Raux, Evelyne Christine; (1999) Biosynthesis of cobalamin (vitamin B12) in Salmonella typhimurium and Bacillus megaterium de Bary; Characterisation of the anaerobic pathway. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The transformation of uroporphyrinogen III into cobalamin (vitamin B12) requires about 25 enzymes and can be performed by either aerobic or anaerobic pathways. The aerobic route is dependent upon molecular oxygen, and cobalt is inserted after the ring contraction process. The anaerobic route occurs in the absence of oxygen and cobalt is inserted into precorrin-2, several steps prior to the ring contraction. A study of the biosynthesis in both S. typhimurium and B. megaterium reveals that two genes, cbiD and cbiG, are essential components of the pathway and constitute genetic hallmarks of the anaerobic pathway. The genes responsible for the cobalt chelation, the S. typhimurium CbiK and the B. megaterium CbiX, were identified within cob operons and were characterised. Moreover, the activity of the multifunctional iron chelatase/dehydrogenase enzymes (E. coli CysG and S. cerevisiae Met8p) involved in sirohaem biosynthesis have been investigated for their ability to act as a cobalt chelatase in corrin biosynthesis. Cobalamin can be produced from the S. typhimurium cob operon with any of these chelatases whereas precorrin-2 dehydrogenase activity is required with the B. megaterium cob operon. The X-ray structure of CbiK has been solved at 2.4A and is highly similar to the structure of the B. subtilis protoporphyrin IX ferrochelatase suggesting a common mechanism. Unlike the P. denitrificans cobalt chelatase complex, which requires three proteins and ATP (similar to the protoporphyrin IX magnesium chelatase), CbiK belongs to the unique protein-ATP independent chelatase family. Conserved amino acids have been characterised as key residues within the CbiK active site. Genomic comparisons of B12-producing organisms highlight divergences between the methyltransferases, which separate into aerobic and anaerobic pathway subgroups. Further insights into the methyltransferases have been gained from the X-ray structure of the B. megaterium CbiF (solved at 2.4A resolution). Finally, the molecular structure of cobyric acid produced from the S. typhimurium cob operon in E. coli has been deduced from a number of spectrometric studies, an approach which could be used in the future to characterise other intermediates along the anaerobic cobalamin pathway. From the results obtained in this thesis, it becomes apparent that the terms "aerobic" and "anaerobic" pathways are misleading and should be replaced by "late-cobalt insertion" and "early-cobalt insertion" pathways respectively.

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