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. 2000 Jul;9(7):1374–1381. doi: 10.1110/ps.9.7.1374

The X-ray structure of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.

A F Monzingo 1, A Breksa 1, S Ernst 1, D R Appling 1, J D Robertus 1
PMCID: PMC2144683  PMID: 10933503

Abstract

Eucaryotes possess one or more NADP-dependent methylene-THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD-dependent enzyme, yMTD. We report X-ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 A resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD-binding domain that has two inserted strands compared with the NADP-binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein.

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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., West M. G. Monofunctional NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae. Methods Enzymol. 1997;281:178–188. doi: 10.1016/s0076-6879(97)81024-5. [DOI] [PubMed] [Google Scholar]
  3. Barlowe C. K., Appling D. R. Isolation and characterization of a novel eukaryotic monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase. Biochemistry. 1990 Jul 31;29(30):7089–7094. doi: 10.1021/bi00482a020. [DOI] [PubMed] [Google Scholar]
  4. Birktoft J. J., Rhodes G., Banaszak L. J. Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5-A resolution. Biochemistry. 1989 Jul 11;28(14):6065–6081. doi: 10.1021/bi00440a051. [DOI] [PubMed] [Google Scholar]
  5. Hoffman D. W., Davies C., Gerchman S. E., Kycia J. H., Porter S. J., White S. W., Ramakrishnan V. Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein. EMBO J. 1994 Jan 1;13(1):205–212. doi: 10.1002/j.1460-2075.1994.tb06250.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Kleywegt G. J., Jones T. A. xdlMAPMAN and xdlDATAMAN - programs for reformatting, analysis and manipulation of biomacromolecular electron-density maps and reflection data sets. Acta Crystallogr D Biol Crystallogr. 1996 Jul 1;52(Pt 4):826–828. doi: 10.1107/S0907444995014983. [DOI] [PubMed] [Google Scholar]
  8. MacKenzie R. E. Mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase. Methods Enzymol. 1997;281:171–177. doi: 10.1016/s0076-6879(97)81023-3. [DOI] [PubMed] [Google Scholar]
  9. Mejia N. R., MacKenzie R. E. NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed by immortal cells. J Biol Chem. 1985 Nov 25;260(27):14616–14620. [PubMed] [Google Scholar]
  10. Monzingo A. F., West M. G., Schelp E., Appling D. R., Robertus J. D. Crystallization of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae. Proteins. 1996 Dec;26(4):481–482. doi: 10.1002/(SICI)1097-0134(199612)26:4<481::AID-PROT10>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  11. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Ramakrishnan V., Biou V. Treatment of multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. Methods Enzymol. 1997;276:538–557. [PubMed] [Google Scholar]
  14. Shen B. W., Dyer D. H., Huang J. Y., D'Ari L., Rabinowitz J., Stoddard B. L. The crystal structure of a bacterial, bifunctional 5,10 methylene-tetrahydrofolate dehydrogenase/cyclohydrolase. Protein Sci. 1999 Jun;8(6):1342–1349. doi: 10.1110/ps.8.6.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Terwilliger T. C., Berendzen J. Automated MAD and MIR structure solution. Acta Crystallogr D Biol Crystallogr. 1999 Apr;55(Pt 4):849–861. doi: 10.1107/S0907444999000839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. West M. G., Horne D. W., Appling D. R. Metabolic role of cytoplasmic isozymes of 5,10-methylenetetrahydrofolate dehydrogenase in Saccharomyces cerevisiae. Biochemistry. 1996 Mar 5;35(9):3122–3132. doi: 10.1021/bi952713d. [DOI] [PubMed] [Google Scholar]
  18. Yang X. M., MacKenzie R. E. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene. Biochemistry. 1993 Oct 19;32(41):11118–11123. doi: 10.1021/bi00092a022. [DOI] [PubMed] [Google Scholar]

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