Mechanistic studies of alkyl hydroperoxide reductase

细菌烷基氢过氧化物还原酶系统可防止氧化应激的毒性和诱变作用。AhpC是基于半胱氨酸的过氧化物酶组分，是普遍存在的“过氧化物酶”（Prx）家族的一员，通过在酶上瞬时生成半胱氨酸磺酸和随后形成亚基间二硫键来还原H2O2和有机氢过氧化物。AhpF是一种含有黄素的还原酶成分，存在于大多数细菌中，但不是所有细菌中，并能有效地将电子从NADH（或NADPH）转移到AhpC。哺乳动物Prxs涉及细胞增殖和分化、免疫反应和细胞信号传导等多种过程。虽然大多数AhpC/Prx同源物都是高表达的，并在氧化防御中发挥重要作用，但已知只有幽门螺杆菌（与胃癌相关的胃溃疡的病原体）的AhpC是该生物体生存所绝对需要的。该提案的第一个具体目标侧重于(1)在过氧化物存在下鼠伤寒沙门氏菌AhpC和哺乳动物Prx II的转化过程中被认为会改变的构象状态、寡聚和膜结合，以及(2)一种假定的通用碱催化剂（Arg119）参与AhpC的过氧化物还原。第二个具体目的是探讨鼠伤寒沙门氏菌AhpF的n端二硫中心和n端二硫中心之间电子转移的机制。该中心（Cys129- Cys132）是AhpF中一个独特的氧化还原结构域的一部分，从我们的研究中得知，该结构域介导电子从蛋白质c端部分的氧化还原中心（FAD和Cys345-Cys348）转移到AhpC。我们最近对AhpF的晶体学分析已经证明了n端结构域（NTD）的独特结构和一个来自嗜热菌的同源物，蛋白质二硫氧化物还原酶（PDO）；NTD和PDO都由两个密切相关的硫氧还蛋白样折叠组成，其前半部分的活性位点谷氨酸在结构域后半段的Cys-X-X-Cys基序上起一般酸碱催化剂的作用。我们对AhpF的晶体学分析也强烈支持AhpF催化循环中大结构域运动的参与。晶体学和荧光方法将用于第三个特定目标，以定义AhpF- ahpc相互作用的性质，以及亚单位内电子转移过程中AhpF内部的域间相互作用。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宿主的氧化应激防御机制和氧化还原调节的细胞信号传导。预防人类退行性疾病、癌症和衰老中的氧化损伤的治疗干预，以及对抗致病防御系统，需要对细菌和人类来源的烷基氢过氧化物还原酶有完整的分子和生物学理解。

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.