Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

High Yield Bacterial Expression and Purification of Active Cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis Membrane Protein

대장균 시스템을 이용한 Arabidopsis 막 단백질 cytochrome P450 p-coumarate-3hydroxylase (C3H) 활성형의 과발현 및 분리정제

  • Yang, Hee-Jung (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Kim, Wan-Yeon (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Yun, Young-Ju (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Yoon, Ji-Won (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Kwon, Tae-Woo (College of Natural Sciences, Department of Biological Sciences, Pusan National University) ;
  • Youn, Hye-Sook (Department of Bioscience & Biotechnology/institute of Bioscience, Sejong University) ;
  • Youn, Bu-Hyun (College of Natural Sciences, Department of Biological Sciences, Pusan National University)
  • 양희정 (부산대학교 자연과학대학 생명과학과) ;
  • 김완연 (부산대학교 자연과학대학 생명과학과) ;
  • 윤영주 (부산대학교 자연과학대학 생명과학과) ;
  • 윤지원 (부산대학교 자연과학대학 생명과학과) ;
  • 권태우 (부산대학교 자연과학대학 생명과학과) ;
  • 윤혜숙 (세종대학교 생명과학대학 생명공학부 생명공학과) ;
  • 윤부현 (부산대학교 자연과학대학 생명과학과)
  • Published : 2009.08.30
Download PDF
⟨ Previous Next ⟩

Abstract

The cytochrome P450s (P450s) metabolizing natural products are among the most versatile biological catalysts known in plants, but knowledge of the structural basis for their broad substrate specificity has been limited. The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants however, all attempts to express and purify the protein corresponding C3H gene have failed. As a result, no conditions suitable for the unambiguous assay of the enzyme are known. The detailed understanding of the mechanism and substrate-specificity of C3Hdemands a method for the production of active protein on the milligram scale. We have developed a bacterial expression and purification system for the plant C3H, which allows for the quick expression and purification of active wild-type C3H via introduction of combinational mutagenesis. The modified cytochrome P450 C3H ($C3H_{mod}$) could be purified in the absence of detergent using immobilized metal affinity chromatography and size exclusion chromatography following extraction from isolated membranes in a high salt buffer and catalytically activated. This method makes the use of isotopic labeling of C3H for NMRstudies and X-ray crystallography practical, and is also applicable to other plant cytochrome P450 proteins.

다양한 천연물의 합성대사에 관여하는 식물 cytochrome P450 (P450s)은 그 기능적 다양성에도 불구하고, 이들 효소의 광범위한 기질 특이성을 설명해 줄 수 있는 구조분석에 대해서는 충분한 연구가 이루어지지 못하고 있는 실정이다. 식물 p-coumarate 3-hydroxylase (C3H)에 의해 매개되는 효소 반응은 lignin 과 다양한 phenylpropanoid 부산물들의 생합성에 매우 중요한 것으로 여겨지지만, 막 단백질인 C3H의 발현 및 정제가 효과적으로 이루어지지 못하여, 활성을 측정하기 위한 분석방법이 체계화 되지 못하고 있다. C3H의 작용기작과 기질특이성에 대해 폭넓은 이해를 위한 구조분석의 선행단계는 활성을 갖는 C3H를 밀리그램 단위로 분리, 정제하는 실험적 방법을 확립하는 것이라 할 수 있다. 이를 위해, 본 연구에서는 다양한 돌연변이 방법을 도입하여 식물 막단백질 C3H를 대장균 시스템에서 효과적으로 발현 및 정제할 수 있는 시스템을 사용하였다. 변형된 cytochrome P450 C3H ($C3H_{mod}$)을 세포막으로부터 고농도의 염완충용액을 이용하여 계면활성제 없이 추출하였으며, 2단계 chromatography를 통해 활성을 유지한 상태로 분리할 수 있었다. 이러한 실험적 기법은 NMR 및 X-ray crystallography와 같은 구조분석을 통한 C3H의 효과적인 분석에 적용될 수 있을 것이며, 또한 다른 식물 cytochrome P450 단백질의 효과적인 분석에도 적용 될 수 있을 것이다.

