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. 1998 Jun 1;332(Pt 2):583–589. doi: 10.1042/bj3320583

Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1) involved in cutin monomers synthesis.

N Tijet 1, C Helvig 1, F Pinot 1, R Le Bouquin 1, A Lesot 1, F Durst 1, J P Salaün 1, I Benveniste 1
PMCID: PMC1219516  PMID: 9601090

Abstract

The chemical tagging of a cytochrome P-450-dependent lauric acid omega-hydroxylase from clofibrate-treated Vicia sativa seedlings with [1-14C]11-dodecynoic acid allowed the isolation of a full-length cDNA designated CYP94A1. We describe here the functional expression of this novel P-450 in two Saccharomyces cerevisiae strains overproducing their own NADPH-cytochrome P-450 reductase or a reductase from Arabidopsis thaliana. The results show a much higher efficiency of the yeast strain overproducing the plant reductase compared with the yeast strain overproducing its own reductase for expressing CYP94A1. The methyl end of saturated (from C-10 to C-16) and unsaturated (C18:1, C18:2 and C18:3) fatty acids was mainly oxidized by CYP94A1. Both E/Z and Z/E configurations of 9, 12-octadecadienoic acids were omega-hydroxylated. Lauric, myristic and linolenic acids were oxidized with the highest turnover rate (24 min-1). The strong regioselectivity of CYP94A1 was clearly shifted with sulphur-containing substrates, since both 9- and 11-thia laurate analogues were sulphoxidized. Similar to animal omega-hydroxylases, this plant enzyme was strongly induced by clofibrate treatment. Rapid CYP94A1 transcript accumulation was detected less than 20 min after exposure of seedlings to the hypolipidaemic drug. The involvement of CYP94A1 in the synthesis of cutin monomers and fatty acid detoxification is discussed.

