The vitamin B₁₂ biosynthetic pathway: structure analysis of Uroporphyrinogen-III C-methyltransferase

J. Vévodová, D. I. Roper, H. L. Schubert, A. A. Brindley, M. J. Warren, K. S. Wilson

Structural Biology Laboratory, University of York, York and Queen Mary and Westfield College, London

The biosynthesis of vitamin B₁₂, “the anti-pernicious anaemia factor”, requires about 30 enzymes, and is further complicated by the appearance in nature of two separate pathways, representing aerobic and anaerobic routes, where the major difference seem to be concerned with the process of cobalamin ring contraction and cobalt chelatation. Pseudomonas aeruginosa is able to synthesise the vitamin in the absence of oxygen. However, the bacterium can also make B₁₂ when grown aerobically. Thus, there must exist a pathway that can operate both in the presence and absence of molecular oxygen.

Uroporphyrinogen (uro’gen) III methyltransferase, a key enzyme in the biosynthetic pathways of vitamin B₁₂ and siroheme, catalyzes the S-adenosyl-L-methionine (SAM)- dependent bismethylation of its substrate, uro’gen III, resulting in the formation of dihydrosirohydrochlorin (precorrin-2). The enzyme exists in at least two forms. One form, encoded by the cobA gene, is required for vitamin B₁₂ synthesis in Pseudomonas denitrificans. The second form, encoded by the cysG gene, is required for siroheme in E. coli. Both forms of the enzyme perform the in vivo synthesis of precorrin-2, but in addition, CysG has NAD⁺-dependent precorrin-2 oxidase and ferrochelatase activities. The CysG enzyme mass is ~52 kDa, whereas the smaller CobA protein mass is of ~30 kDa and is homologous only to the C-terminal region of CysG.

CobA is a key regulatory enzyme in the branched tetrapyrrole biosynthetic pathway, and is sensitive to both substrate and product inhibition. To gain some molecular insight into how this enzyme exerts its control, we have crystallised the CobA protein and collected data on the SRS synchrotron in Daresbury. The molecular replacement method (AMoRe) has been used for the phase problem solution with the C-terminal domain of CysG as a search model. Structure refinement is currently under way.

We acknowledge Elizabeth D. Getzoff and Beth Stroupe from The Scripps Research Institute, La Jolla, Canada for providing the coordinates of CysG protein.

E. Raux, H.L. Schubert, J.M. Roper, K.S. Wilson, M.H. Warren Bioorganic chemistry 27 (1999) 100-118.

E. Raux, H.L. Schubert, M.J. Warren Cell. Mol. Life Sci. 57 (2000) 1880-1893.