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BluB cannibalizes flavin to form the lower ligand of vitamin B12

Nature volume 446, pages 449–453 (2007)Cite this article

Abstract

Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products, and the only vitamin synthesized exclusively by microorganisms1. The biosynthesis of the lower ligand of vitamin B12, 5,6-dimethylbenzimidazole (DMB), is poorly understood1,2,3. Recently, we discovered that a Sinorhizobium meliloti gene, bluB, is necessary for DMB biosynthesis4. Here we show that BluB triggers the unprecedented fragmentation and contraction of the bound flavin mononucleotide cofactor and cleavage of the ribityl tail to form DMB and d-erythrose 4-phosphate. Our structural analysis shows that BluB resembles an NAD(P)H-flavin oxidoreductase, except that its unusually tight binding pocket accommodates flavin mononucleotide but not NAD(P)H. We characterize crystallographically an early intermediate along the reaction coordinate, revealing molecular oxygen poised over reduced flavin. Thus, BluB isolates and directs reduced flavin to activate molecular oxygen for its own cannibalization. This investigation of the biosynthesis of DMB provides clarification of an aspect of vitamin B12 that was otherwise incomplete, and may contribute to a better understanding of vitamin B12-related disease.

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Figure 1: BluB catalyses DMB production.
Figure 2: Structure of BluB.
Figure 3: Active site of BluB.
Figure 4: Mutation of Asp 32 or Ser 167 eliminates DMB production.

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Acknowledgements

This work was supported by NIH grants to G.C.W. and C.T.W. and postdoctoral fellowships from the Jane Coffin Childs Memorial Fund for Medical Research to M.E.T. and N.A.L. G.C.W. is an American Cancer Society Research Professor. We are grateful to E. Yeh for providing purified SsuE and H. Zhang for purified riboflavin kinase. We thank C. Sheahan and G. Heffron for their assistance with 31P-NMR, and A. Haykov for assistance with protein purification. We acknowledge the Advanced Light Source for beam time. We thank S. Harrison, T. Begley, C. Drennan and members of the Walsh and Walker laboratories for helpful discussions.

The coordinates and structure factors for BluB-FMN, BluB-FMNA (flavin anion) and BluB-FMNH2 have been deposited in the Protein Data Bank under accession codes 2ISJ, 2ISK and 2ISL, respectively.

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Author notes

  1. Michiko E. Taga and Nicholas A. Larsen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA,

    Michiko E. Taga & Graham C. Walker

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA,

    Nicholas A. Larsen, Annaleise R. Howard-Jones & Christopher T. Walsh

Authors
  1. Michiko E. Taga
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Correspondence to Christopher T. Walsh or Graham C. Walker.

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Supplementary Information

This file contains Supplementary Figures 1-7 with Legends, Supplementary Tables 1-4 with Legends, Supplementary Discussion, Supplementary Scheme 1, Supplementary Methods and additional references. (PDF 661 kb)

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Taga, M., Larsen, N., Howard-Jones, A. et al. BluB cannibalizes flavin to form the lower ligand of vitamin B12. Nature 446, 449–453 (2007). https://doi.org/10.1038/nature05611

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