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

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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.