ENZYMES INVOLVED IN COENZYME B12 SYNTHESIS

维生素B12是自然界最重要的辅因子，是B型维生素中最大的一种。它含有一个由四个吡咯氮基和两个轴向配体配位的中心钴离子八面体的科林环系。这项建议的总体目标是在详细的分子水平上了解在鼠伤寒沙门氏菌中组装辅酶B12的轴向配体所涉及的酶的结构/功能方面。这一过程需要至少七种酶将5‘-脱氧腺苷部分添加到Corin环上，合成“核苷酸环”，并组装钴胺的较低配体。将使用结构和生化方法的组合来提供对这些酶系统的详细描述。近期的目标是确定ATP：腺苷转移酶(COBA)如何催化钴-碳键形成的结构机制，以及激酶/腺苷钴酰胺磷酸鸟苷转移酶(COBU)如何在组装较低配体之前激活腺苷钴酰胺。同时，计划确定负责低配体生物合成的磷酸核糖转移酶(CobT)的结构机制。实现这些目标所需的实验系统已经建立得很好。这一提议的长期目标是结晶，然后确定钴胺生物合成最后阶段涉及的另外四种酶的结构，即CobD、CbiB、COBC和COBS，以了解组装这种复杂辅酶下层配体的完整结构框架。总之，这项研究将揭示形成钴胺生物合成途径的酶之间的结构关系和生化耦合的可能性。它还将提供对它们潜在的化学机制的洞察。此外，这项研究将阐明那些影响皮质醇识别和利用的因素，这反过来将为理解维生素B12依赖的酶提供更广泛的框架。

Vitamin B12 is nature's most magnificent cofactor and represents the largest of the B-type vitamins. It contains a corrin ring system with a central cobalt ion octahedrally coordinated by the four pyrrole nitrogens of the corrin and two axial ligands. The overall goal of this proposal is to understand, on a detailed molecular level, the structural/functional aspects of the enzymes involved in assembling the axial ligands of coenzyme B12 in Salmonella typhimurium. This process requires at least seven enzymes to add the 5'-deoxyadenosyl moiety to the corrin ring, synthesize the "nucleotide loop" and assemble the lower ligand of cobalamin. A combination of structural and biochemical approaches will be utilized to provide a detailed description of these enzymatic systems. The immediate aims are to define the structural mechanism for how ATP:corrinoid adenosyl transferase (CobA) catalyzes the formation of a cobalt-carbon bond and how kinase/adenosylcobinamide phosphate guanylytransferase (CobU) activates adenosyl cobinamide prior to assembly of the lower ligand. In parallel it is planned to define the structural mechanism of the phosphoribosyltransferase (CobT) responsible for biosynthesis of the lower ligand. The experimental systems necessary to accomplish these goals are well established. The longer term goal of this proposal is to crystallize and then determine the structures of the four other enzymes involved in the final stages of cobalamin biosynthesis, CobD, CbiB, CobC and CobS, to understand the complete structural framework for assembling the lower ligand of this complex coenzyme. Together this study will reveal the structural relationships and potential for biochemical coupling between the enzymes that form the biosynthetic pathway for cobalamin. It will also provide insight into their underlying chemical mechanisms. In addition this study will clarify those factors that influence corrinoid recognition and utilization which will in turn provide a broader framework for understanding vitamin B12 dependent enzymes.