Cell Biochemistry and Biophysics

, Volume 65, Issue 1, pp 57–68

Key Residues at the Riboflavin Kinase Catalytic Site of the Bifunctional Riboflavin Kinase/FMN Adenylyltransferase From Corynebacterium ammoniagenes

  • Ana Serrano
  • Susana Frago
  • Beatriz Herguedas
  • Marta Martínez-Júlvez
  • Adrián Velázquez-Campoy
  • Milagros Medina
Original Paper


Many known prokaryotic organisms depend on a single bifunctional enzyme, encoded by the RibC of RibF gene and named FAD synthetase (FADS), to convert Riboflavin (RF), first into FMN and then into FAD. The reaction occurs through the sequential action of two activities present on a single polypeptide chain where the N-terminus is responsible for the ATP:FMN adenylyltransferase (FMNAT) activity and the C-terminus for the ATP: riboflavin kinase (RFK) activity. Sequence and structural analysis suggest that T208, N210 and E268 at the C-terminus RFK module of Corynebacterium ammoniagenes FADS (CaFADS) might be key during RF phosphorylation. The effect of site-directed mutagenesis on the RFK activity, as well as on substrates and products binding, indicates that T208 and N210 provide the RFK active-site geometry for binding and catalysis, while E268 might be involved in the catalytic step as catalytic base. These data additionally suggest concerted conformational changes at the RFK module of CaFADS during its activity. Mutations at the RFK site also modulate the binding parameters at the FMNAT active site of CaFADS, altering the catalytic efficiency in the transformation of FMN into FAD. This observation supports the hypothesis that the hexameric assembly previously revealed by the crystal structure of CaFADS might play a functional role during catalysis.


FAD synthetase ATP:riboflavin kinase ATP:FMN adenylyltransferase Site-directed mutagenesis Substrate binding Catalytic activity 



FAD synthetase




Flavin mononucleotide


Flavin adenine dinucleotide


Adenosine 5′-triphosphate


ATP:riboflavin kinase


ATP:FMN adenylyltransferase


1,4-Piperazine diethane sulphonic acid


Isothermal titration calorimetry


High-performance liquid chromatography




Circular dichroism




FAD and FMN fluorescence constants

Supplementary material

12013_2012_9403_MOESM1_ESM.pdf (686 kb)
Additional information includes; oligonucleotides for site-directed mutagenesis and methods for determination of kinetic and binding parameters; Table SD.1 with crystallographic data, Tables SD.2, SD.3 and SD.4 with thermodynamic parameters; Fig. SD.1 with scheme of the RFK and FMNAT activities of CaFADS; Fig. SD.2 with overall folding, topology and logo of sequence at the RFK consensus sequences; Fig. SD.3 with difference spectra; Fig. SD.4 with structural comparison of RFK modules; Fig. SD.5 with the trimeric structure of CaFADS. This material is available free of charge via the Internet at (pdf 686 kb)


