Membrane-associated Steps in Coenzyme B12 Biosynthesis

描述（由申请人提供）：辅酶B12（又名腺苷钴胺素（adenosylcobalamin），简称ACBl）仅由原核生物合成，对许多病原体的生存至关重要。因此，对支撑这一主要途径的生物化学（约25个反应）的精确了解，以及对所涉及酶的结构特性的理解，对于设计可用于靶向致病细菌或任何其他具有社会重要性的原核生物的抑制剂至关重要。 我将应用多学科方法来研究由β-Cbl-P磷酸酶（CobC）催化的β-Cbll生物合成的最后一步。我的建议有两个部分：i）CobC的功能和拓扑学研究; ii）分析CobC与其他蛋白质的相互作用，这些蛋白质被怀疑包含定位于细胞膜的多蛋白复合物。 我将与Ivan Rayment（生物化学系，UW-Madison）领导的结构生物学家合作，深入了解CobC的功能机制，并推进我们对膜相关生物合成复合物存在的意义的理解。 公共卫生相关性：辅酶B12只由原核生物合成，包括许多病原体，它对动物的生存至关重要，包括人类。更好地了解辅酶B12生物合成的生物化学将有助于设计可用于靶向致病细菌的抗菌剂。在与其他人的合作中，我们的实验室使用了从辅酶B12合成研究中获得的知识来对抗癌症。将采用多方面的方法来了解更多关于该途径的最后一步，在肠道沙门氏菌中，它可能发生在细胞外。

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. PUBLIC HEALTH RELEVANCE: Coenzyme B12 is only synthesized by prokaryotes, including many pathogens, and it is essential to the survival of animals, including humans. A better understanding of the biochemistry of coenzyme B12 biosynthesis will facilitate the design of antimicrobials that could be used to target disease-causing bacteria. In collaboration with others, our laboratory used knowledge gained from the studies of coenzyme B12 synthesis in our fight against cancer. A multifaceted approach will be applied to learn more about the last step of the pathway, which, in Salmonella enterica, it may occur outside the cell.