Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The X-ray structure of a cobalamin biosynthetic enzyme, cobalt-precorrin-4 methyltransferase

Nature Structural Biology volume 5, pages 585–592 (1998)Cite this article

Abstract

Biosynthesis of the corrin ring of vitamin B12 requires the action of six S-adenosyl-L-methionine (AdoMet) dependent transmethylases, closely related in sequence. The first X-ray structure of one of these, cobalt-precorrin-4 transmethylase, CbiF, from Bacillus megaterium has been determined to a resolution of 2.4 Å. CbiF contains two α/β domains forming a trough in which S-adenosyl-L-homocysteine (AdoHcy) binds. The location of AdoHcy and a number of conserved residues, helps define the precorrin binding site. A second crystal form determined at 3.1 Å resolution highlights the flexibility of two loops around this site. CbiF employs a unique mode of AdoHcy binding and represents a new class of transmethylase.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The vitamin B12 biosynthetic pathway from uro'gen III, highlighting the structures of precorrin-4 and -5 in the anaerobic and aerobic pathways.
Figure 2: S-Adenosyl-L-homocysteine (AdoHcy) bound to CbiF.
Figure 3: Sequence alignment of CysG, CobM and CbiF.
Figure 4: The AdoHcy binding pocket.
Figure 5: Comparison of CbiF in the presence and absence of phosphate.
Figure 6: CbiF topology and alignments to the DNA transmethylase HhaI.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Stubbe, J. Binding site revealed for Nature's most beautiful cofactor. Science 266, 1663 (1994).

    Article  CAS  Google Scholar 

  2. Scott, I. How nature synthesizes vatamin B12 - a survey of the last four billion years. Angew. Chem. 32, 1223-1376 ( 1993).

    Article  Google Scholar 

  3. Battersby, A.R. How nature builds the pigments of life - The conquest of vitamin B12 . Science 264, 1551-1557 ( 1994).

    Article  CAS  Google Scholar 

  4. Blanche, F. et al.,Vitamin B12 - How the problem of biosynthesis was solved. Angew. Chem. Int. Ed. Engl. 32, 1651-1653 (1995).

    Article  Google Scholar 

  5. Debussche, L., Thibaut, D., Cameron, B., Crouzet, J. & Blanche, F. Biosynthesis of the corrin macrocycle of coenzyme-B 12 in Pseudomonas denitrificans. J. Bacteriol. 175, 7430-7440 (1993).

    Article  CAS  Google Scholar 

  6. Lawerence, J.G. & Roth, J.R. The cobalamin (coenzyme B12 ) biosynthetic genes of Escherichia coli. J. Bacteriol. 177, 6371-6380 (1995 ).

    Article  Google Scholar 

  7. Roth, J.R., Lawerence, J.G., Rubenfield, M., Kieffer-Higgins, S. & Church, G.M. Characterization of the cobalamin (vitamin B 12) biosynthetic genes of Salmonella typhimurium. J. Bacteriol. 175, 3303–3316 (1993).

    Article  CAS  Google Scholar 

  8. Jordan, P.M. Highlights in haem biosynthesis. Curr. Opin. Struc. Biol. 4, 902–911 (1994).

    Article  CAS  Google Scholar 

  9. Blanche, F., Parallels and decisive differences in vitamin B12 biosyntheses. Angew. Chem. Int. Ed. Engl. 32, 1651–1653 (1993).

    Article  Google Scholar 

  10. Raux, E., Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: Functional studies in S. typhiumurium and Escherichia coli. J. Bacteriol. 178, 753–767 (1996).

    Article  CAS  Google Scholar 

  11. Raux, E., Thermes, C., Heathcote, P., Rambach, A. & Warren, M.J. A role for the Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis. J. Bacteriol. 179, 3203–3212 (1997).

    Article  Google Scholar 

  12. Scott, I.A.,et al.Biosynthesis of vitamin B12: Factor IV, a new intermediate in the anarobic pathway. Proc. Natl. Acad. Sci. USA 93 , 14316–14319 (1996).

    Article  CAS  Google Scholar 

  13. Martin-Verstraete, I., Debarbouille, M., Klier, A. & Rapoport, G. Levanase operon of Bacillus subtilus includes a fructose-specific phosphotransferase system regulating the expression of the operon. J. Mol. Biol. 214, 657–669 (1990).

    Article  CAS  Google Scholar 

  14. Raux, E., Woodcock, S.C., Schubert, H.L., Wilson, K.S. & Warren, M.J. Cobalamin (vitamin B12) biosynthesis; Cloning, expression and crystallisation of the Bacillus megaterium S-adenosyl-L-methionine dependent cobalt-precorrin-4 transmethylase CbiF. Euro. J. Bacteriol. in the press (1998).

  15. Oldfield, T.J. Real space refinement as a tool for model building. CCP4 Study Weekend: Macromolecular refinement (Dodson, E.J., Moore, M.H., Ralph, A. & Bailey, S., eds.) 67–74 (SERC Daresbury Laboratory, Warrington, UK.;1996).

    Google Scholar 

  16. Malone, T., Blumenthal, R.M. & Cheng, X. Structure-guided analysis reveals nine sequence motifs conserved among DNA amino-methyl-transferases, and suggests a catalytic mechanism for these enzymes . J. Mol. Biol. 253, 618– 632 (1995).

    Article  CAS  Google Scholar 

  17. Hodel, A.E., Gershon, P.D., Shi, X. & Quiocho, F.A. The 1.85 Å structure of Vaccinia protein VP39: A bifunctional enzyme that participates in the modification of both mRNA ends. Cell 85, 247–256 (1996).

