Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Cloning and Characterization of Zebrafish Microsomal Epoxide Hydrolase Based on Bioinformatics

생물정보학을 이용한 Zebrafish Microsomal Epoxide Hydrolase 클로닝 및 특성연구

  • Lee Eun-Yeol (Department of Food Science and Technology, Kyungsung University) ;
  • Kim Hee-Sook (Department of Food Science and Technology, Kyungsung University)
  • Published : 2006.06.01
Download PDF
⟨ Previous Next ⟩

Abstract

A gene encoding for a putative microsomal epoxide hydrolase (mEH) of a zebrafish, Danio rerio, was cloned and characterized. The putative mEH protein of D. rerio exhibited sequence similarity with mammalian mEH and some other bacterial EHs. A structural model for the putative mEH was constructed using homology modeling based on the crystallographic templates, 1 qo7 and 1 ehy. The catalytic triad consisting of $Asp^{233}$, $Glu^{413}$, and $His^{440}$ was identified, and the characteristic features such as two tyrosine residues and oxyanion hole were found to be highly conserved. Based on bioinformatic analysis together with EH activity assay, the putative protein was annotated as mEH of D. rerio. Enantiopure styrene oxide with enantiopurity of 99%ee and yield of 33.5% was obtained from racemic styrene oxide by the enantioselective hydrolysis activity of recombinant mEH of D. rerio for 45 min.

Zebrafish (Danio rerio)의 microsomal epoxide hydrolase(mEH)로 추정되는 유전자를 클로닝하고 그 특성을 연구하였다. D. rerio의 mEH 추정단백질은 포유동물의 mEH 및 세균의 EH들과 아미노산서열 상동성을 보였으므로 결정분자구조(1qo7 및 1ehy)를 template로 하여 homology modelling을 행하였다. 클로된 단백질은 $Asp^{233}$, $Glu^{413}$ 및 $His^{440}$으로 구성된 catalytic triad와 2개의 tyrosine 잔기 및 oxyanion hole이 보존되어 있었다. 생물정보학적인 분석 및 EH 활성시험은 추정단백질이 D. rerio의 mEH라는 것을 보여주었다. Racemic styrene oxide를 기질로 하여 활성시험을 행한 결과, 재조합 D. rerio mEH는 (R)-styrene oxide을 입체선택적으로 가수분해하였으며 45분 반응시간에 99%ee의 광학순도를 가진 (S)-styrene oxide를 33.5% 얻을 수 있었다.

