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BluB cannibalizes flavin to form the lower ligand of vitamin B12


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.


  1. Roth, J. R., Lawrence, J. G. & Bobik, T. A. Cobalamin (coenzyme B12): synthesis and biological significance. Annu. Rev. Microbiol. 50, 137–181 (1996)

    Article  CAS  Google Scholar 

  2. Warren, M. J., Finding the final pieces of the vitamin B12 biosynthetic jigsaw. Proc. Natl Acad. Sci. USA 103, 4799–4800 (2006); published online 27 March 2006.

    Article  ADS  CAS  Google Scholar 

  3. Renz, P. in Chemistry and Biochemistry of B12 (ed. Banerjee, R.) 557–575 (John Wiley & Sons, New York, 1999)

    Google Scholar 

  4. Campbell, G. R. O. et al. Sinorhizobium meliloti bluB is necessary for production of 5,6-dimethylbenzimidazole, the lower ligand of B12 . Proc. Natl Acad. Sci. USA 103, 4634–4639 (2006)

    Article  ADS  CAS  Google Scholar 

  5. Renz, P. Riboflavin as precursor in the biosynthesis of the 5,6-dimethylbenzimidazole-moiety of vitamin B12 . FEBS Lett. 6, 187–189 (1970)

    Article  CAS  Google Scholar 

  6. Renz, P. & Weyhenmeyer, R. Biosynthesis of 5,6-dimethylbenzimidazole from riboflavin. Transformation of C-1′ of riboflavin into C-2 of 5,6-dimethylbenzimidazole. FEBS Lett. 22, 124–126 (1972)

    Article  CAS  Google Scholar 

  7. Rodionov, D. A., Vitreschak, A. G., Mironov, A. A. & Gelfand, M. S., Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes. J. Biol. Chem. 278, 41148–41159 (2003); published online 17 July 2003.

  8. Fraaije, M. W. & Mattevi, A. Flavoenzymes: diverse catalysts with recurrent features. Trends Biochem. Sci. 25, 126–132 (2000)

    Article  CAS  Google Scholar 

  9. Tu, S. C. Reduced flavin: donor and acceptor enzymes and mechanisms of channeling. Antioxid. Redox Signal. 3, 881–897 (2001)

    Article  CAS  Google Scholar 

  10. Horig, J. A. & Renz, P. Biosynthesis of vitamin B12. Some properties of the 5,6-dimethylbenzimidazole-forming system of Propionibacterium freudenreichii and Propionibacterium shermanii.. Eur. J. Biochem. 105, 587–592 (1980)

    Article  CAS  Google Scholar 

  11. Eichhorn, E., van der Ploeg, J. R. & Leisinger, T. Characterization of a two-component alkanesulfonate monooxygenase from Escherichia coli. J. Biol. Chem. 274, 26639–26646 (1999)

    Article  CAS  Google Scholar 

  12. Lei, B. & Tu, S. C. Mechanism of reduced flavin transfer from Vibrio harveyi NADPH-FMN oxidoreductase to luciferase. Biochemistry 37, 14623–14629 (1998)

    Article  CAS  Google Scholar 

  13. Bochner, B. R. & Ames, B. N. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J. Biol. Chem. 257, 9759–9769 (1982)

    CAS  PubMed  Google Scholar 

  14. Lingens, B., Schild, T. A., Vogler, B. & Renz, P. Biosynthesis of vitamin B12. Transformation of riboflavin 2H-labeled in the 1′R position of 1′S position into 5,6-dimethylbenzimidazole. Eur. J. Biochem. 207, 981–985 (1992)

    Article  CAS  Google Scholar 

  15. Keck, B., Munder, M. & Renz, P. Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety. Arch. Microbiol. 171, 66–68 (1998)

    Article  CAS  Google Scholar 

  16. Kolonko, B., Horig, J. A. & Renz, P. Transformation of [5′-3H]riboflavin into 5,6-dimethylbenzimidazole. Z. Naturforsch. C 47, 171–176 (1992)

    Article  CAS  Google Scholar 

  17. Maggio-Hall, L. A., Dorrestein, P. C., Escalante-Semerena, J. C. & Begley, T. P. Formation of the dimethylbenzimidazole ligand of coenzyme B12 under physiological conditions by a facile oxidative cascade. Org. Lett. 5, 2211–2213 (2003)

    Article  CAS  Google Scholar 

  18. Tanner, J. T., Lei, B., Tu, S. C. & Krause, K. L. Flavin reductase P: structure of a dimeric enzyme that reduces flavin. Biochemistry 35, 13531–13539 (1996)

    Article  CAS  Google Scholar 

  19. Koike, H. et al. 1.8 Å crystal structure of the major NAD(P)H:FMN oxidoreductase of a bioluminescent bacterium, Vibrio fischeri: overall structure, cofactor and substrate-analog binding, and comparison with related flavoproteins. J. Mol. Biol. 280, 259–273 (1998)

    Article  CAS  Google Scholar 

  20. Hecht, H. J., Erdmann, H., Park, H. J., Sprinzl, M. & Schmid, R. D. Crystal structure of NADH oxidase from Thermus thermophilus. Nature Struct. Mol. Biol. 2, 1109–1114 (1995)

    Article  CAS  Google Scholar 

  21. Lovering, A. L., Hyde, E. I., Searle, P. F. & White, S. A. The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 Å, 1.8 Å and 2.4 Å resolution. J. Mol. Biol. 309, 203–213 (2001)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Larsen, N. A., Lin, H., Wei, R., Fischbach, M. A. & Walsh, C. T. Structural characterization of enterobactin hydrolase IroE. Biochemistry 45, 10184–10190 (2006)

    Article  CAS  Google Scholar 

  24. Eswaramoorthy, S., Bonanno, J. B., Burley, S. K. & Swaminathan, S. Mechanism of action of a flavin-containing monooxygenase. Proc. Natl Acad. Sci. USA 103, 9832–9837 (2006)

    Article  ADS  CAS  Google Scholar 

  25. Gatti, D. L., Entsch, B., Ballou, D. P. & Ludwig, M. L. pH-dependent structural changes in the active site of p-hydroxybenzoate hydroxylase point to the importance of proton and water movements during catalysis. Biochemistry 35, 567–578 (1996)

    Article  CAS  Google Scholar 

  26. Malito, E., Alfieri, A., Fraaije, M. W. & Mattevi, A., Crystal structure of a Baeyer–Villiger monooxygenase. Proc. Natl Acad. Sci. USA 101, 13157–13162 (2004); published online 24 August 2004.

  27. Dong, C. et al. Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination. Science 309, 2216–2219 (2005)

    Article  ADS  CAS  Google Scholar 

  28. Berkovitch, F., Nicolet, Y., Wan, J. T., Jarrett, J. T. & Drennan, C. L. Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 303, 76–79 (2004)

    Article  ADS  CAS  Google Scholar 

  29. Cicchillo, R. M. & Booker, S. J. Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide. J. Am. Chem. Soc. 127, 2860–2861 (2005)

    Article  CAS  Google Scholar 

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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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Correspondence to Christopher T. Walsh or Graham C. Walker.

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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).

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