ENZYMES INVOLVED IN COENZYME B12 SYNTHESIS

参与辅酶 B12 合成的酶

• 批准号: 6519925
• 负责人: IVAN RAYMENT
• 金额: $ 20.8万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519925
• 关键词: Salmonella typhimurium; X ray crystallography; active sites; adenosine triphosphate; alkyltransferase; cobalamin; cobalt; cobamide; crystallization; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; guanosine triphosphate; microorganism metabolism; nucleotidyltransferase; pentosyltransferase; phosphorylation; protein purification; reduction; site directed mutagenesis; vitamin B12 coenzyme; vitamin B12 compound; vitamin biosynthesis

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.

维生素 B12 是自然界最重要的辅助因子，也是最大的 B 型维生素的代表。 它包含一个咕啉环系统，其中心钴离子由咕啉的四个吡咯氮和两个轴向配体以八面体配位。 该提案的总体目标是在详细的分子水平上了解鼠伤寒沙门氏菌中参与组装辅酶 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”并组装钴胺素的下配体。 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。直接目标是确定 ATP：类咕啉腺苷转移酶 (CobA) 如何催化钴-碳键形成的结构机制，以及激酶/腺苷酸鸟苷酸转移酶 (CobU) 如何在下配体组装之前激活腺苷酸酰胺。 与此同时，计划定义负责低级配体生物合成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完善。该提案的长期目标是结晶并确定参与钴胺素生物合成最后阶段的其他四种酶（CobD、CbiB、CobC 和 CobS）的结构，以了解组装该钴胺素下配体的完整结构框架复合辅酶。 这项研究将共同​​揭示形成钴胺素生物合成途径的酶之间的结构关系和生化偶联的潜力。 它还将提供对其潜在化学机制的深入了解。 此外，这项研究将阐明影响类咕啉识别和利用的因素，从而为理解维生素 B12 依赖性酶提供更广泛的框架。