title: Arabidopsis thaliana CYP77A4 is the first cytochrome P450 able to catalyze the epoxidation of free fatty acids in plants.
abstract: An approach based on an in silico analysis predicted that CYP77A4, a cytochrome P450 that so far has no identified function, might be a fatty acid-metabolizing enzyme. CYP77A4 was heterologously expressed in a Saccharomyces cerevisiae strain (WAT11) engineered for cytochrome P450 expression. Lauric acid (C(12:0)) was converted into a mixture of hydroxylauric acids when incubated with microsomes from yeast expressing CYP77A4. A variety of physiological C(18) fatty acids were tested as potential substrates. Oleic acid (cis-Delta(9)C(18:1)) was converted into a mixture of omega-4- to omega-7-hydroxyoleic acids (75%) and 9,10-epoxystearic acid (25%). Linoleic acid (cis,cis-Delta(9),Delta(12)C(18:2)) was exclusively converted into 12,13-epoxyoctadeca-9-enoic acid, which was then converted into diepoxide after epoxidation of the Delta(9) unsaturation. Chiral analysis showed that 9,10-epoxystearic acid was a mixture of 9S/10R and 9R/10S in the ratio 33 : 77, whereas 12,13-epoxyoctadeca-9-enoic acid presented a strong enantiomeric excess in favor of 12S/13R, which represented 90% of the epoxide. Neither stearic acid (C(18:0)) nor linolelaidic acid (trans,trans-Delta(9),Delta(12)C(18:2)) was metabolized, showing that CYP77A4 requires a double bond, in the cis configuration, to metabolize C(18) fatty acids. CYP77A4 was also able to catalyze the in vitro formation of the three mono-epoxides of alpha-linolenic acid (cis,cis,cis-Delta(9),Delta(12),Delta(15)C(18:3)), previously described as antifungal compounds. Epoxides generated by CYP77A4 are further metabolized to the corresponding diols by epoxide hydrolases located in microsomal and cytosolic subcellular fractions from Arabidopsis thaliana. The concerted action of CYP77A4 with epoxide hydrolases and hydroxylases allows the production of compounds involved in plant-pathogen interactions, suggesting a possible role for CYP77A4 in plant defense.
authors.affiliation: Institut de Biologie Moléculaire des Plantes, University of Strasbourg, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Sauveplane
authors.forename: Sylvie
authors.initials: S
authors.lastname: Kandel
authors.forename: Pierre-Edouard
authors.initials: PE
authors.lastname: Kastner
authors.forename: Jürgen
authors.initials: J
authors.lastname: Ehlting
authors.forename: Vincent
authors.initials: V
authors.lastname: Compagnon
authors.forename: Danièle
authors.initials: D
authors.lastname: Werck-Reichhart
authors.forename: Franck
authors.initials: F
authors.lastname: Pinot
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004852:Epoxy Compounds / Q000378:metabolism*; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D006868:Hydrolysis; D008861:Microsomes / Q000378:metabolism; D015394:Molecular Structure
chemical_list: D029681:Arabidopsis Proteins; D004852:Epoxy Compounds; D005227:Fatty Acids; D003577:Cytochrome P-450 Enzyme System; C534693:CYP77A4 protein, Arabidopsis