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An approach based on anin silico analysis predicted that CYP77A4, a cytochrome P450 that so far has no identified function, might be a fatty acid-metabolizing enzyme. CYP77A4was heterologously expressed in a Saccharomyces cerevisiaestrain (WAT11) engineered for cytochrome P450 expression. Lauric acid (C12:0 ) was converted into a mixture of hydroxy-lauric acids when incubated with microsomes from yeast expressing CYP77A4. | ỊFEBS Journal Arabidopsis thaliana CYP77A4 is the first cytochrome P450 able to catalyze the epoxidation of free fatty acids in plants Vincent Sauveplane1 Sylvie Kandel2 Pierre-Edouard Kastner1 Jurgen Ehlting1 Vincent Compagnon1 Daniele Werck-Reichhart1 and Franck Pinot1 1 Institut de Biologie Moleculaire des Plantes University of Strasbourg France 2 Department of PharmaceuticalChemistry University of California San Francisco CA USA Keywords cytochrome P450 defense epoxide fatty acid plant Correspondence F. Pinot IBMP-CNRS UPR 2357 Institut de Botanique 28 rue Goethe F-67083 Strasbourg Cedex France Fax 33 3 90 24 19 21 Tel 33 3 90 24 18 37 E-mail franck.pinot@ibmp-ulp.u-strasbg.fr Received 4 September 2008 revised 20 November 2008 accepted 26 November 2008 doi 10.1111 j.1742-4658.2008.06819.x 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 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-A9C18 1 was converted into a mixture of ro-4-to ro-7-hydroxyoleic acids 75 and 9 10-epoxystearic acid 25 . Linoleic acid cis cis-A9 A12C18 2 was exclusively converted into 12 13-epoxyocta-deca-9-enoic acid which was then converted into diepoxide after epoxidation of the A9 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-A9 A12C18 2 was metabolized showing that CYP77A4 requires a double bond in the cis .
