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:

Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferases

Abstract

Chalcone O-methyltransferase (ChOMT) and isoflavone O-methyltransferase (IOMT) are S-adenosyl-l-methionine (SAM) dependent plant natural product methyltransferases involved in secondary metabolism in Medicago sativa (alfalfa). Here we report the crystal structure of ChOMT in complex with the product S-adenosyl-l-homocysteine and the substrate isoliquiritigenin (4,2′,4′-trihydroxychalcone) refined to 1.8 Å as well as the crystal structure of IOMT in complex with the products S-adenosyl-l-homocysteine and isoformononetin (4′-hydroxy-7-methoxyisoflavone) refined to 1.4 Å. These two OMTs constitute the first plant methyltransferases to be structurally characterized and reveal a novel oligomerization domain and the molecular determinants for substrate selection. As such, this work provides a structural basis for understanding the substrate specificity of the diverse family of plant OMTs and facilitates the engineering of novel activities in this extensive class of natural product biosynthetic enzymes.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Minimal phenylpropanoid biosynthetic pathway in M. sativa L (alfalfa)48.
Figure 2: Architecture of the ChOMT and IOMT monomers.
Figure 3: Architecture of the ChOMT and IOMT dimers and active sites.
Figure 4: Structural and sequence comparisons of representative OMTs.
Figure 5: ChOMT and IOMT active sites.
Figure 6: Thin layer chromatography assay of ChOMT and IOMT catalytic His mutants.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Hartwig, U.A., Maxwell, C.A., Joseph, C.M. & Phillips, D.A. Effects of alfalfa nod gene-inducing flavonoids on nodABC transcription in Rhizobium meliloti strains containing different nodD genes. J. Bacteriol. 172, 2769–2773 (1990).

    Article  CAS  PubMed Central  Google Scholar 

  2. Peters, N.K., Frost, J.W. & Long, S.R. A plant flavone, luteolin, induces expression of Rhizobium meliloti genes. Science 233, 977–980 (1986).

    Article  CAS  Google Scholar 

  3. Smith, D.A. & Banks, S.W. Formation and biological properties of isoflavonoid phytoalexins. Prog. Clin. Biol. Res. 213, 113–124 (1986).

    CAS  PubMed  Google Scholar 

  4. Ingham, J.L. & Millar, R.L. Sativin: an induced isoflavan from the leaves of Medicago sativa L. Nature 242, 125–126 (1973).

    Article  CAS  Google Scholar 

  5. Dobson, H.E.M. Floral volatiles in insect biology. In Insect-plant interactions. (ed., Bernays, E.) 47–81 (CRC Press, Boca Raton, Florida; 1993).

    Google Scholar 

  6. Lewis, N.G., Davin, L.B. & Sarkanen, S. The nature and function of lignins. In Comprehensive natural products chemistry 3 (eds Barton, D. & Nakanishi, K.) (Elsevier Science Ltd., Amsterdam; 1999).

    Google Scholar 

  7. Barton, D. & Nakanishi, K. Comprehensive Natural Products Chemistry 1 (Elsevier Science Ltd., Amsterdam; 1999).

    Google Scholar 

  8. Maxwell, C.A., Harrison, M.J. & Dixon, R.A. Molecular characterization and expression of alfalfa isoliquiritigenin 2′-O-methyltransferase, an enzyme specifically involved in the biosynthesis of an inducer of Rhizobium meliloti nodulation genes. Plant J. 4, 971–981 (1993).

    Article  CAS  Google Scholar 

  9. Maxwell, C.A., Edwards, R. & Dixon, R.A. Identification, purification, and characterization of S-adenosyl-L-methionine: isoliquiritigenin 2'-O-methyltransferase from alfalfa (Medicago sativa L.). Arch. Biochem. Biophys. 293, 158–166 (1992).

    Article  CAS  Google Scholar 

  10. Jez, J.M., Bowman, M.E., Dixon, R.A. & Noel, J.P. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase. Nature Struct. Biol. 7, 786–791 (2000).

    Article  CAS  Google Scholar 

  11. He, X-Z. & Dixon, R.A. Affinity chromatography, substrate/product specificity, and amino acid sequence analysis of an isoflavone O-methyltransferase from alfalfa (Medicago sativa L.). Arch. Biochem. Biophys. 336, 121–129 (1996).

    Article  CAS  Google Scholar 

  12. He, X-Z., Reddy, J.T. & Dixon, R.A. Stress response in alfalfa (Medicago sativa L). XXII. cDNA cloning and characterization of an elicitor-inducible isoflavone 7-O-methyltranferase. Plant Mol. Biol. 36, 43–54 (1998).

    Article  CAS  Google Scholar 

  13. 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 

  14. Cheng, X., Kumar, S., Posfai, J., Pflugrath, J.W. & Roberts, R.J. Crystal structure of the HhaI DNA methyltranferase complexed with S-adenosyl-L-methionine. Cell 74, 299–307 (1993).

    Article  CAS  Google Scholar 

  15. Schroeder, S.G. & Samudzi, C.T. Structural studies of EcoRII methylase: exploring similarities among methylases. Protein Eng. 10, 1385–1393 (1997).

    Article  CAS  Google Scholar 

  16. Djordjevic, S. & Stock, A.M. Crystal structure of the chemotaxis receptor methyltransferase CheR suggests a conserved structural motif for binding S-adenosylmethionine. Structure 5, 545–558 (1997).

    Article  CAS  Google Scholar 

  17. Schluckebier, G., Zhong, P., Stewart, K.D., Kavanaugh, T.J. & Abad-Zapatero, C. The 2.2 Å structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism. J. Mol. Biol. 289, 277–291 (1999).

    Article  CAS  Google Scholar 

  18. Tran, P.H., Korszun, Z.R., Cerritelli, S., Springhorn, S.S. & Lacks, S.A. Crystal structure of the DpnM DNA adenine methyltransferase from the DpnII restriction system of Streptococcus pneumoniae bound to S-adenosylmethionine. Structure 6, 1563–1575 (1998).

    Article  CAS  Google Scholar 

  19. Schluckebier, G., Kozak, M., Bleimling, N., Weinhold, E. & Saenger, W. Differential binding of S-adenosylmethionine S-adenosylhomocysteine and sinefungin to the adenine-specific DNA methyltransferase M.TaqI. J. Mol. Biol. 265, 56–67 (1997).

    Article  CAS  Google Scholar 

  20. Gong, W., O'Gara, M., Blumenthal, R.M. & Cheng, X. Structure of PvuII DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment. Nucleic Acids Res. 25, 2702–2715 (1997).

    Article  CAS  PubMed Central  Google Scholar 

  21. Hashimoto, H., Inoue, T., Nishioka, M., Fujiwara, S., Takagi, M., Imanaka, T. & Kai, Y. Hyperthermostable protein structure maintained by intra and inter-helix ion-pairs in archaeal O6-methylguanine-DNA methyltransferase. J. Mol. Biol. 292, 707–716 (1999).

    Article  CAS  Google Scholar 

  22. Vidgren, J., Svensson, L. & Liljas, A. Crystal structure of catechol O-methyltransferase. Nature 368, 354–358 (1994).

    Article  CAS  Google Scholar 

  23. Pattanayek, R., Newcomer, M.E. & Wagner, C. Crystal structure of apo-glycine N-methyltransferase (GNMT). Protein Sci. 7, 1326–1331 (1998).

    Article  CAS  PubMed Central  Google Scholar 

  24. Zhang, X., Zhou, L. & Cheng, X. Crystal structure of the conserved core of protein arginine methyltransferase PRMT3. EMBO J. 19, 3509–3519 (2000).

    Article  CAS  PubMed Central  Google Scholar 

  25. Kagan, R.M. & Clarke, S. Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. Arch. Biochem. Biophys. 310, 417–427 (1994).

    Article  CAS  Google Scholar 

  26. O'Gara, M., McCloy, K., Malone, T. & Cheng, X. Structure-based alignment of three Ado-Met-dependent DNA methyltransferases. Gene 157, 135–138 (1995).

    Article  CAS  Google Scholar 

  27. 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 

  28. Rossmann, M.G., Moras, D. & Olsen, K.W. Chemical and biological evolution of a nucleotide-binding protein. Nature 250, 194–199 (1974).

    Article  CAS  Google Scholar 

  29. Frick, S. & Kutchan, T.M. Molecular cloning and functional expression of O-methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis. Plant J. 17, 329–339 (1999).

    Article  CAS  Google Scholar 

  30. He, X-Z. & Dixon, R.A. Genetic manipulation of isoflavone 7-O-methyltransferase enhances the biosynthesis of 4′-O-methylated isoflavonoid phytoalexins and disease resistance in alfalfa. Plant Cell 12, 1689–1702 (2000).

    Article  CAS  PubMed Central  Google Scholar 

  31. Steele, C.L., Gijzen, M., Qutob, D. & Dixon, R.A. Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis. Arch. Biochem. Biophys. 367, 146–150 (1999).

    Article  CAS  Google Scholar 

  32. Akashi, T., Aoki, T. & Ayabe, S. Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice. Plant Physiol. 121, 821–828 (1999).

    Article  CAS  PubMed Central  Google Scholar 

  33. Jung, W., Yu, O., Lau, S.M.C., O'Keefe, D.P., Odell, J., Fader, G. & McGonigle, B. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes. Nature Biotechnol. 18, 208–212 (2000).

    Article  CAS  Google Scholar 

  34. Hashim, M. F., Hakamatsuka, T., Ebizuka, Y. & Sankawa U. Reaction mechanism of oxidative rearrangement of flavanone in isoflavone biosynthesis. FEBS Lett. 271, 219–222 (1990).

    Article  CAS  Google Scholar 

  35. Kochs, G. & Grisebach, H. Enzymatic synthesis of isoflavones. Eur. J. Biochem. 155, 311–318 (1986).

    Article  CAS  Google Scholar 

  36. Walsh, C. Enzymatic reaction mechanisms. (W.H. Freeman and Company, San Francisco; 1979).

    Google Scholar 

  37. Ibrahim, R.K., Bruneau, A. & Bantignies, B. Plant O-methyltransferases: molecular analysis, common signature and classification. Plant Mol. Biol. 36, 1–10 (1998).

    Article  CAS  Google Scholar 

  38. Ibrahim, R.K. Plant O-methyltransferase signatures. Trends Plant Sci. 2, 249–250 (1997).

    Article  Google Scholar 

  39. Jez, J.M., Ferrer, J.L., Bowman, M.E., Dixon, R.A. & Noel, J.P. Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry 39, 890–902 (2000).

    Article  CAS  Google Scholar 

  40. Doublie, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997).

    Article  CAS  Google Scholar 

  41. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed Central  Google Scholar 

  42. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999).

    Article  CAS  Google Scholar 

  43. de la Fourtelle, E. & Bricogne, G. Maximum likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997).

    Article  Google Scholar 

  44. Abrahams, J.P. & Leslie, A.G.W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 49, 148–157 (1996).

    Google Scholar 

  45. Brunger, A.T. et al. Crystallography and NMR system: a new software suite for macromolecular structure dertermination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  46. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. D 49, 148–157 (1993).

    Google Scholar 

  47. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thrornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  48. Dixon, R.A. & Paiva, N.L. Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085–1097 (1995).

    Article  CAS  PubMed Central  Google Scholar 

  49. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  51. Amundsen, S. et al. X-POV-Team POV-Ray: persistence of vision ray-tracer. http://www.povray.org (1997).

Download references

Acknowledgements

We acknowledge the assistance provided by members of the Noel group and the staff of beamlines 7-1 and 9-2 at the Stanford Synchrotron Radiation Facility. The SSRL Biotechnology Program is supported by the NIH, National Center for Research Resources, Biomedical Technology Program, and the DOE, Office of Biological and Environmental Research. This work was supported by funds from the Salk Institute and the National Science Foundation awarded to J.P.N. C.Z. was supported by funds from the NIH Molecular Biophysics Training Grant administered by the University of California, San Diego and funds from the Samuel Roberts Noble Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Joseph P. Noel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zubieta, C., He, XZ., Dixon, R. et al. Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferases. Nat Struct Mol Biol 8, 271–279 (2001). https://doi.org/10.1038/85029

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/85029

This article is cited by

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