Keywords

References

  1. Alibert, G., R. Ranjeva, and A. Boudet. 1972. Studies on enzymes catalyzing phenolic acids formation in Quercus penduculata (Ehrh.). II. Intracellular location of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and 'benzoate synthase'. Biochim. Biophys. Acta. 279, 282-289 https://doi.org/10.1016/0304-4165(72)90144-4
  2. Chapple, C. 1998. Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 311-343 https://doi.org/10.1146/annurev.arplant.49.1.311
  3. Davin, L. B., M. Jourdes, A. M. Patten, K. W. Kim, D. G. Vassao, and N. G. Lewis. 2008. Dissection of lignin macromolecular configuration and assembly: comparison to related biochemical processes in allyl/propenyl phenol and lignan biosynthesis. Nat. Prod Rep. 25, 1015-1090 https://doi.org/10.1039/b510386j
  4. Dick, R. A., and T. W. Kensler. 2004. The catalytic and kinetic mechanisms of NADPH-dependent alkenal/one oxidoreductase. J. Biol. Chem. 279, 17269-17277 https://doi.org/10.1074/jbc.M400427200
  5. Ford, J. D., L. B. Davin, and N. G. Lewis. 1999. Plant lignans and health: cancer chemoprevention and biotechnological opportunities. Basic Life Sci. 66, 675-694
  6. Franke, R., J. M. Humphreys, M. R. Hemm, J. W. Denault, M. O. Ruegger, J. C. Cusumano, and C. Chapple. 2002. The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J. 30, 33-45 https://doi.org/10.1046/j.1365-313X.2002.01266.x
  7. Fromm, H. J. 1983. Contemporary Enzyme Kinetics and Mechanism, pp. 233-251, In Purich, D. L (ed.), Academic Press, New York
  8. Gabriac, B., D. Werck-Reichhart, H. Teutsch, and F. Durst. 1991. Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase. Arch. Biochem. Biophys. 288, 302-309 https://doi.org/10.1016/0003-9861(91)90199-S
  9. Heller, W. and T. Kuhnl. 1985. Elicitor induction of a microsomal 5-O-(4-coumaroyl)shikimate 3'-hydroxylase in parsley cell suspension cultures. Arch. Biochem. Biophys. 241, 453-460 https://doi.org/10.1016/0003-9861(85)90570-3
  10. Hotze, M., G. Schroder, and J. Schroder. 1995. Cinnamate 4-hydroxylase from Catharanthus roseus, and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli. FEBS Lett 374, 345-350 https://doi.org/10.1016/0014-5793(95)01141-Z
  11. Kneusel, R. E., U. Matern, and K. Nicolay. 1989. Formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA by $Zn2^+$-dependent enzymes in cultured plant cells and its activation by an elicitor-induced pH shift. Arch. Biochem. Biophys. 269, 455-462 https://doi.org/10.1016/0003-9861(89)90129-X
  12. Kraus, P. F. and T. M. Kutchan. 1995. Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera. Proc. Natl. Acad. Sci. USA 92, 2071-2075 https://doi.org/10.1073/pnas.92.6.2071
  13. Lewis, D. F., M. Dickins, B. G. Lake, and P. S. Goldfarb. 2003. Investigation of enzyme selectivity in the human CYP2C subfamily: homology modelling of CYP2C8, CYP2C9 and CYP2C19 from the CYP2C5 crystallographic template. Drug Metabol. Drug Interact 19, 257-285
  14. Meyer, K., J. C. Cusumano, C. Somerville, and C. C. Chapple. 1996. Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases. Proc. Natl. Acad. Sci. USA 93, 6869-6874 https://doi.org/10.1073/pnas.93.14.6869
  15. Mizutani, M., E. Ward, J. DiMaio, D. Ohta, J. Ryals, and R. Sato. 1993. Molecular cloning and sequencing of a cDNA encoding mung bean cytochrome P450 (P450C4H) possessing cinnamate 4-hydroxylase activity. Biochem. Biophys. Res. Commun. 190, 875-880 https://doi.org/10.1006/bbrc.1993.1130