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Selected References

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  1. Alterman M. A., Chaurasia C. S., Lu P., Hanzlik R. P. Heteroatom substitution shifts regioselectivity of lauric acid metabolism from omega-hydroxylation to (omega-1)-oxidation. Biochem Biophys Res Commun. 1995 Sep 25;214(3):1089–1094. doi: 10.1006/bbrc.1995.2397. [DOI] [PubMed] [Google Scholar]
  2. Bafor M., Smith M. A., Jonsson L., Stobart K., Stymne S. Biosynthesis of vernoleate (cis-12-epoxyoctadeca-cis-9-enoate) in microsomal preparations from developing endosperm of Euphorbia lagascae. Arch Biochem Biophys. 1993 May 15;303(1):145–151. doi: 10.1006/abbi.1993.1265. [DOI] [PubMed] [Google Scholar]
  3. Bajar A., Podila G. K., Kolattukudy P. E. Identification of a fungal cutinase promoter that is inducible by a plant signal via a phosphorylated trans-acting factor. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8208–8212. doi: 10.1073/pnas.88.18.8208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benveniste I., Salaün J. P., Simon A., Reichhart D., Durst F. Cytochrome P-450-Dependent omega-Hydroxylation of Lauric Acid by Microsomes from Pea Seedlings. Plant Physiol. 1982 Jul;70(1):122–126. doi: 10.1104/pp.70.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bolwell G. P., Bozak K., Zimmerlin A. Plant cytochrome P450. Phytochemistry. 1994 Dec;37(6):1491–1506. doi: 10.1016/s0031-9422(00)89567-9. [DOI] [PubMed] [Google Scholar]
  6. Broun P., Somerville C. Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean. Plant Physiol. 1997 Mar;113(3):933–942. doi: 10.1104/pp.113.3.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown D. G., Riederer S. J., Jack C. R., Farzaneh F., Ehman R. L. MR angiography with oblique gradient-recalled echo technique. Radiology. 1990 Aug;176(2):461–466. doi: 10.1148/radiology.176.2.2367661. [DOI] [PubMed] [Google Scholar]
  8. CaJacob C. A., Chan W. K., Shephard E., Ortiz de Montellano P. R. The catalytic site of rat hepatic lauric acid omega-hydroxylase. Protein versus prosthetic heme alkylation in the omega-hydroxylation of acetylenic fatty acids. J Biol Chem. 1988 Dec 15;263(35):18640–18649. [PubMed] [Google Scholar]
  9. Chandra S., Heinstein P. F., Low P. S. Activation of Phospholipase A by Plant Defense Elicitors. Plant Physiol. 1996 Mar;110(3):979–986. doi: 10.1104/pp.110.3.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conconi A., Miquel M., Browse J. A., Ryan C. A. Intracellular Levels of Free Linolenic and Linoleic Acids Increase in Tomato Leaves in Response to Wounding. Plant Physiol. 1996 Jul;111(3):797–803. doi: 10.1104/pp.111.3.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Croteau R., Kolattukudy P. E. Biosynthesis of hydroxyfatty acid polymers. Enzymatic epoxidation of 18-hydroxyoleic acid to 18-hydroxy-cis-9,10-epoxystearic acid by a particulate preparation from spinach (Spinacia oleracea). Arch Biochem Biophys. 1975 Sep;170(1):61–72. doi: 10.1016/0003-9861(75)90097-1. [DOI] [PubMed] [Google Scholar]
  12. Duggleby R. G. Regression analysis of nonlinear Arrhenius plots: an empirical model and a computer program. Comput Biol Med. 1984;14(4):447–455. doi: 10.1016/0010-4825(84)90045-3. [DOI] [PubMed] [Google Scholar]
  13. Durst F., Nelson D. R. Diversity and evolution of plant P450 and P450-reductases. Drug Metabol Drug Interact. 1995;12(3-4):189–206. doi: 10.1515/dmdi.1995.12.3-4.189. [DOI] [PubMed] [Google Scholar]
  14. Helvig C., Alayrac C., Mioskowski C., Koop D., Poullain D., Durst F., Salaün J. P. Suicide inactivation of cytochrome P450 by midchain and terminal acetylenes. A mechanistic study of inactivation of a plant lauric acid omega-hydroxylase. J Biol Chem. 1997 Jan 3;272(1):414–421. doi: 10.1074/jbc.272.1.414. [DOI] [PubMed] [Google Scholar]
  15. Johnson E. F., Palmer C. N., Griffin K. J., Hsu M. H. Role of the peroxisome proliferator-activated receptor in cytochrome P450 4A gene regulation. FASEB J. 1996 Sep;10(11):1241–1248. doi: 10.1096/fasebj.10.11.8836037. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lake B. G. Mechanisms of hepatocarcinogenicity of peroxisome-proliferating drugs and chemicals. Annu Rev Pharmacol Toxicol. 1995;35:483–507. doi: 10.1146/annurev.pa.35.040195.002411. [DOI] [PubMed] [Google Scholar]
