Covalent Flavin Coenzyme in Flavoenzyme Catalysis

单胺氧化酶A和B （MAO A和B）是线粒体外膜结合酶，在神经递质如血清素、多巴胺和去甲肾上腺素的降解中起作用，并且在治疗各种病理疾病如抑郁症、多动症和帕金森病方面具有重要的药理学意义。每种酶都含有一个共价的8 α - s -半胱氨酸fad作为功能性辅酶。该项目寻求继续支持，通过x射线晶体学确定每种酶的结构，以研究催化中H+抽象的详细机制。为了促进这些研究，我们开发了在毕赤酵母中高水平表达每种酶的方法。通过诱变破坏共价FAD附着位点，表达和纯化突变酶，并将其与WT酶的动力学特性进行比较，研究共价FAD在MAO B中的作用。通过制备MAO A和MAO B的截断形式和纯化突变体与WT酶特性的比较，研究c端疏水结构域在酶结构和功能中的作用。初步研究旨在研究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。这些研究的结果将为这些黄蛋白胺氧化酶的结构和机制提供深入的见解，并导致开发新的，治疗神经疾病和可能的结核病感染的特异性药物。

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.