Expression, Purification, and Crystallization of D-Psicose 3-Epimerase from Agrobacterium tumefaciens

  • Kim Kwang-Soo (School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University) ;
  • Kim Hye-Jung (Department of Molecular Biotechnology, Konkuk University) ;
  • Oh Deok-Kun (Department of Molecular Biotechnology, Konkuk University) ;
  • Cheong Jong-Joo (School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University) ;
  • Rhee Sang-Kee (School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University)
  • Published : 2006.04.01

Abstract

D-Psicose 3-epimerase (DPE) catalyzes the interconversion of D-fructose to D-psicose by epimerizing the carbon-3 position. The DPE from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by affinity chromatography on an IMAC, gel filtration chromatography on a Sephacryl S-300 HR, and anion-exchange chromatography on a RESOURCE Q. The molecular mass of the purified enzyme was estimated to be about 135 kDa by Superdex 200 gel filtration chromatography, corresponding to a homotetramer. The enzyme produced crystals suitable for X-ray diffraction to a $2.0{\AA}$ resolution at 100 K. The crystals were found to belong to the orthorhombic space group $P2_12_12_1$, with unit-cell parameters a=102.4, b=113.0, and $c=131.8{\AA}$. In addition, the calculated packing parameter $(V_m)$ was $2.79{\AA}^3/Da$, the solvent content was 55.92%, and an asymmetric unit consisted of four monomers.

Keywords

References

  1. Baek, D. H., Y. J. Lee, H. S. Sin, and D. K. Oh. 2004. A new thermophile strain of Geobacillus thermodenitrificans having L-arabinose isomerase activity for tagatose production. J. Microbiol. Biotechnol. 14: 312-316
  2. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Cree, G. M. and A. S. Perlin. 1968. O-Isopropylidene derivatives of D-allulose (D-psicose) and D-erythro-hexopyranose-2, 3- diulose. Can. J. Biochem. 46: 765-770 https://doi.org/10.1139/o68-117
  4. Ishida, Y., T. Kamiya, H. Itoh, Y. Kimura, and K. Izumori. 1997. Cloning and characterization of the D-tagatose 3- epimerase gene from Pseudomonas cichorii ST-24. J. Ferment. Bioeng. 83: 529-534 https://doi.org/10.1016/S0922-338X(97)81132-4
  5. Itoh, H., H. Okaya, A. R. Khan, S. Tajima, S. Hayakawa, and K. Izumori. 1994. Purification and characterization of Dtagatose 3-epimerase from Pseudomonas sp. ST-24. Biosci. Biotech. Biochem. 58: 2168-2171 https://doi.org/10.1271/bbb.58.2168
  6. Itoh, H., T. Sato, and K. Izumori. 1995. Preparation of $_D$-psicose from $_D$-fructose by immobilized D-tagatose 3- epimerase. J. Ferment. Bioeng. 80: 101-103 https://doi.org/10.1016/0922-338X(95)98186-O
  7. Itoh, H., T. Sato, T. Takeuchi, R. K. Anisur, and K. Izumori. 1995. Preparation of D-sorbose from D-tagatose by immobilized $_D$-tagatose 3-epimerase. J. Ferment. Bioeng. 70: 184-185
  8. Kantardjieff, K. A. and B. Rupp. 2003. Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA, and protein-nucleic acid complex crystals. Protein Sci. 12: 1865-1871 https://doi.org/10.1110/ps.0350503
  9. Kim, H. J., E. K. Hyun, Y. S. Kim, Y. J. Lee, and D. K. Oh. 2006. Characterization of an Agrobacterium tumefaciens Dpsicose 3-epimerase that converts $_D$-fructose to $_D$-psicose. Appl. Envir. Microbiol. 72: 981-985 https://doi.org/10.1128/AEM.72.2.981-985.2006
  10. Matsuo, T., H. Suzuki, M. Hashiguchi, and K. Izumori. 2002. D-Psicose is a rare sugar that provides no energy to growing rats. J. Nutr. Sci. Vitaminol. 48: 77-80 https://doi.org/10.3177/jnsv.48.77
  11. Matsuo, T. and K. Izumori. 2004. $_D$-Psicose, a rare sugar that provides no energy and additionally beneficial effects for clinical nutrition. Asia Pac. J. Clin. Nutr. 13: S127
  12. Matsuo, T., Y. Baba, M. Hashiguchi, K. Takeshita, K. Izumori, and H. Suzuki. 2001. Dietary D-psicose, a C-3 epimer of D-fructose, suppresses the activity of hepatic lipogenic enzymes in rats. Asia Pac. J. Clin. Nutr. 10: 233- 237 https://doi.org/10.1046/j.1440-6047.2001.00246.x
  13. Matthews, B. W. 1968. Solvent content of protein crystals. J. Mol. Biol. 33: 491-497 https://doi.org/10.1016/0022-2836(68)90205-2
  14. Otwinowski, Z. and W. Minor. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 267: 307-326
  15. Takeshita, K., A. Suga, G. Takada, and K. Izumori. 2000. Mass production of $_D$-psicose from $_D$-fructose by a continuous bioreactor system using immobilized $_D$-tagatose 3-epimerase. J. Biosci. Bioeng. 90: 453-455 https://doi.org/10.1016/S1389-1723(01)80018-9
  16. Terwilliger, T. C. and J. Berendzen. 1999. Automated MAD and MIR structure solution. Acta Crystallog D Biol. Crystallogr. 55: 849-861 https://doi.org/10.1107/S0907444999000839