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

维生素B12，即钴胺素，是一种高度修饰的四吡咯，是人类饮食中必不可少的成分，尽管摄入量非常少。B12结构的复杂性支撑着一些诱人的化学反应，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。可能B12形成过程中最有趣的一步是碳C-20从四吡咯环上消失，这一反应是由一种叫做CobG的酶催化的。然而，人们对这种重要酶的结构和催化功能了解甚少。在本应用中，将建立一种新的酶分析方法。利用高端光谱（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。催化重要的氨基酸残基被相应基因的定点诱变靶向。底物识别和结合将通过荧光测定法（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形成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.