Covalent Flavin Coenzyme in Flavoenzyme Catalysis

黄素酶催化中的共价黄素辅酶

• 批准号: 6729030
• 负责人: Dale E. Edmondson
• 金额: $ 35.54万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6729030
• 关键词: Ascomycetes; Mycobacterium tuberculosis; X ray crystallography; amine oxidase (flavin); chemical kinetics; cofactor; crystallization; electron spin resonance spectroscopy; enzyme activity; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate analog; enzyme substrate complex; flavin adenine dinucleotide; gene mutation; intermolecular interaction; mass spectrometry; model design /development; oxidation reduction reaction; protein purification; recombinant proteins; stop flow technique; structural biology

Monoamine oxidases A and B (MAO A and B) are outer mitochondrial membrane-bound enzymes that function in the degradation of neurotransmitters such as serotonin, dopamine, and norepinephrine and have been pharmacologically important in the treatment of various pathological disorders such as depression, addition, and Parkinson's Disease. Each enzyme contains a covalent 8alpha-S-cysteinylFAD as a functional coenzyme. This project seeks continued support to determine the structure of each enzyme by x-ray crystallography to investigate the detailed mechanisms of H+ abstraction in catalysis. To facilitate these studies, we have developed methods for high level expression of each enzyme in Pichia pastrois. The role of the covalent FAD in MAO B will be investigated by disrupting the site for covalent FAD attachment by mutagenesis, followed by expression and purification of the mutant enzyme and comparison of its kinetic properties with WT enzyme The role of the C-terminal hydrophobic domain in enzyme structure and function for MAO A and B will be investigated in preparing the truncated forms and comparison of the purified mutants with WT enzyme properties. Initial studies are proposed to investigat3e the substrate specificity, inhibitor sensitivity, and structural properties of an expressed amine oxidase from Mycobacterium tuberculosis. Results from these studies should provide insights into the structures and mechanisms of these flavoprotein amine oxidases and lead to the development of new, specific drugs for the treatment of neuro-disorders and possibly tuberculosis infections.

单胺氧化酶A和B（MAO A和B）是外线粒体膜结合酶，其在神经递质如5-羟色胺、多巴胺和去甲肾上腺素的降解中起作用，并且在各种病理性疾病如抑郁症、加法症和帕金森病的治疗中非常重要。每种酶都含有共价的8 α-S-半胱氨酰FAD作为功能性辅酶。该项目寻求持续的支持，通过X射线晶体学确定每种酶的结构，以研究催化中H+提取的详细机制。为了促进这些研究，我们已经开发了在巴斯德毕赤酵母中高水平表达每种酶的方法。共价FAD在MAO B中的作用将通过诱变破坏共价FAD连接位点来研究，然后表达和纯化突变酶，并将其动力学性质与WT酶进行比较。在制备截短形式和纯化突变体与WT酶的比较中，将研究MAO A和B的酶结构和功能中的末端疏水结构域特性.初步研究拟阐明结核分枝杆菌表达的胺氧化酶的底物特异性、抑制剂敏感性和结构特性。这些研究的结果应该可以深入了解这些黄蛋白胺氧化酶的结构和机制，并导致开发用于治疗神经疾病和可能的结核病感染的新型特异性药物。