Mechanistic basis for the low O2-dependent activity of a nicotine-degrading enzyme

项目总结/摘要 尽管人们普遍认识到烟草使用对健康的负面影响， 烟草使用者停止使用烟草产品，因为尼古丁具有成瘾性。一种新型 药物代谢动力学策略，包括给予尼古丁降解酶，NicA 2， 恶臭假单胞菌S16，最近已被证明是非常有效的逆转尼古丁的症状 啮齿动物模型中的依赖性。NicA 2是含黄素的胺氧化酶家族的成员， 它们总是使用O2作为氧化剂，因此也假定NicA 2是氧化酶。但 酶显示出非常差的O2依赖性催化活性，这种低活性需要使用 不可接受的高剂量NicA 2（10 mg/kg或更高），以实现尼古丁寻求的症状缓解 动物模型中的行为。我们发现O2不是NicA 2的天然氧化剂，这解释了为什么 酶显示出如此低的活性与O2; NicA 2实际上是一种脱氢酶，使用一种新的细胞色素 c蛋白（CycN）作为其直接电子受体，使NicA 2成为“氧化酶”的一个显着例外。 含黄素的胺氧化酶类酶的范例。本项目旨在阐明 NicA 2使用CycN而不是O2的机制基础。第一个目标是实验进化 结合遗传选择将用于确定负责限制NicA 2的结构特征， 使用O2作为电子受体的能力。第二个目的是确定电子的作用机理 NicA 2和CycN之间的转移。将使用定点诱变和预稳态动力学来定义 两种蛋白质中氧化还原中心之间的电子传递途径和位点特异性化学物质 交联将用于确定NicA 2和CycN之间的结合界面。第三个目标将确定 在恶臭假单胞菌S16中再氧化CycN的下游末端电子受体。基础知识 通过这些努力获得的成果将为继续开发基于NicA 2的 戒烟疗法，这可能最终减少与烟草相关的巨大健康负担 使用.该项目还将加强区域本科院校的研究环境， 从历史上看，它没有从NIH获得大量资金，并将使本科生接触到 涉及一系列遗传、生物化学和微生物学技术的有价值的研究。

PROJECT SUMMARY/ABSTRACT Despite widespread knowledge of the negative health consequences of tobacco use, it is very challenging for tobacco users to cease using tobacco products due to the addictive properties of nicotine. A novel pharmacokinetic strategy that involves the administration of the nicotine-degrading enzyme, NicA2 from Pseudomonas putida S16, has recently been shown to be highly effective at reversing the symptoms of nicotine dependence in rodent models. NicA2 is a member of the flavin-containing amine oxidase family of enzymes, which invariably use O2 as an oxidant, and NicA2 is therefore also assumed to be an oxidase. However, the enzyme has displayed very poor O2-dependent catalytic activity, and this low activity has required the use of unacceptably high doses of NicA2 (10 mg/kg or greater) in order to achieve symptomatic relief of nicotine seeking behavior in animal models. We have discovered that O2 is not the natural oxidant for NicA2, explaining why the enzyme has displayed such low activity with O2; NicA2 is actually a dehydrogenase that uses a novel cytochrome c protein (CycN) as its immediate electron acceptor, making NicA2 a remarkable exception to the “oxidase” paradigm of the flavin-containing amine oxidase class of enzymes. This project is designed to elucidate the mechanistic underpinnings of NicA2’s use of CycN instead of O2. In the first aim, experimental evolution combined with genetic selections will be used to identify the structural features responsible for restricting NicA2’s capacity to use O2 as an electron acceptor. The second aim is designed to determine the mechanism of electron transfer between NicA2 and CycN. Site-directed mutagenesis and pre-steady state kinetics will be used to define the electron transfer pathway between the redox centers in the two proteins and site-specific chemical crosslinking will be used to determine the binding interface between NicA2 and CycN. The third aim will identify the downstream terminal electron acceptor(s) that reoxidize CycN in P. putida S16. The fundamental knowledge obtained through these efforts will provide a strong framework for the continued development of NicA2-based tobacco-cessation therapies, which could ultimately reduce the massive health burden associated with tobacco use. This project will also strengthen the research environment at a regional undergraduate institution that historically has not received substantial funding from the NIH and will expose undergraduate students to meritorious research involving an array of genetic, biochemical and microbiological techniques.