  16. Richardson, T. H., M. H. Hsu, T. Kronbach, H. J. Barnes, G. Chan, M. R. Waterman, B. Kemper, and E. F. Johnson. 1993. Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b. Arch. Biochem. Biophys. 300, 510-516 https://doi.org/10.1006/abbi.1993.1069
  17. Russell, D. W. 1971. The metabolism of aromatic compounds in higer plants. X. Properties of the cinnamic acid 4-hydroxylase of pea seedlings and some aspects of its metabolic and developmental control. J. Biol. Chem. 246, 3870-3878
  18. Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  19. Scheller, U., R. Kraft, K. L. Schroder, and W. H. Schunck. 1994. Generation of the soluble and functional cytosolic domain of microsomal cytochrome P450 52A3. J. Biol. Chem. 269, 12779-12783
  20. Schoch, G. A., J. K. Yano, M. R. Wester, K. J. Griffin, C. D. Stout, and E. F. Johnson. 2004. Structure of human microsomal cytochrome P450 2C8. Evidence for a peripheral fatty acid binding site. J. Biol. Chem. 279, 9497-9503 https://doi.org/10.1074/jbc.M312516200
  21. Scott, E. E., Y. A. He, M. R. Wester, M. A. White, C. C. Chin, J. R. Halpert, E. F. Johnson, and C. D. Stout. 2003. An open conformation of mammalian cytochrome P450 2B4 at 1.6-A resolution. Proc. Natl. Acad. Sci. USA 100, 13196- 13201 https://doi.org/10.1073/pnas.2133986100
  22. Strauss, A., F. Bitsch, B. Cutting, G. Fendrich, P. Graff, J. Liebetanz, M. Zurini, and W. Jahnke. 2003. Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies. J. Biomol. NMR 26, 367-372 https://doi.org/10.1023/A:1024013111478
  23. Teutsch, H. G., M. P. Hasenfratz, A. Lesot, C. Stoltz, J. M. Garnier, J. M. Jeltsch, F. Durst, and D. Werck-Reichhart. 1993. Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway. Proc. Natl. Acad. Sci. USA 90, 4102-4106 https://doi.org/10.1073/pnas.90.9.4102
  24. von Wachenfeldt, C., T. H. Richardson, J. Cosme, and E. F. Johnson. 1997. Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modification of its N-terminus. Arch. Biochem. Biophys. 339, 107-114 https://doi.org/10.1006/abbi.1996.9859
  25. Wester, M. R., J. K. Yano, G. A. Schoch, C. Yang, K. J. Griffin, C. D. Stout, and E. F. Johnson. 2004. The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-A resolution. J. Biol. Chem. 279, 35630-35637 https://doi.org/10.1074/jbc.M405427200
  26. Williams, P. A., J. Cosme, V. Sridhar, E. F. Johnson, and D. E. McRee. 2000. Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol. Cell 5, 121-131 https://doi.org/10.1016/S1097-2765(00)80408-6
  27. Wiseman, T., S. Williston, J. F. Brandts, and L. N. Lin. 1989. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131-137 https://doi.org/10.1016/0003-2697(89)90213-3
  28. Yano, J. K., M. H. Hsu, K. J. Griffin, C. D. Stout, and E. F. Johnson. 2005. Structures of human microsomal cytochrome P450 2A6 complexed with coumarin and methoxsalen. Nat. Struct. Mol. Biol. 12, 822-823 https://doi.org/10.1038/nsmb971
  29. Youn, B., S. G. Moinuddin, L. B. Davin, N. G. Lewis, and C. Kang. 2005. Crystal structures of apo-form and binary/ternary complexes of Podophyllum secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans. J. Biol. Chem. 280, 12917-12926 https://doi.org/10.1074/jbc.M413266200