Mechanistic and structural investigations into the unique monooxygenase CobG involved in vitamin B12 (cobalamin) biosynthesis

对参与维生素 B12（钴胺素）生物合成的独特单加氧酶 CobG 的机理和结构研究

• 批准号: 58324451
• 负责人: Dr. Rebekka Biedendieck
• 金额: --
• 依托单位: Department of Biosciences
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/58324451?language=en
• 关键词: Mechanistic; structural; investigations; into; unique

Vitamin B12, cobalamin, is a highly modified tetrapyrrole that is an essential component of the human diet albeit in very small quantities. The structural complexity of B12 underpins some beguiling chemistry that allows it to mediate a number of challenging biochemical reactions. The biosynthesis of vitamin B12 requires around 30 enzymatic steps. Probably the most interesting step of B12 formation is the elimination of carbon C-20 from the tetrapyrrole ring in a reaction that is catalysed by an enzyme called CobG. However, the structure and catalytic function of this important enzyme are only poorly understood. In this application, a novel enzyme assay will be established. The function of the observed Fe-S-cluster and bound nonhaem iron will be investigated using high end spectroscopy (ICP-MS, FTIR, IR, NMR, EPR, ENDOR). Catalytically important amino acid residues are targeted by site directed mutagenesis of the corresponding gene. Substrate recognition and binding will be tested by fluorometric assays (FS, FRET). Finally, anaerobic crystallisation of the oxygen sensitive protein will be attempted. The results from this investigation will help compose a molecular overture of events that allow ring contraction to take place.

维生素 B12（钴胺素）是一种高度改性的四吡咯，虽然含量很少，但却是人类饮食的重要组成部分。 B12 的结构复杂性支撑着一些令人着迷的化学反应，使其能够介导许多具有挑战性的生化反应。维生素 B12 的生物合成需要大约 30 个酶促步骤。 B12 形成过程中最有趣的步骤可能是在 CobG 酶催化的反应中消除四吡咯环中的碳 C-20。然而，人们对这种重要酶的结构和催化功能知之甚少。在本申请中，将建立一种新的酶测定方法。将使用高端光谱（ICP-MS、FTIR、IR、NMR、EPR、ENDOR）研究观察到的 Fe-S 簇和结合的非血红素铁的功​​能。通过相应基因的定点诱变来靶向催化重要的氨基酸残基。底物识别和结合将通过荧光测定（FS、FRET）进行测试。最后，将尝试氧敏感蛋白的厌氧结晶。这项研究的结果将有助于构成允许环收缩发生的分子序曲。