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Epoxyalcohol Synthase RjEAS (CYP74A88) from the Japanese Buttercup (Ranunculus japonicus): Cloning and Characterization of Catalytic Properties

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Abstract

Cytochromes P450 of the CYP74 family play a key role in the lipoxygenase cascade generating oxylipins (products of polyunsaturated fatty acid oxidation). The CYP74 family includes allene oxide synthases, hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases. In this work, we cloned the CYP74A88 gene from the Japanese buttercup (Ranunculus japonicus) and studied the properties of the encoded recombinant protein. The CYP74A88 enzyme specifically converts linoleic acid 9-and 13-hydroperoxides to oxiranyl carbinols 9,10-epoxy-11-hydroxy-12-octadecenoic acid and 11-hydroxy-12,13-epoxy-9-octadecenoic acid, respectively, which was confirmed by GC-MS analysis and kinetic studies. Therefore, the CYP74A88 enzyme is a specific epoxyalcohol synthase.

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Abbreviations

GC-MS:

gas chromatography/mass spectrome-try

HPLC:

high-performance liquid chromatography

9-H(P)OD:

(9S,10E,12Z)-9-hydro(pero)xy-10,12-octadeca-dienoic acid

9-H(P)OT:

(9S,10E,12Z,15Z)-9-hydro(pero)xy-10,12,15-octadecatrienoic acid

13-H(P)OD:

(9Z,11E,13S)-13-hydro(pero)xy-9,11-octadecadienoic acid

13-H(P)OT:

(9Z,11E,13S,15Z)-13-hydro(pero)xy-9,11,15-octadecatrienoic acid

Me:

methyl

ORF:

open reading frame

TMS:

trimethylsilyl

References

  1. Blee, E. (1998) Phytooxylipins and plant defense reactions, Prog. Lipid Res., 37, 33–72.

    Article  CAS  PubMed  Google Scholar 

  2. Grechkin, A. N. (1998) Recent developments in biochem–istry of the plant lipoxygenase pathway, Prog. Lipid Res., 37, 317–352.

    Article  CAS  PubMed  Google Scholar 

  3. Grechkin, A. N. (2002) Hydroperoxide lyase and divinyl ether synthase, Prostaglandins Other Lipid Mediat., 68–69, 457–470.

    Google Scholar 

  4. Stumpe, M., and Feussner, I. (2006) Formation of oxylip–ins by CYP74 enzymes, Phytochem. Rev., 5, 347–357.

    Article  CAS  Google Scholar 

  5. Brash, A. R. (2009) Mechanistic aspects of CYP74 allene oxide synthases and related cytochrome P450 enzymes, Phytochemistry, 70, 1522–1531.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Toporkova, Y. Y., Smirnova, E. O., Gorina, S. S., Mukhtarova, L. S., and Grechkin, A. N. (2018) Detection of the first higher plant epoxyalcohol synthase: molecular cloning and characterization of the CYP74M2 enzyme of spike moss Selaginella moellendorffii, Phytochemistry, 156, 73–82.

    Article  CAS  PubMed  Google Scholar 

  7. Toporkova, Y. Y., Gorina, S. S., Bessolitsyna, E. K., Smirnova, E. O., Fatykhova, V. S., Bruhlmann, F., Ilyina, T. M., Mukhtarova, L. S., and Grechkin, A. N. (2018) Double function hydroperoxide lyases/epoxyalcohol syn–thases (CYP74C) of higher plants: identification and con–version into allene oxide synthases by site–directed mutage–nesis, Biochim. Biophys. Acta, 1863, 369–378.

    Article  CAS  Google Scholar 

  8. Nelson, D. R., Goldstone, J. V., and Stegeman, J. J. (2013) The cytochrome P450 genesis locus: the origin and evolu–tion of animal cytochrome P450s, Philos. Trans. R. Soc. Lond. B Biol. Sci., 368, 20120474.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lee, D.–S., Nioche, P., Hamberg, M., and Raman, C. S. (2008) Structural insights into the evolutionary paths of oxylipin biosynthesis enzymes, Nature, 455, 363–370.

