Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Jun;8(6):1342–1349. doi: 10.1110/ps.8.6.1342

The crystal structure of a bacterial, bifunctional 5,10 methylene-tetrahydrofolate dehydrogenase/cyclohydrolase.

B W Shen 1, D H Dyer 1, J Y Huang 1, L D'Ari 1, J Rabinowitz 1, B L Stoddard 1
PMCID: PMC2144347  PMID: 10386884

Abstract

The structure of a bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase from Escherichia coli has been determined at 2.5 A resolution in the absence of bound substrates and compared to the NADP-bound structure of the homologous enzyme domains from a trifunctional human synthetase enzyme. Superposition of these structures allows the identification of a highly conserved cluster of basic residues that are appropriately positioned to serve as a binding site for the poly-gamma-glutamyl tail of the tetrahydrofolate substrate. Modeling studies and molecular dynamic simulations of bound methylene-tetrahydrofolate and NADP shows that this binding site would allow interaction of the nicotinamide and pterin rings in the dehydrogenase active site. Comparison of these enzymes also indicates differences between their active sites that might allow the development of inhibitors specific to the bacterial target.

Full Text

The Full Text of this article is available as a PDF (312.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allaire M., Li Y., MacKenzie R. E., Cygler M. The 3-D structure of a folate-dependent dehydrogenase/cyclohydrolase bifunctional enzyme at 1.5 A resolution. Structure. 1998 Feb 15;6(2):173–182. doi: 10.1016/s0969-2126(98)00019-7. [DOI] [PubMed] [Google Scholar]
  2. Appling D. R., Rabinowitz J. C. Evidence for overlapping active sites in a multifunctional enzyme: immunochemical and chemical modification studies on C1-tetrahydrofolate synthase from Saccharomyces cerevisiae. Biochemistry. 1985 Jul 2;24(14):3540–3547. doi: 10.1021/bi00335a023. [DOI] [PubMed] [Google Scholar]
  3. Brünger A. T. Assessment of phase accuracy by cross validation: the free R value. Methods and applications. Acta Crystallogr D Biol Crystallogr. 1993 Jan 1;49(Pt 1):24–36. doi: 10.1107/S0907444992007352. [DOI] [PubMed] [Google Scholar]
  4. Butterworth C. E., Jr, Bendich A. Folic acid and the prevention of birth defects. Annu Rev Nutr. 1996;16:73–97. doi: 10.1146/annurev.nu.16.070196.000445. [DOI] [PubMed] [Google Scholar]
  5. Cheung E., D'Ari L., Rabinowitz J. C., Dyer D. H., Huang J. Y., Stoddard B. L. Purification, crystallization, and preliminary x-ray studies of a bifunctional 5,10-methenyl/methylene-tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli. Proteins. 1997 Feb;27(2):322–324. doi: 10.1002/(sici)1097-0134(199702)27:2<322::aid-prot19>3.0.co;2-o. [DOI] [PubMed] [Google Scholar]
  6. Cohen L., Mackenzie R. E. Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase from porcine liver. Interaction between the dehydrogenase and cyclohydrolase activities of the multifunctional enzyme. Biochim Biophys Acta. 1978 Feb 10;522(2):311–317. doi: 10.1016/0005-2744(78)90065-7. [DOI] [PubMed] [Google Scholar]
  7. D'Ari L., Rabinowitz J. C. Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli. J Biol Chem. 1991 Dec 15;266(35):23953–23958. [PubMed] [Google Scholar]
  8. Drummond D., Smith S., MacKenzie R. E. Methylenetetrahydrofolate dehydrogenase - methenyltetrahydrofolate cyclohydrolase - formyltetrahydrofolate synthetase from porcine liver: evidence to support a common dehydrogenase-cyclohydrolase site. Can J Biochem Cell Biol. 1983 Nov;61(11):1166–1171. doi: 10.1139/o83-150. [DOI] [PubMed] [Google Scholar]