  1. 1.
    Manstein, D. J., & Pai, E. F. (1986). Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes. Journal of Biological Chemistry, 261, 16169–16173.PubMedGoogle Scholar
  2. 2.
    Mack, M., van Loon, A. P., & Hohmann, H. P. (1998). Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC. Journal of Bacteriology, 180, 950–955.PubMedGoogle Scholar
  3. 3.
    McCormick, D. B. (1989). Two interconnected B vitamins: riboflavin and pyridoxine. Physiological Reviews, 69, 1170–1198.PubMedGoogle Scholar
  4. 4.
    Powers, H. J. (2003). Riboflavin (vitamin B2) and health. American Journal of Clinical Nutrition, 77, 1352–1360.PubMedGoogle Scholar
  5. 5.
    Bacher, A. (1991). Biosynthesis of flavins. In F. Müller (Ed.), Chemistry and biochemistry of flavoproteins (pp. 215–259). Boca Raton, FL: CRC Press.Google Scholar
  6. 6.
    Bacher, A. (1991). Riboflavin kinase and FAD synthetase. In F. Müller (Ed.), Chemistry and biochemistry of flavoproteins (pp. 349–370). Boca Raton, FL: CRC Press.Google Scholar
  7. 7.
    Eisenreich, W., Schwarzkopf, B., & Bacher, A. (1991). Biosynthesis of nucleotides, flavins, and deazaflavins in Methanobacterium thermoautotrophicum. Journal of Biological Chemistry, 266, 9622–9631.PubMedGoogle Scholar
  8. 8.
    Volk, R., & Bacher, A. (1991). Biosynthesis of riboflavin. Studies on the mechanism of l-3,4-dihydroxy-2-butanone 4-phosphate synthase. Journal of Biological Chemistry, 266, 20610–20618.PubMedGoogle Scholar
  9. 9.
    Efimov, I., Kuusk, V., Zhang, X., & McIntire, W. S. (1998). Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli. Biochemistry, 37, 9716–9723.PubMedCrossRefGoogle Scholar
  10. 10.
    Barile, M., Brizio, C., Valenti, D., De Virgilio, C., & Passarella, S. (2000). The riboflavin/FAD cycle in rat liver mitochondria. European Journal of Biochemistry, 267, 4888–4900.PubMedCrossRefGoogle Scholar
  11. 11.
    Giancaspero, T. A., Locato, V., de Pinto, M. C., De Gara, L., & Barile, M. (2009). The occurrence of riboflavin kinase and FAD synthetase ensures FAD synthesis in tobacco mitochondria and maintenance of cellular redox status. FEBS Journal, 276, 219–231.PubMedCrossRefGoogle Scholar
  12. 12.
    Sandoval, F. J., Zhang, Y., & Roje, S. (2008). Flavin nucleotide metabolism in plants: monofunctional enzymes synthesize FAD in plastids. Journal of Biological Chemistry, 283, 30890–30900.PubMedCrossRefGoogle Scholar
  13. 13.
    Pallotta, M. L., Brizio, C., Fratianni, A., De Virgilio, C., Barile, M., & Passarella, S. (1998). Saccharomyces cerevisiae mitochondria can synthesise FMN and FAD from externally added riboflavin and export them to the extramitochondrial phase. FEBS Letters, 428, 245–249.PubMedCrossRefGoogle Scholar
  14. 14.
    Mashhadi, Z., Xu, H., Grochowski, L. L., & White, R. H. (2010). Archaeal ribL: a new FAD synthetase that is air sensitive. Biochemistry, 49, 8748–8755.PubMedCrossRefGoogle Scholar
  15. 15.
    Mashhadi, Z., Zhang, H., Xu, H., & White, R. H. (2008). Identification and characterization of an archaeon-specific riboflavin kinase. Journal of Bacteriology, 190, 2615–2618.PubMedCrossRefGoogle Scholar
  16. 16.
    Yruela, I., Arilla-Luna, S., Medina, M., & Contreras-Moreira, B. (2010). Evolutionary divergence of chloroplasts FAD synthetase proteins. BMC Evolutionary Biology, 10, 311.PubMedCrossRefGoogle Scholar
  17. 17.
    Torchetti, E. M., Bonomi, F., Galluccio, M., Gianazza, E., Giancaspero, T. A., Lametti, S., et al. (2011). Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins. FEBS Journal, 278, 4434–4449.PubMedCrossRefGoogle Scholar
  18. 18.
    Torchetti, E. M., Brizio, C., Colella, M., Galluccio, M., Giancaspero, T. A., Indiveri, C., et al. (2010). Mitochondrial localization of human FAD synthetase isoform 1. Mitochondrion, 10, 263–273.PubMedCrossRefGoogle Scholar
  19. 19.
    Wu, M., Repetto, B., Glerum, D. M., & Tzagoloff, A. (1995). Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae. Molecular and Cellular Biology, 15, 264–271.PubMedGoogle Scholar
  20. 20.
    Santos, M. A., Jimenez, A., & Revuelta, J. L. (2000). Molecular characterization of FMN1, the structural gene for the monofunctional flavokinase of Saccharomyces cerevisiae. Journal of Biological Chemistry, 275, 28618–28624.PubMedCrossRefGoogle Scholar
  21. 21.
    Herguedas, B., Martínez-Júlvez, M., Frago, S., Medina, M., & Hermoso, J. A. (2010). Oligomeric state in the crystal structure of modular FAD synthetase provides insights in its sequential catalysis in prokaryotes. Journal of Molecular Biology, 400, 218–230.PubMedCrossRefGoogle Scholar
  22. 22.
    Frago, S., Velázquez-Campoy, A., & Medina, M. (2009). The puzzle of ligand binding to Corynebacterium ammoniagenes FAD synthetase. Journal of Biological Chemistry, 284, 6610–6619.PubMedCrossRefGoogle Scholar
  23. 23.
    Frago, S., Martínez-Júlvez, M., Serrano, A., & Medina, M. (2008). Structural analysis of FAD synthetase from Corynebacterium ammoniagenes. BMC Microbiology, 8, 160.PubMedCrossRefGoogle Scholar
  24. 24.
    Krupa, A., Sandhya, K., Srinivasan, N., & Jonnalagadda, S. (2003). A conserved domain in prokaryotic bifunctional FAD synthetases can potentially catalyze nucleotide transfer. Trends in Biochemical Sciences, 28, 9–12.PubMedCrossRefGoogle Scholar
  25. 25.
    Huerta, C., Borek, D., Machius, M., Grishin, N. V., & Zhang, H. (2009). Structure and mechanism of a eukaryotic FMN adenylyltransferase. Journal of Molecular Biology, 389, 388–400.PubMedCrossRefGoogle Scholar
  26. 26.
    Wang, W., Kim, R., Yokota, H., & Kim, S. H. (2005). Crystal structure of flavin binding to FAD synthetase of Thermotoga maritima. Proteins, 58, 246–248.PubMedCrossRefGoogle Scholar
  27. 27.
    Leulliot, N., Blondeau, K., Keller, J., Ulryck, N., Quevillon-Cheruel, S., & van Tilbeurgh, H. (2010). Crystal structure of yeast FAD synthetase (Fad1) in complex with FAD. Journal of Molecular Biology, 398, 641–646.PubMedCrossRefGoogle Scholar
  28. 28.
    Karthikeyan, S., Zhou, Q., Osterman, A. L., & Zhang, H. (2003). Ligand binding-induced conformational changes in riboflavin kinase: structural basis for the ordered mechanism. Biochemistry, 42, 12532–12538.PubMedCrossRefGoogle Scholar
  29. 29.
    Bauer, S., Kemter, K., Bacher, A., Huber, R., Fischer, M., & Steinbacher, S. (2003). Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold. Journal of Molecular Biology, 326, 1463–1473.PubMedCrossRefGoogle Scholar
  30. 30.
    Herguedas, B., Martínez-Júlvez, M., Frago, S., Medina, M., & Hermoso, J. A. (2009). Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes. Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 65, 1285–1288.CrossRefGoogle Scholar
  31. 31.
    Karthikeyan, S., Zhou, Q., Mseeh, F., Grishin, N. V., Osterman, A. L., & Zhang, H. (2003). Crystal structure of human riboflavin kinase reveals a beta barrel fold and a novel active site arch. Structure, 11, 265–273.PubMedCrossRefGoogle Scholar
  32. 32.
    Cheek, S., Ginalski, K., Zhang, H., & Grishin, N. V. (2005). A comprehensive update of the sequence and structure classification of kinases. BMC Structural Biology, 5, 6.PubMedCrossRefGoogle Scholar
  33. 33.
    Leskovac, V. (2003). Comprehensive enzyme kinetics. New York: Kluwer Adacemic/Plenum Publishers.Google Scholar
  34. 34.
    Kabsch, W. (2010). XDS. Acta Crystallographica. Section D, Biological Crystallography, 66, 125–132.PubMedCrossRefGoogle Scholar
  35. 35.
    CCP4: Collaborative Computational Project, N. (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallographica. Section D, Biological Crystallography, 50, 760–763.CrossRefGoogle Scholar
  36. 36.
    Vagin, A. A., & Teplyakov, A. (1997). MOLREP: an automated program for molecular replacement. Journal of Applied Crystallography, 30, 1022–1025.CrossRefGoogle Scholar
  37. 37.
    Murshudov, G. N., Vagin, A. A., & Dodson, E. J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallographica. Section D, Biological Crystallography, 53, 240–255.PubMedCrossRefGoogle Scholar
  38. 38.
    Emsley, P., & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallographica. Section D, Biological Crystallography, 60, 2126–2132.PubMedCrossRefGoogle Scholar
  39. 39.
    Chen, V. B., Arendall, W. B., I. I. I., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., et al. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica. Section D, Biological Crystallography, 66, 12–21.PubMedCrossRefGoogle Scholar
  40. 40.
    Hagihara, T., Fujio, T., & Aisaka, K. (1995). Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production. Applied Microbiology and Biotechnology, 42, 724–729.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ana Serrano
    • 1
    • 2
  • Susana Frago
    • 1
    • 2
  • Beatriz Herguedas
    • 1
    • 2
  • Marta Martínez-Júlvez
    • 1
    • 2
  • Adrián Velázquez-Campoy
    • 1
    • 2
    • 3
  • Milagros Medina
    • 1
    • 2
  1. 1.Departamento de Bioquímica y Biología Molecular y Celular, Facultad de CienciasUniversidad de ZaragozaSaragossaSpain
  2. 2.Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit BIFI-IQFR (CSIC)Universidad de ZaragozaSaragossaSpain
  3. 3.Fundación ARAIDDiputación General de AragónSaragossaSpain

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