    Article  CAS  Google Scholar 

  18. Blanche, F., Debussche, L., Thibaut, D., Crouzet, J. & Cameron, B. Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans. J. Bacteriol. 171, 4222–4231 (1989).

    Article  CAS  Google Scholar 

  19. Drennan, C.L., Huang, S., Drummond, J.T., Matthews, R.G. & Ludwig, M.L. How a protein binds B12: A 3.0 Å X-ray structure of B12-binding domains of methionine synthase. Science 266, 1669–1674 ( 1994).

    Article  CAS  Google Scholar 

  20. Roessner, C.A., et al.Expression of 9 Salmonella typhimurium enzymes for cobalamide synthesis . FEBS letters 301, 73– 78 (1992).

    Article  CAS  Google Scholar 

  21. Woodcock, S.C. & Warren, M.J. Evidence for a covalent intermediate in the S-adenosyl-L-methionine-dependent transmethylation reaction caused by sirohaem synthase. Biochem. J. 313, 415 –421 (1996).

    Article  CAS  Google Scholar 

  22. Woodcock, S.C., et al.The contribution of the CysGA and CysGB domains of siroheam synthase (CysG) towards cobalamin (vitamin B12) biosynthesis. Biochem. J. 330, 121–129 ( 1998).

    Article  CAS  Google Scholar 

  23. Schluckebier, G., O'Gara, M., Saenger, W. & Cheng, X. Universal catalytic domain structure of AdoMet-dependent methyltransferases. J. Mol. Biol. 247, 16–20 ( 1995).

    Article  CAS  Google Scholar 

  24. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    Article  CAS  Google Scholar 

  25. Bernstein, F.C., Koetzle, T.F., Williams, G.J.B., Meyer, E.J., Brice, M.D., Rogers, J.K., Kennard, O., Shimanouchi, T. & Tasumi, M. (1977).The protein data bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535–542.

  26. Freyman, D.M., Keenan, R.J., Stoud, R.M. & Walter, P. The structure of the conserved GTPase domain of the signal recognition particle. Nature 385, 361–365 ( 1997).

    Article  Google Scholar 

  27. Orengo, C.A., Michie, A.D., Jones, S., Jones, D.T., Swindells, M.B. & Thornton, J.M. CATH- a hierarchic classification of protein domain structures. Structure 5, 1093–1108 (1997).

    Article  CAS  Google Scholar 

  28. Dixon, M.M., Huang, S., Matthews, R.G. & Ludwig, M. The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylastion of B12. Structure 4, 1263–1275 (1996).

    Article  CAS  Google Scholar 

  29. Otwinowski, Z. processing of X-ray diffraction data collected in ossilation mode. Meth. Enz . 276, 307–326 ( 1991).

    Article  Google Scholar 

  30. Otwinowski, Z. Maximum likelihood refinement of heavy atom parameters. Proceedings of the CCP4 Study Weekend (Wolf, W., Evans, P.R. & Leslie, A.G.W., eds) 80- 88 (SERC Daresbury Laboratory, Warrington, UK; 1991 ).

    Google Scholar 

  31. Cowtan, K. in CCP4 & ESF-EACBM Newsletter on Protein Crystallography 34- 38 (1994).

  32. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. Improved methods for building protein models in electron density maps and location of errors in these models. Acta Crystallogr. A 47, 110-119 (1991).

    Article  Google Scholar 

  33. Brunger, A.T. X-PLOR Version 3.1: A system for X-ray Crystallography and NMR (Yale University Press, New Haven, Connecticut, USA; 1992).

    Google Scholar 

  34. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum likelihood method. Acta Crystallogr. D 53, 240-255 (1997).

    Article  CAS  Google Scholar 

  35. Ramachandran, S. Conformations of polypeptides and proteins. Adv. Prot. Chem. 23, 283-437 (1968).

    CAS  Google Scholar 

  36. Laskowski, R.A., MacAuthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK - a program to check the sterochemical quality of protein structures . J. Appl. Crystallogr. 26, 283- 291 (1993).

    Article  CAS  Google Scholar 

  37. Navaza, J. AMORE - an automated package for molecular replacement. Acta Crystallogr. A 50, 157-163 (1994).

    Article  Google Scholar 

  38. Esnouf, R.M. An extensively modified version of MolScript that includes greatly enhanced coloring capabilities . J. Mol. Graph. 15, 133-138 (1997).

    Google Scholar 

  39. Kraulis, P.J. MOLSCRIPT - a program to produce both detailed and schematic plots of proteins structures . J. Appl. Crystallogr. 24, 946- 950 (1991).

    Article  Google Scholar 

  40. Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281-296 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge funding from the National Institutes of Health, the Wellcome Trust and the Biotechnology and Biological Sciences Research Council. We thank the Central Laboratory of the Research Council and the staff of the Daresbury Laboratory for the provision of synchrotron radiation facilities and the BBSRC for support of such usage through the Rolling Project Mode Time allocation to York.

Author information

Authors and Affiliations

  1. Department of Chemistry, Protein Structure Group, University of York, Heslington, YO1 5DD, York, UK

    Heidi L. Schubert & Keith S. Wilson

  2. Department of Molecular Genetics, Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK

    Evelyne Raux, Sarah C. Woodcock & Martin J. Warren

Authors
  1. Heidi L. Schubert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Keith S. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Evelyne Raux
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sarah C. Woodcock
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martin J. Warren
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Keith S. Wilson.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schubert, H., Wilson, K., Raux, E. et al. The X-ray structure of a cobalamin biosynthetic enzyme, cobalt-precorrin-4 methyltransferase. Nat Struct Mol Biol 5, 585–592 (1998). https://doi.org/10.1038/846

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/846

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing