ENZYMES INVOLVED IN COENZYME B12 SYNTHESIS

维生素B12是自然界中最重要的辅助因子，也是最大的B型维生素。 它包含一个咕啉环系统，中心钴离子八面体协调的四个吡咯氮的咕啉和两个轴向配体。 本提案的总体目标是在详细的分子水平上了解参与组装鼠伤寒沙门氏菌中辅酶B12轴向配体的酶的结构/功能方面。 这个过程需要至少七种酶将5 '-脱氧腺苷部分添加到咕啉环上，合成“核苷酸环”并组装钴胺素的低级配体。 将利用结构和生物化学方法的组合来提供这些酶系统的详细描述。近期的目标是确定ATP：corrinoid腺苷转移酶（CobA）如何催化钴-碳键的形成，以及激酶/腺苷可宾酰胺磷酸鸟苷酰转移酶（CobU）如何在组装低级配体之前激活腺苷可宾酰胺的结构机制。 与此同时，计划确定负责生物合成较低配体的磷酸核糖基转移酶（CobT）的结构机制。 实现这些目标所需的实验系统已经建立。该提案的长期目标是结晶，然后确定钴胺素生物合成最后阶段涉及的其他四种酶的结构，CobD，CbiB，CobC和CobS，以了解组装这种复杂辅酶的低级配体的完整结构框架。 这项研究将揭示构成钴胺素生物合成途径的酶之间的结构关系和生化偶联潜力。 它还将提供深入了解其潜在的化学机制。 此外，这项研究将澄清那些影响corrinoid识别和利用的因素，这将反过来为理解维生素B12依赖性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.