Keywords

References

  1. Arand, M., H. Wagner, and F. Oesch. 1996. Asp(333), Asp(495), and His(523) form the catalytic triad of rat soluble epoxide hydrolase. J. Biol. Chem. 271: 4223-4229 https://doi.org/10.1074/jbc.271.8.4223
  2. Armstrong, R. N. 1987. Enzyme-catalyzed detoxication reactions: mechanisms and stereochemistry. CRC Crit. Rev. Biochem. 22: 39-88 https://doi.org/10.3109/10409238709082547
  3. Besse, P. and H. Veschambre. 1994. Chemical and biological synthesis of chiral epoxides. Tetrahedron 50: 8885-8927 https://doi.org/10.1016/S0040-4020(01)85362-X
  4. Botes, A. L., J. Lotter, O. H. Rhode, and A. Botha. 2005. Interspecies differences in the enantioselectivity of epoxide hydro lases in Cryptococcus laurentU (Kufferath) C.E. Skinner and Cryptococcus podzolicus (BabJeva & Reshetova) Golubev. Syst. Appl. Microbiol. 28: 27-33
  5. Choi, W. J., E. C. Huh, H. J. Park, E. Y. Lee, and C. Y. Choi. 1998. Kinetic resolution for optically active epoxides by microbial enantioselective hydrolysis. Biotechnol. Tech. 12: 225-228 https://doi.org/10.1023/A:1008825508904
  6. Choi, W. J., E. Y. Lee, S. J. Yoon, and C. Y. Choi. 1999. Biocatalytic production of chiral epichlorohydrin in organic solvent. J. Biosci. Bioeng. 88: 339-341 https://doi.org/10.1016/S1389-1723(00)80022-5
  7. Cleij, M., A. Archelas, and R. Furstoss. 1998. Microbiological transformations 42. A two-phase preparative scale process for an epoxide hydrolase catalysed resolution of parabromo-a-methyl-styrene oxide. Occurrence of a surprising enantioselectivity enhancement. Tetrahedron: Asymmetry 9: 1839-1842 https://doi.org/10.1016/S0957-4166(98)00180-3
  8. Elfstrom, L. T. and M. Widersten. 2005. The Saccharomyces cerevisiae ORF YNR064c protein has characteristics of an 'orphaned' epoxide hydrolase. Biochim. Biophys. Acta 1748: 213-221
  9. Gong, P. F., J. H. Xu, Y. F. Tang, and H. Y. Wu. 2003. Improved catalytic performance of Bacillus megaterium epoxide hydrolase in a medium containing Tween-80. Biotechnol. Prog. 19: 652-654 https://doi.org/10.1021/bp020293v
  10. Hopmann, K. H., B. M. Hallberg, and F. Himo. 2005. Catalytic mechanism of limonene epoxide hydrolase, a theoretical study. J. Am. Chem. Soc. 127: 14339-14347 https://doi.org/10.1021/ja050940p
  11. Kasai, N., T. Suzuki, and Y. Furukawa. 1998. Chiral C3 epoxides and halohydrins: their preparation and synthetic applicaticm. J. Mol. Catal. B: Enzym. 4: 237-252 https://doi.org/10.1016/S1381-1177(97)00034-9
  12. Kim, H. S., J. H. Lee, S. Park, and E. Y. Lee. 2004. Biocatalytic preparation of chiral epichlorohydrins using recombinant Pichia pastoris expressing epoxide hydrolase of Rhodotorula glutinis. Biotechnol. Biopro. Eng. 9: 62-64 https://doi.org/10.1007/BF02949324
  13. Kim, H. S., S. J. Lee, E. J. Lee, J. W. Hwang, S. Park, S. J. Kim, and E. Y. Lee. 2005. Cloning and characterization of a fish microsomal epoxide hydrolase of Danio rerio and application to kinetic resolution of racemic styrene oxide. J. Mol. Celtal. B: Enzym. 37: 30-35
  14. Lee, E. Y., S.-S. Yoo, H. S. Kim, S. J. Lee, Y.-K. Oh, and S. Park. 2004. Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotoru!a g!utinis. Enzyme Microb. Technol. 35: 624-631 https://doi.org/10.1016/j.enzmictec.2004.08.016
  15. Marti-Renom, M. A., A. Stuart, A. Fiser, R. Sanchez, F. Melo, and A. Sali. 2000, Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 29: 291-325 https://doi.org/10.1146/annurev.biophys.29.1.291
  16. Nardini, M., J. S. Ridder, H. J. Rozeboom, K. H. Kalk, R. Rink, D. B. Janssen, and B. W. Dijkstra. 1999. The X-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1. J. Biol. Chem. 274: 14579-14596 https://doi.org/10.1074/jbc.274.21.14579
  17. Qing, G, L. -C. Ma, A. Khorchid, G. V. T. Swapna, T. K. Mal, M. M. Takayama, B. Xia, S. Phadtare, H. Ke, T. Acton, G. T. Montelione, M. Ikura, and M. Inouye. 2004. Coldshock induced high-yield protein production in Escherichia coli. Nature Biotechnol. 22: 877-882 https://doi.org/10.1038/nbt984
  18. Rink, R., J. H. L. Spelberg, R. J. Pieters, J. Kingma, M. Nardini, R. M. Kellogg, B. W. Dijkstra, and D. B. Janssen. 1999. Mutation of tyrosine residues involved in the alkylation half reaction of epoxide hydrolase from Agrobacterium radiobacter ADI results in improved enantioselectivity. J. Am. Chem. Soc. 121: 7417-7418 https://doi.org/10.1021/ja990501o
  19. Roger A. S. and E. J. Milner-White 1995. RasMol: Biomolecular graphics for all. Trends Biochem. Sci. 20: 374-376 https://doi.org/10.1016/S0968-0004(00)89080-5
  20. Tokunaga, M., J. F. Larrow, F. Kakiuchi, and E. N. Jacobsen. 1997. Asymmetric catalysis with water: efficient kinetic resolution of terminal epoxides by means of catalytic hydrolysis. Science 277: 936-938 https://doi.org/10.1126/science.277.5328.936
  21. Weijers, C. A. G. M. and J. A. M. de Bont. 1999. Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis. J. Mol. Catal. B: Enzym. 6: 199-214 https://doi.org/10.1016/S1381-1177(98)00123-4
  22. Xu, Y, J.-H. Xu, J. Pan, L. Zhao, and S.-L. Zhang. 2004. Biocatalytic resolution of nitro-sunstituted phenoxypropylene oxides with Trichosporon loubierii epoxide hydrolase and prediction of their enantiopurity variation with reaction time. J. Mol. Catal. B: Enzym. 27: 155-159 https://doi.org/10.1016/j.molcatb.2003.11.006
  23. Zou, J., B. M. Hallberg, T. Bergfors, F. Oesch, M. Arand, S. L. Mowbray, and T. A. Jones. 2000. Structure of Aspergillus niger epoxide hydrolase at 1.8 resolution: implication for the structure and function of the mammalian microsomal class of epoxide hydrolases. Structure 8: 111-122 https://doi.org/10.1016/S0969-2126(00)00087-3