Arabidopsisthaliana CYP77A4 is the first cytochrome P450 able to catalyze the epoxidation of free fatty acids in plants

• Published in: The FEBS journal Vol. 276; no. 3; pp. 719 - 735
• Main Authors: Sauveplane, Vincent, Kandel, Sylvie, Kastner, Pierre-Edouard, Ehlting, Juergen, Compagnon, Vincent, Werck-Reichhart, Daniele, Pinot, Franck
• Format: Journal Article
• Language: English
• Published: 01.02.2009
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2008.06819.x

An approach based on an insilico 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 Saccharomycescerevisiae strain (WAT11) engineered for cytochrome P450 expression. Lauric acid (C12:0) was converted into a mixture of hydroxylauric acids when incubated with microsomes from yeast expressing CYP77A4. A variety of physiological C18 fatty acids were tested as potential substrates. Oleic acid (cis- Delta 9C18:1) 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 12C18:2) 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 (C18:0) nor linolelaidic acid (trans,trans- Delta 9, Delta 12C18:2) was metabolized, showing that CYP77A4 requires a double bond, in the cis configuration, to metabolize C18 fatty acids. CYP77A4 was also able to catalyze the invitro formation of the three mono-epoxides of alpha -linolenic acid (cis,cis,cis- Delta 9, Delta 12, Delta 15C18:3), 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 Arabidopsisthaliana. 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.