    Article  CAS  PubMed  Google Scholar 

  10. Toporkova, Y. Y., Fatykhova, V. S., Gogolev, Y. V., Khairutdinov, B. I., Mukhtarova, L. S., and Grechkin, A. N. (2017) Epoxyalcohol synthase of Ectocarpus siliculosus. First CYP74–related enzyme of oxylipin biosynthesis in brown algae, Biochim. Biophys. Acta, 1862, 167–175.

    Article  CAS  Google Scholar 

  11. Toporkova, Y. Y., Gorina, S. S., Mukhitova, F. K., Hamberg, M., Ilyina, T. M., Mukhtarova, L. S., and Grechkin, A. N. (2017) Identification of CYP443D1 (CYP74 clan) of Nematostella vectensis as a first cnidarian epoxyalcohol synthase and insights into its catalytic mech–anism, Biochim. Biophys. Acta, 1862, 1099–1109.

    Article  CAS  Google Scholar 

  12. Wilson, R. A., Gardner, H. W., and Keller, N. P. (2001) Cultivar–dependent expression of a maize lipoxygenase responsive to seed infesting fungi, Mol. Plant Microbe Iinteract., 14, 980–987.

    Article  CAS  Google Scholar 

  13. Chechetkin, I. R., Osipova, E. V., Tarasova, N. B., Mukhitova, F. K., Hamberg, M., Gogolev, Y. V., and Grechkin, A. N. (2009) Specificity of oxidation of linoleic acid homologs by plant lipoxygenases, Biochemistry (Moscow), 74, 855–861.

    Article  CAS  Google Scholar 

  14. Glover, D. M. (1988) Gene Cloning. The Mechanics of DNA Manipulation, Springer.

    Google Scholar 

  15. Maniatis, T., Fritsch, E., and Sambrook, J. (1984) The Methods of Genetic Engineering. Molecular Cloning [Russian translation], Mir, Moscow.

    Google Scholar 

  16. Schenkman, J. B., and Jansson, I. (2006) Spectral analyses of cytochromes P450, Meth. Mol. Biol., 320, 11–18.

    CAS  Google Scholar 

  17. Grechkin, A. N., Bruhlmann, F., Mukhtarova, L. S., Gogolev, Y. V., and Hamberg, M. (2006) Hydroperoxide lyases (CYP74C and CYP74B) catalyze the hemolytic iso–merization of fatty acid hydroperoxides into hemiacetals, Biochim. Biophys. Acta, 1761, 1419–1428.

    Article  CAS  PubMed  Google Scholar 

  18. Gogolev, Y. V., Gorina, S. S., Gogoleva, N. E., Toporkova, Y. Y., Chechetkin, I. R., and Grechkin, A. N. (2012) Green leaf divinyl ether synthase: gene detection, molecular cloning and identification of a unique CYP74B subfamily member, Biochim. Biophys. Acta, 1821, 287–294.

    Article  CAS  PubMed  Google Scholar 

  19. Mukhtarova, L. S., Mukhitova, F. K., Gogolev, Y. V., and Grechkin, A. N. (2011) Hydroperoxide lyase cascade in pea seedlings: non–volatile oxylipins and their age and stress dependent alterations, Phytochemistry, 72, 356–364.

    Article  CAS  PubMed  Google Scholar 

  20. Hamberg, M., and Hamberg, G. (1996) Peroxygenase–cat–alyzed fatty acid epoxidation in cereal seeds (sequential oxidation of linoleic acid into 9(S),12(S),13(S)–trihydroxy–10(E)–octadecenoic acid), Plant Physiol., 110, 807–815.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hamberg, M., and Hamberg, G. (1990) Hydroperoxide–dependent epoxidation of unsaturated fatty acids in the broad bean (Vicia faba L.), Arch. Biochem. Biophys., 283, 409–416.

    Article  CAS  PubMed  Google Scholar 

  22. Blee, E., Wilcox, A. L., Marnett, L. J., and Schuber, F. (1993) Mechanism of reaction of fatty acid hydroperoxides with soybean peroxygenase, J. Biol. Chem., 268, 1708–1715.

    CAS  PubMed  Google Scholar 

  23. Hamberg, M., and Olsson, U. (2011) Efficient and specific conversion of 9–lipoxygenase hydroperoxides in the beet–root, formation of pinellic acid, Lipids, 46, 873–878.

