Engineered flavin-dependent enzymes for probing redox environment and regulation

摘要 各种细胞区室中氧化还原环境的紊乱与许多病理学有关， 包括神经变性疾病、癌症、心血管疾病和衰老。由于氧化物与 还原的烟酰胺腺嘌呤二核苷酸是细胞氧化还原环境的主要贡献者， 工程工具来扰乱这一比例将使研究氧化还原不平衡的作用，在驱动这些 病理学在这项工作中，我们研究了一些微生物用来控制 它们的最佳氧化还原环境，以及这些机制在哺乳动物细胞中的可能应用。 许多微需氧细菌和原生动物缺乏常规的多复合呼吸链， 而是依赖于催化形成H2O的NADH氧化酶反应的酶来再循环NAD+， 消除环境中的有毒氧气。由于该反应的产物是良性水， 过氧化氢（H2 O2），这些酶代表干扰哺乳动物代谢的有吸引力的试剂 细胞在这项工作中，我们提出了一个细菌的H2O形成的NADH氧化酶对NADPH特异性工程。 然后，这种NADPH氧化酶将在哺乳动物细胞的不同区室中表达，并且其对哺乳动物细胞的影响将在哺乳动物细胞的不同区室中表达。 将系统地评价细胞活力和代谢。由于以前没有工具报告给 安全地增加NADP+/NADPH在细胞中的比例，在一个区室特异性的方式，我们的工作将提供 对NADPH代谢及其调节的基本见解，以及对NAD（P）H池的大小， 不同的细胞区室。我们的工作还探讨了微需氧的人 原生动物寄生虫，如贾第鞭毛虫，阴道毛滴虫和溶组织内阿米巴，缺乏 常规呼吸链控制它们的氧化还原环境以支持能量代谢。我们 已经在T. vagulovirus中鉴定了一种天然蛋白质，其代表了黄素二铁核心蛋白与 它的氧化还原伙伴：红氧还蛋白和红氧还蛋白氧化还原酶。这种融合蛋白催化一个四电子 使用还原当量的NAD（P）H将氧还原成水。我们的研究结构和 这种融合蛋白的机制将提供这些人类寄生虫如何能够维持其 最佳氧化还原环境。这些机制可以是治疗干预的有吸引力的靶点， 人类原生动物寄生虫引起的疾病。

Abstract Disturbances of the redox environment in various cellular compartments are linked to many pathologies, including neurodegenerative diseases, cancer, cardiovascular disease and aging. Since the ratio of oxidized to reduced nicotinamide adenine dinucleotides is a major contributor to the cellular redox environment, engineering tools to perturb this ratio would enable the study the role of redox imbalances in driving these pathologies. In this work we examine the fundamental mechanisms used by some microorganisms to control their optimal redox environment, as well as the possible applications of these mechanisms in mammalian cells. A number of microaerophilic bacteria and protozoa lack a conventional multi-complex respiratory chain, and instead rely on enzymes which catalyze a H2O-forming NADH oxidase reaction to recycle NAD+ and to eliminate toxic oxygen from the environment. Since the product of this reaction is benign water and not hydrogen peroxide (H2O2), these enzymes represent attractive reagents to perturb metabolism in mammalian cells. In this work we propose to engineer a bacterial H2O-forming NADH oxidase towards NADPH specificity. This NADPH oxidase will then be expressed in different compartments of mammalian cells, and its effects on cell viability and metabolism will be systematically evaluated. Since no tool has previously been reported to safely increase the NADP+/NADPH ratio in cells in a compartment specific manner, our work will provide fundamental insights into NADPH metabolism and its regulation, as well as into the sizes of NAD(P)H pools in different cellular compartments. Our work also explores the mechanisms of how microaerophilic human protozoan parasites like Giardia intestinalis, Trichomonas vaginalis and Entamoeba histolytica, which lack conventional respiratory chains, control their redox environments in order to support energy metabolism. We have identified in T.vaginalis a natural protein which represents a fusion between a flavodiiron core protein with its redox partners: rubredoxin and rubredoxin oxidoreductase. This fusion protein catalyzes a four-electron reduction of oxygen to water using reducing equivalents of NAD(P)H. Our studies of the structure and mechanism of this fusion protein will provide insights into how these human parasites are able to maintain their optimal redox environment. These mechanisms can be attractive targets for therapeutic intervention, allowing us to combat diseases caused by human protozoan parasites.