Covalent Flavin Coenzyme in Flavoenzyme Catalysis

单胺氧化酶A和B(MAO A和B)是线粒体膜外酶，在5-羟色胺、多巴胺和去甲肾上腺素等神经递质的降解中起作用，在治疗抑郁症、成瘾和帕金森病等各种病理疾病中具有重要的药理作用。每个酶都含有一个共价的8α-S半胱氨酸基FAD作为功能辅酶。本项目寻求继续支持，通过X射线结晶学确定每个酶的结构，以研究催化中H提取的详细机制。为了便于这些研究，我们开发了在巴斯德毕赤酵母中高效表达每种酶的方法。共价Fad在MAO B中的作用将通过突变破坏共价Fad的结合位点，然后表达和纯化突变酶并与WT酶进行动力学性质的比较来研究。在制备截短形式的MAO A和MAO B的过程中，将研究C端疏水结构域在MAO A和MAO B的酶结构和功能中的作用，并将纯化的突变体与WT酶的性质进行比较。初步研究是为了研究结核分枝杆菌表达的胺氧化酶的底物专一性、抑制物敏感性和结构性质。这些研究的结果将提供对这些黄素蛋白胺氧化酶的结构和机制的洞察，并导致开发用于治疗神经疾病和可能的结核病感染的新的、特定的药物。

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.