    Article  CAS  PubMed  Google Scholar 

  24. Gardner, H. W., Weisleder, D., and Nelson, E. C. (1984) Acid catalysis of a linoleic acid hydroperoxide: formation of epoxides by an intramolecular cyclization of the hydroper–oxide group, J. Org. Chem., 49, 508–515.

    Article  CAS  Google Scholar 

  25. Gardner, H. W., Nelson, E. C., Tjarks, L. W., and England, R. E. (1984) Acid–catalyzed transformation of 13(S)–hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids, Chem. Phys. Lipids, 35, 87–101.

    Article  CAS  Google Scholar 

  26. Gardner, H. W., Kleiman, R., and Weisleder, D. (1974) Homolytic decomposition of linoleic acid hydroperoxide: identification of fatty acid products, Lipids, 9, 696–706.

    Article  CAS  Google Scholar 

  27. Gardner, H. W. (1975) Decomposition of linoleic acid hydroperoxides. Enzymic reactions compared with nonen–zymic, J. Agr. Food Chem., 23, 129–136.

    Article  CAS  Google Scholar 

  28. Gardner, H. W., and Jursinic, P. A. (1981) Degradation of linoleic acid hydroperoxides by a cysteine FeCl3 catalyst as a model for similar biochemical reactions: I. Study of oxy–gen requirement, catalyst and effect of pH, Biochim. Biophys. Acta, 665, 100–112.

    Article  CAS  PubMed  Google Scholar 

  29. Gardner, H. W., and Kleiman, R. (1981) Degradation of linoleic acid hydroperoxides by a cysteine FeCl3 catalyst as a model for similar biochemical reactions: II. Specificity in formation of fatty acid epoxides, Biochim. Biophys. Acta, 665, 113–125.

    Article  CAS  PubMed  Google Scholar 

  30. Dix, T. A., and Marnett, L. J. (1985) Conversion of linole–ic acid hydroperoxide to hydroxy, keto, epoxyhydroxy, and trihydroxy fatty acids by hematin, J. Biol. Chem., 260, 5351–5357.

    CAS  PubMed  Google Scholar 

  31. Gardner, H. W. (1989) Oxygen radical chemistry of polyun–saturated fatty acids, Free Rad. Biol. Med., 7, 65–86.

    Article  CAS  PubMed  Google Scholar 

  32. Hamberg, M., and Gotthammar, B. (1973) A new reaction of unsaturated fatty acid hydroperoxides: formation of 11–hydroxy–12,13–epoxy–9–octadecenoic acid from 13–hydroperoxy–9,11–octadecadienoic acid, Lipids, 8, 737–744.

    Article  CAS  Google Scholar 

  33. Gorina, S. S., Smirnova, E.O., Mukhtarova, L. S., Toporkova, Y. Y., and Grechkin, A. N. (2018) Conversion of tomato allene oxide synthase LeAOS3 (CYP74C3) into epoxyalcohol synthase by site–directed mutagenesis, Dokl. Biochem. Biophys, 483, 329–332.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center of Russian Academy of Sciences, 420111, Kazan, Tatarstan, Russia

    Y. Y. Toporkova, V. S. Fatykhova, S. S. Gorina, L. S. Mukhtarova & A. N. Grechkin

Authors
  1. Y. Y. Toporkova
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  2. V. S. Fatykhova
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  3. S. S. Gorina
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  4. L. S. Mukhtarova
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  5. A. N. Grechkin
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Corresponding author

Correspondence to Y. Y. Toporkova.

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Published in Russian in Biokhimiya, 2019, Vol. 84, No. 2, pp. 269–280.

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Toporkova, Y.Y., Fatykhova, V.S., Gorina, S.S. et al. Epoxyalcohol Synthase RjEAS (CYP74A88) from the Japanese Buttercup (Ranunculus japonicus): Cloning and Characterization of Catalytic Properties. Biochemistry Moscow 84, 171–180 (2019). https://doi.org/10.1134/S0006297919020081

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  • DOI: https://doi.org/10.1134/S0006297919020081

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