Crystallization and Preliminary X-Ray Diffraction Analysis of 5,10-Methylenetetrahydrofolate Dehydrogenase/Cyclohydrolase from Thermoplasma acidophilum DSM 1728

  • Kim, Jae-Hee (Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University) ;
  • Sung, Min-Woo (Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University) ;
  • Lee, Eun-Hye (Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University) ;
  • Nam, Ki-Hyun (Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University) ;
  • Hwang, Kwang-Yeon (Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University)
  • Published : 2008.02.29

Abstract

The methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFDC) from the thermoacidophilic archaeon Thermoplasma acidophilum is a 30.6kDa molecular-mass enzyme that sequentially catalyzes the conversion of formyltetrahydrofollate to methylenetetrahydrofolate, with a preference for NADP as a cofactor, rather than NAD. In order to elucidate the functional and structural features of MTHFDC from archaeons at a molecular level, it was overexpressed in Escherichia coli and crystallized in the presence of its cofactor, NADP, at 295K using polyethylene glycol (PEG) 4000 as a precipitant. The crystal is a member of the monoclinic space group $P2_1$, with the following unit cell parameters: $a=66.333{\AA},\;b=52.868{\AA},\;c=86.099{\AA},\;and\;{\beta}=97.570^{\circ}$, and diffracts to a resolution of at least $2.40{\AA}$ at the synchrotron. Assuming a dimer in the crystallographic asymmetric unit, the calculated Matthews parameter $(V_M)\;was\;2.44{\AA}^3/Da$ and the solvent content was 49.7%.

Keywords

References

  1. Allaire, M., Y. Li, R. E. MacKenzie, and M. Cygler. 1998. The 3-D structure of a folate-dependent dehydrogenase/cyclohydrolase bifunctional enzyme at 1.5 $\AA$ resolution. Structure 6: 173-182 https://doi.org/10.1016/S0969-2126(98)00019-7
  2. Collaborative Computational Project, Number 4. 1994. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D50: 760-763
  3. Choi, J.-H., T.-K. Kim, Y.-M. Kim, W.-C. Kim, G.-J. Joo, K. Y. Lee, and I.-K. Rhee. 2005. Cloning and characterization of cyclohexanol dehydrogenase gene from Rhodococcus sp. TK6. J. Microbiol. Biotechnol. 15: 1189-1196
  4. Choi, J. J., J. W. Park, H. Shim, S. Lee, M. Kwon, J.-S. Yang, H. Hwang, and S.-T. Kwon. 2006. Cloning, expression, and characterization of a hyperalkaline phosphatase from the thermophilic bacterium Thermus sp. T351. J. Microbiol. Biotechnol. 16: 272-279
  5. D'Ari, L. and J. C. Rabinowitz. 1991. 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. 266: 23953-23958
  6. Green, J. M., R. F. MacKenzie, and R. G. Matthews. 1988. 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 27: 8014-8022 https://doi.org/10.1021/bi00421a007
  7. Hum, D. W., A. W. Bell, R. Rozen, and R. E. MacKenzie. 1988. Primary structure of a human trifunctional enzyme: Isolation of a cDNA encoding methylenetetrahydrofolate dehydrogenasemethenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. J. Biol. Chem. 263: 15946-15950
  8. Kim, K. and H. Kim. 2006. Expression, purification, and crystallization of D-psicose 3-epimerase from Agrobacterium tumefaciens. J. Microbiol. Biotechnol. 16: 647-650
  9. Langsenlehner, U., P. Krippl, W. Renner, B. Yazdani-Biuki, G. Wolf, T. C. Wascher, B. Paulweber, W. Weitzer, and H. Samonigg. 2003. The common 677C>T gene polymorphism of methylenetetrahydrofolate reductase gene is not associated with breast cancer risk. Breast Cancer Res. Treat. 81: 169-172 https://doi.org/10.1023/A:1025752420309
  10. Lee, J. H., Y. J. So, and S. H. Choi. 2006. Regulatory characteristics of the Vibrio vulnificus putAP operon encoding proline dehydrogenase and proline permease. J. Microbiol. Biotechnol. 16: 1285-1291
  11. Lucock, M. 2000. Folic acid: Nutritional biochemistry, molecular biology, and role in disease processes. Molec. Gene. Metab. 71: 121-138 https://doi.org/10.1006/mgme.2000.3027
  12. MacKenzie, R. E. 1984. In Blakley, R. and S. Benkovic (eds.). Folates and Pterins: Chemistry and Biochemistry of Folates. John Wiley and Sons, New York. pp. 255-306
  13. Matthews, B. W. 1968. Solvent content of protein crystals. J. Mol. Biol. 33: 6491-6493
  14. Monzingo, A. F., A. Breksa, S. Ernst, D. R. Appling, and J. D. Robertus. 2000. The X-ray structure of the NADdependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae. Protein Sci. 9: 1374-1381 https://doi.org/10.1110/ps.9.7.1374
  15. Otwinowski, Z. and W. Minor. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276: 307-326 https://doi.org/10.1016/S0076-6879(97)76066-X
  16. Pawelek, P. D. and R. E. MacKenzie. 1998. Methenyltetrahydrofolate cyclohydrolase is rate limiting for the enzymatic conversion of 10-formyltetrahydrofolate to 5,10-methylenetetrahydrofolate in bifunctional dehydrogenasecyclohydrolase enzyme. Biochemistry 37: 1109-1115 https://doi.org/10.1021/bi971906t
  17. Pelletier, J. N. and R. E. MacKenzie. 1994. Binding to the 2', 5'-ADP subsite stimulates cyclohydrolase activity of human $NADP^{+} -dependent$ methylenetetrahydrofolate dehydrogenase/ cyclohydrolase. Biochemistry 33: 1900-1906 https://doi.org/10.1021/bi00173a037
  18. Pelletier, J. N. and R. E. MacKenzie. 1995. Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/ cyclohydrolase. Biochemistry 34: 12673-12680 https://doi.org/10.1021/bi00039a025
  19. Ruepp, R. A., W. Graml, M. L. Santos-Martinez, K. K. Koretke, C. Volker, H. W. Mewes, D. Frishman, S. Stocker, A. N. Lupas, and W. Baumeister. 2000. The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum. Nature 407: 508-513 https://doi.org/10.1038/35035069
  20. Schmidt, A., H. Wu, R. E. MacKenzie, V. J. Chen, J. R. Bewly, J. E. Ray, J. E. Toth, and M. Cygler. 2000. Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/ cyclohydrolase. Biochemistry 39: 6325-6335 https://doi.org/10.1021/bi992734y
  21. Shen, B. W., D. H. Dyer, J. Y. Huang, L. D'Ari, J. Rabinowitz, and B. L. Stoddard. 1999. The crystal structure of a bacterial, bifunctional 5,10 methylene-tetrahydrofolate dehydrogenase/ cyclohydrolase Protein Sci. 8: 1342-1349 https://doi.org/10.1110/ps.8.6.1342