  9. Green J. M., MacKenzie R. E., Matthews R. G. Substrate flux through methylenetetrahydrofolate dehydrogenase: predicted effects of the concentration of methylenetetrahydrofolate on its partitioning into pathways leading to nucleotide biosynthesis or methionine regeneration. Biochemistry. 1988 Oct 18;27(21):8014–8022. doi: 10.1021/bi00421a007. [DOI] [PubMed] [Google Scholar]
  10. Hum D. W., MacKenzie R. E. Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli. Protein Eng. 1991 Apr;4(4):493–500. doi: 10.1093/protein/4.4.493. [DOI] [PubMed] [Google Scholar]
  11. Jacobsen D. W. Homocysteine and vitamins in cardiovascular disease. Clin Chem. 1998 Aug;44(8 Pt 2):1833–1843. [PubMed] [Google Scholar]
  12. 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 A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  13. Kamb A., Finer-Moore J. S., Stroud R. M. Cofactor triggers the conformational change in thymidylate synthase: implications for an ordered binding mechanism. Biochemistry. 1992 Dec 29;31(51):12876–12884. doi: 10.1021/bi00166a024. [DOI] [PubMed] [Google Scholar]
  14. Matthews D. A., Villafranca J. E., Janson C. A., Smith W. W., Welsh K., Freer S. Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate. J Mol Biol. 1990 Aug 20;214(4):937–948. doi: 10.1016/0022-2836(90)90347-O. [DOI] [PubMed] [Google Scholar]
  15. Montfort W. R., Perry K. M., Fauman E. B., Finer-Moore J. S., Maley G. F., Hardy L., Maley F., Stroud R. M. Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate. Biochemistry. 1990 Jul 31;29(30):6964–6977. doi: 10.1021/bi00482a004. [DOI] [PubMed] [Google Scholar]
  16. Ohlsson I., Nordström B., Brändén C. I. Structural and functional similarities within the coenzyme binding domains of dehydrogenases. J Mol Biol. 1974 Oct 25;89(2):339–354. doi: 10.1016/0022-2836(74)90523-3. [DOI] [PubMed] [Google Scholar]
  17. Oshiro C. M., Kuntz I. D., Dixon J. S. Flexible ligand docking using a genetic algorithm. J Comput Aided Mol Des. 1995 Apr;9(2):113–130. doi: 10.1007/BF00124402. [DOI] [PubMed] [Google Scholar]
  18. Pawelek P. D., MacKenzie R. E. Methenyltetrahydrofolate cyclohydrolase is rate limiting for the enzymatic conversion of 10-formyltetrahydrofolate to 5,10-methylenetetrahydrofolate in bifunctional dehydrogenase-cyclohydrolase enzymes. Biochemistry. 1998 Jan 27;37(4):1109–1115. doi: 10.1021/bi971906t. [DOI] [PubMed] [Google Scholar]
  19. Pelletier J. N., MacKenzie R. E. Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase. Biochemistry. 1995 Oct 3;34(39):12673–12680. doi: 10.1021/bi00039a025. [DOI] [PubMed] [Google Scholar]
  20. Schirch L. Formyl-methenyl-methylenetetrahydrofolate synthetase from rabbit liver (combined). Evidence for a single site in the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate. Arch Biochem Biophys. 1978 Aug;189(2):283–290. doi: 10.1016/0003-9861(78)90214-x. [DOI] [PubMed] [Google Scholar]
  21. Song J. M., Rabinowitz J. C. The N-terminal, dehydrogenase/cyclohydrolase domain of yeast cytoplasmic trifunctional C1-tetrahydrofolate synthase requires the C-terminal, synthetase domain for the catalytic activity in vitro. FEBS Lett. 1995 Dec 4;376(3):229–232. doi: 10.1016/0014-5793(95)01288-9. [DOI] [PubMed] [Google Scholar]
  22. Wasserman G. F., Benkovic P. A., Young M., Benkovic S. J. Kinetic relationships between the various activities of the formyl-methenyl-methylenetetrahydrofolate synthetase. Biochemistry. 1983 Mar 1;22(5):1005–1013. doi: 10.1021/bi00274a002. [DOI] [PubMed] [Google Scholar]
  23. West M. G., Barlowe C. K., Appling D. R. Cloning and characterization of the Saccharomyces cerevisiae gene encoding NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase. J Biol Chem. 1993 Jan 5;268(1):153–160. [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES