Mechanistic studies of alkyl hydroperoxide reductase

细菌烷基过氧化氢还原酶系统用于防止氧化应激的毒性和致突变作用。 AhpC是基于半胱氨酸的过氧化物酶组分，是普遍存在的“过氧化物氧还蛋白”（Prx）家族的成员，通过在酶上瞬时产生半胱氨酸次磺酸和随后的亚基间二硫键形成来减少H2 O2和有机氢过氧化物。 AhpF是含有黄素的还原酶组分，存在于大多数但不是所有的细菌中，并有效地将电子从NADH（或NADPH）转移到AhpC。 哺乳动物Prxs参与细胞增殖和分化、免疫应答和细胞信号传导等多种过程。 虽然大多数AhpC/Prx同源物高度表达，并在氧化防御中发挥重要作用，但已知只有来自幽门螺杆菌（与胃癌相关的胃溃疡的病原体）的AhpC是该生物体生存所必需的。该提案的第一个具体目标集中在（1）在过氧化物存在下鼠伤寒沙门氏菌AhpC和哺乳动物Prx II周转期间被认为会改变的构象状态、低聚化和膜缔合，以及（2）推定的通用碱催化剂（Arg 119）参与AhpC的过氧化物还原。 第二个具体目标是探索S的N-末端二硫键中心的电子转移机制。鼠伤寒AhpF. 该中心（Cys 129-Cys 132）是我们研究中已知的AhpF中独特的氧化还原结构域的一部分，介导从蛋白质C末端部分的氧化还原中心（FAD和Cys 345-Cys 348）到AhpC的电子转移。我们最近对AhpF的晶体学分析表明，它的N-末端结构域（NTD）具有独特的结构，而其同源物--来自嗜热菌的蛋白质二硫键氧化还原酶（PDO）的特征不明显; NTD和PDO都由两个紧密相关的硫氧还蛋白样折叠组成，其中来自前半部分的推定活性位点谷氨酸盐在Cys-X-X-处充当化学的一般酸碱催化剂。结构域的后半部分的Cys基序。 我们对AhpF的晶体学分析也强烈支持AhpF的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具体目标，以定义亚基内电子转移期间AhpF-AhpC相互作用以及AhpF内结构域间相互作用的性质。理解细菌AhpF和细菌和哺乳动物AhpC同系物的催化作用将有助于我们了解病原体和哺乳动物宿主中的氧化应激防御机制和氧化还原调节的细胞信号传导。 在预防参与人类退行性疾病，癌症和衰老的氧化损伤，以及在打击致病性防御系统的治疗干预需要从细菌和人类来源的烷基氢过氧化物还原酶的完整的分子和生物学的理解。

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.