  18. Lemberger T., Desvergne B., Wahli W. Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology. Annu Rev Cell Dev Biol. 1996;12:335–363. doi: 10.1146/annurev.cellbio.12.1.335. [DOI] [PubMed] [Google Scholar]
  19. Moody D. E., Gibson G. G., Grant D. F., Magdalou J., Rao M. S. Peroxisome proliferators, a unique set of drug-metabolizing enzyme inducers: commentary on a symposium. Drug Metab Dispos. 1992 Nov-Dec;20(6):779–791. [PubMed] [Google Scholar]
  20. Ohlrogge J. B. Design of New Plant Products: Engineering of Fatty Acid Metabolism. Plant Physiol. 1994 Mar;104(3):821–826. doi: 10.1104/pp.104.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Palma J. M., Garrido M., Rodríguez-García M. I., del Río L. A. Peroxisome proliferation and oxidative stress mediated by activated oxygen species in plant peroxisomes. Arch Biochem Biophys. 1991 May 15;287(1):68–74. doi: 10.1016/0003-9861(91)90389-z. [DOI] [PubMed] [Google Scholar]
  23. Pierrel M. A., Batard Y., Kazmaier M., Mignotte-Vieux C., Durst F., Werck-Reichhart D. Catalytic properties of the plant cytochrome P450 CYP73 expressed in yeast. Substrate specificity of a cinnamate hydroxylase. Eur J Biochem. 1994 Sep 15;224(3):835–844. doi: 10.1111/j.1432-1033.1994.00835.x. [DOI] [PubMed] [Google Scholar]
  24. Pinot F., Bosch H., Alayrac C., Mioskowski C., Vendais A., Durst F., Salaun J. P. [omega]-Hydroxylation of Oleic Acid in Vicia sativa Microsomes (Inhibition by Substrate Analogs and Inactivation by Terminal Acetylenes). Plant Physiol. 1993 Aug;102(4):1313–1318. doi: 10.1104/pp.102.4.1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pinot F., Salaün J. P., Bosch H., Lesot A., Mioskowski C., Durst F. omega-Hydroxylation of Z9-octadecenoic, Z9,10-epoxystearic and 9,10-dihydroxystearic acids by microsomal cytochrome P450 systems from Vicia sativa. Biochem Biophys Res Commun. 1992 Apr 15;184(1):183–193. doi: 10.1016/0006-291x(92)91176-q. [DOI] [PubMed] [Google Scholar]
  26. Pompon D., Louerat B., Bronine A., Urban P. Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol. 1996;272:51–64. doi: 10.1016/s0076-6879(96)72008-6. [DOI] [PubMed] [Google Scholar]
  27. Salaün J. P., Benveniste I., Reichhart D., Durst F. A microsomal (cytochrome P-450)-linked lauric-acid-monooxygenase from aged Jerusalem-artichoke-tuber tissues. Eur J Biochem. 1978 Sep 15;90(1):155–159. doi: 10.1111/j.1432-1033.1978.tb12586.x. [DOI] [PubMed] [Google Scholar]
  28. Salaün J. P., Helvig C. Cytochrome P450-dependent oxidation of fatty acids. Drug Metabol Drug Interact. 1995;12(3-4):261–283. doi: 10.1515/dmdi.1995.12.3-4.261. [DOI] [PubMed] [Google Scholar]
  29. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  30. Stahl D. J., Schäfer W. Cutinase is not required for fungal pathogenicity on pea. Plant Cell. 1992 Jun;4(6):621–629. doi: 10.1105/tpc.4.6.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Urban P., Cullin C., Pompon D. Maximizing the expression of mammalian cytochrome P-450 monooxygenase activities in yeast cells. Biochimie. 1990 Jun-Jul;72(6-7):463–472. doi: 10.1016/0300-9084(90)90070-w. [DOI] [PubMed] [Google Scholar]
  32. Weissbart D., Salaün J. P., Durst F., Pflieger P., Mioskowski C. Regioselectivity of a plant lauric acid omega hydroxylase. Omega hydroxylation of cis and trans unsaturated lauric acid analogs and epoxygenation of the terminal olefin by plant cytochrome P-450. Biochim Biophys Acta. 1992 Mar 4;1124(2):135–142. doi: 10.1016/0005-2760(92)90089-e. [DOI] [PubMed] [Google Scholar]
  33. Zimmerlin A., Salaün J. P., Durst F., Mioskowski C. Cytochrome p-450-dependent hydroxylation of lauric Acid at the subterminal position and oxidation of unsaturated analogs in wheat microsomes. Plant Physiol. 1992 Oct;100(2):868–873. doi: 10.1104/pp.100.2.868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. del Río L. A., Palma J. M., Sandalio L. M., Corpas F. J., Pastori G. M., Bueno P., López-Huertas E. Peroxisomes as a source of superoxide and hydrogen peroxide in stressed plants. Biochem Soc Trans. 1996 May;24(2):434–438. doi: 10.1042/bst0240434. [DOI] [PubMed] [Google Scholar]
  35. van de Loo F. J., Broun P., Turner S., Somerville C. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6743–6747. doi: 10.1073/pnas.92.15.6743. [DOI] [PMC free article] [PubMed] [Google Scholar]

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