Membrane-associated Steps in Coenzyme B12 Biosynthesis

描述(申请人提供)：辅酶B12(又名，腺苷钴胺，Adobe Cbl)只由原核生物合成，对许多病原体的生存是必不可少的。因此，准确了解这一主要途径(~25个反应)的生物化学基础，并了解所涉及酶的结构性质，对于设计可用于靶向致病细菌或任何其他具有社会意义的原核生物的抑制剂至关重要。我将应用多学科的方法来研究由ADOCbl-P磷酸酶(COBC)酶催化的ADOCbl生物合成的最后一步。我的建议包括两个部分：i)COBC的功能和拓扑研究；ii)COBC与其他疑似构成定位于细胞膜的多蛋白复合体的蛋白质相互作用的分析。我将与伊万·雷蒙特(威斯康星大学麦迪逊分校生物化学系)领导的结构生物学家合作，深入了解COBC的作用机制，并促进我们对存在与膜相关的生物合成复合体的影响的理解。

DESCRIPTION (provided by applicant): Coenzyme B12 (aka, adenosylcobalamin, AdoCbl) is synthesized only by prokaryotes, and is essential to the survival of many pathogens. Hence, precise knowledge of the biochemistry underpinning this major pathway (~25 reactions), and an understanding of the structural properties of the enzymes involved, is critical to the design of inhibitors that could be used to target disease-causing bacteria or any other prokaryote of societal importance. I will apply a multidisciplinary approach to study the last step of AdoCbl biosynthesis, which is catalyzed by the AdoCbl-P phosphatase (CobC) enzyme. There are two parts to my proposal: i) Functional and topological studies of CobC; ii) analysis of interactions of CobC with other proteins suspected to comprise a multiprotein complex localized to the cell membrane. I will collaborate with structural biologists led by Ivan Rayment (Department of Biochemistry, UW-Madison) to gain insights into the mechanism of function of CobC, and to advance our understanding of the implications of the existence of a membrane associated biosynthetic complex.