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

维生素B12，钴胺，是一种高度修饰的四吡咯，是人类饮食的基本成分，尽管数量很少。B12的结构复杂性支撑了一些令人着迷的化学物质，使其能够调节一些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形成的最有趣的步骤可能是在一种名为CobG的酶的催化下，从四吡咯环上消除碳C-20。然而，人们对这种重要酶的结构和催化功能知之甚少。在这一应用中，将建立一种新的酶分析方法。用高端光谱(ICPMS、FTIR、IR、核磁共振、EPR、ENDOR)研究观察到的Fe-S团簇和结合的非血红铁的作用。具有催化作用的重要氨基酸残基通过相应基因的定点突变而成为靶点。底物识别和结合将通过荧光分析(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建允许环收缩发生的事件的分子序曲。

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.