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

尼古丁降解酶低 O2 依赖性活性的机制基础

• 批准号: 10201301
• 负责人: Frederick Stull
• 金额: $ 44.87万
• 依托单位: WESTERN MICHIGAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10201301
• 关键词: American; Animal Model; Area; Awareness; Binding; Binding Sites; Biochemical; Biochemistry; Brain; Cessation of life; Chemicals; Chemistry; Cytochrome a; Data; Development; Dose; Drug Kinetics; Drug Targeting; Electron Transport; Electrons; Environment; Enzymes; Escherichia coli; Evolution; Family; Flavins; Foundations; Funding; Genetic; Health; Healthcare; Ice; Institution; Intervention; Kinetics; Knowledge; Membrane; Michigan; Microbiological Techniques; Monoamine Oxidase; Mutagenesis; Nicotine; Nicotine Dependence; Oxidants; Oxidases; Oxidation-Reduction; Oxides; Oxidoreductase; Pathway interactions; Patients; Physiological; Property; Proteins; Pseudomonas putida; Public Health; Rattus; Relapse; Reporting; Research; Rodent Model; Site; Site-Directed Mutagenesis; Structure; Symptoms; Therapeutic; Tobacco Use Cessation; Tobacco use; Toxic effect; United States; United States National Institutes of Health; Universities; Variant; amine oxidase; base; cigarette smoking; citrate carrier; cofactor; cost; crosslink; cytochrome c; cytochrome c oxidase; design; experimental study; genetic selection; heme C; improved; insight; member; nicotine oxidase; nicotine seeking behavior; novel; novel strategies; phenotypic data; prevent; scaffold; smoking cessation; tobacco products; tobacco user; undergraduate research experience; undergraduate student; unnatural amino acids

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.

项目概要/摘要 尽管人们普遍了解烟草使用对健康的负面影响，但对于 由于尼古丁的成瘾特性，烟草使用者停止使用烟草制品。 药代动力学策略涉及使用尼古丁降解酶 NicA2 最近已证明恶臭假单胞菌 S16 在逆转尼古丁症状方面非常有效 NicA2 是含黄素胺氧化酶家族的成员， 它们总是使用 O2 作为氧化剂，因此 NicA2 也被认为是一种氧化酶。 酶表现出非常差的 O2 依赖性催化活性，这种低活性需要使用 为了缓解寻求尼古丁的症状，使用不可接受的高剂量 NicA2（10 毫克/千克或更高） 我们发现 O2 不是 NicA2 的天然氧化剂，这解释了为什么 NicA2 酶对 O2 表现出如此低的活性，实际上是一种使用新型细胞色素的脱氢酶； c 蛋白（CycN）作为其直接电子受体，使 NicA2 成为“氧化酶”的显着例外 含黄素胺氧化酶类酶的范例 该项目旨在阐明 NicA2 使用 CycN 代替 O2 的机制基础 第一个目标是实验进化。 与遗传选择相结合将用于识别负责限制 NicA2 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机制。 NicA2 和 CycN 之间的转移将用于定义定点诱变和前稳态动力学。 两种蛋白质的氧化还原中心和位点特异性化学物质之间的电子传递途径 交联将用于确定 NicA2 和 CycN 之间的结合界面。 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基于 NicA2 的持续发展提供一个强有力的框架 戒烟疗法，最终可以减轻与烟草相关的巨大健康负担 该项目还将加强区域本科机构的研究环境。 历史上从未获得过 NIH 的大量资助，并且将使本科生接触到 涉及一系列遗传、生化和微生物技术的杰出研究。

项目成果

The enzyme pseudooxynicotine amine oxidase from Pseudomonas putida S16 is not an oxidase, but a dehydrogenase.

来自恶臭假单胞菌 S16 的假氧烟碱胺氧化酶不是氧化酶，而是脱氢酶。

• 发表时间: 2022-08
• 作者: Choudhary, Vishakha;Wu, Kevin;Zhang, Zhiyao;Dulchavsky, Mark;Barkman, Todd;Bardwell, James C A;Stull, Frederick
• 通讯作者: Stull, Frederick

Binding Interface and Electron Transfer Between Nicotine Oxidoreductase and Its Cytochrome c Electron Acceptor.

尼古丁氧化还原酶与其细胞色素 c 电子受体之间的结合界面和电子转移。

• DOI: 10.1021/acs.biochem.2c00472
• 发表时间: 2022-10-18
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Mumby, Elizabeth J.;Willoughby, Jamin A., Jr.;Vasquez, Cristian;Delavari, Niusha;Zhang, Zhiyao;Clark, Christopher T.;Stull, Frederick
• 通讯作者: Stull, Frederick