还原酶(PDO)具有独特的结构；NTD和PDO都由两个密切相关的硫氧还蛋白样折叠组成，推测其活性部位为上半段的谷氨酸，在该结构域的后半部分的Cys-X-X-Cys基序上充当化学的一般酸碱催化剂。我们对AhpF的结晶学分析也有力地支持了AhpF催化循环中的大结构域运动。结晶学和荧光方法将用于第三个具体目标，以确定AhpF-AhpC相互作用的性质以及AhpF内亚单位内电子转移过程中的域间相互作用。了解细菌AhpF以及细菌和哺乳动物AhpC同源物的催化作用将有助于我们了解病原体和哺乳动物体内的氧化应激防御机制和氧化还原调节的细胞信号。在预防涉及人类退行性疾病、癌症和衰老的氧化损伤以及对抗致病防御系统方面，治疗干预需要对细菌和人类来源的烷基过氧化氢还原酶有完整的分子和生物学了解。

The bacterial alkyl hydroperoxide reductase system serves to protect against the toxic and mutagenic effects of oxidative stress. AhpC, the cysteine-based peroxidase component, is a member of the ubiquitous "peroxiredoxin" (Prx) family and reduces H2O2 and organic hydroperoxides through transient generation of a cysteine sulfenic acid on the enzyme and subsequent intersubunit disulfide bond formation. AhpF, the flavin-containing reductase component, is present in most, but not all, bacteria and efficiently transfers electrons from NADH (or NADPH) to AhpC. Mammalian Prxs have been implicated in such diverse processes as cellular proliferation and differentiation, immune responses and cell signaling. While most AhpC/Prx homologues are highly expressed and play an important role in oxidative defense, only the AhpC from Helicobacter pylori (the causative agent of gastric ulcers linked to stomach cancer) is known to be absolutely required for viability of that organism. The first specific aim of the proposal focuses on (1) the conformational states, oligomerization and membrane association thought to change during turnover of Salmonella typhimurium AhpC and mammalian Prx II in the presence of peroxides, and (2) the participation of a putative general base catalyst (Arg119) in peroxide reduction by AhpC. The second specific aim explores the mechanism of electron transfers to and from the N-terminal disulfide center of S. typhimurium AhpF. This center (Cys129- Cys132) is part of a distinct redox domain in AhpF known from our studies to mediate electron transfer from redox centers (FAD and Cys345-Cys348) in the C-terminal portion of the protein to AhpC. Our recent crystallographic analyses of AhpF have demonstrated a unique architecture for the N-terminal domain (NTD) and a poorly- characterized homologue, protein disulfide oxidoreductase (PDO), from a thermophile; both NTD and PDO are composed of two intimately-associated thioredoxin-like folds with a putative active site glutamate from the first half acting as a general acid-base catalyst for chemistry at the Cys-X-X-Cys motif of the second half of the domain. Our crystallographic analyses of AhpF also strongly support the involvement of large domain movements in the catalytic cycle of AhpF. Crystallographic and fluorescence approaches will be used in the third specific aim to define the nature of AhpF-AhpC interactions as well as inter- domain interactions within AhpF during intrasubunit electron transfer. Understanding of catalysis by bacterial AhpF and both bacterial and mammalian AhpC homologues will contribute to our knowledge of oxidative stress defense mechanisms and redox-regulated cell signaling in both pathogens and mammalian hosts. Therapeutic intervention in preventing oxidative damage involved in human degenerative diseases, cancer and aging as well as in combating pathogenic defense systems requires a complete molecular and biological understanding of the alkyl hydroperoxide reductase enzymes from both bacterial and human sources.