Beck, Richard; (1997) Concerning the Biosynthesis of Sirohaem in Bacillus megaterium: The Relationship with Cobalamin Metabolism. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Sirohaem and vitamin B12 (cyanocobalamin) share a significant part of their biosynthetic pathways, the last common intermediate being precorrin-2 (dihydrosirohydrochlorin). Dehydrogenation of precorrin-2 leads to sirohaem formation, whereas methylation at C-20 directs production of vitamin B12. A novel enzyme has been identified which may play a crucial role in regulating sirohaem and cobalamin biosynthesis at the precorrin-2 branch-point. In vivo, the CbiX protein from Bacillus megaterium has been shown to catalyse the final two steps of sirohaem synthesis, namely dehydrogenation and ferrochelation. CbiX is encoded within a cobalamin biosynthetic (cob) operon by cbiX. The protein consists of 301 amino acids with a Mr of 34,297. The C-terminus of the protein contains a novel poly-histidine motif and this has enabled the purification of CbiX to homogeneity by metal chelate chromatography. The protein was found to be soluble but relatively unstable, being broken down to give a 24kDa polypeptide. Although the native Mr of CbiX is approximately 62,000, suggesting a dimeric form, the purified protein was found to aggregate, forming multimers which could be visualised by PAGE. The poly-histidine motif is involved in metal binding: deletion from the protein inhibits sirohaem synthesis but truncation, retaining 7 of the 19 histidines, maintains function. In vivo assays have shown that cobalt (the metal associated with vitamin B12) inhibits the function of CbiX in sirohaem synthesis. Other metals do not show this ability. Deletion of the cbiX gene from the cob operon greatly reduces cobalamin production. The position of the cbiX gene within a cob operon in B. megaterium, deletion experiments with the cbiX gene, and the apparent ability of the CbiX protein to bind cobalt, all suggest regulation of sirohaem and vitamin B12 production by CbiX at the precorrin-2 branch-point of tetrapyrrole modification.

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