title: Characterization of a methyl jasmonate and wounding-responsive cytochrome P450 of Arabidopsis thaliana catalyzing dicarboxylic fatty acid formation in vitro.
abstract: A fatty-acid-metabolizing enzyme from Arabidopsis thaliana, CYP94C1, belonging to the cytochrome P450 family was cloned and characterized. CYP94C1 was heterologously expressed in a Saccharomyces cerevisiae strain (WAT11) engineered for P450 expression. When recombinant yeast microsomes were incubated with lauric acid (C12:0) for 15 min, one major metabolite was formed. The product was purified and identified by GC/MS as 12-hydroxylauric acid. Longer incubation (40 min) led to the formation of an additional metabolite identified by GC/MS as dodecadioic acid. This diacid was also produced by incubation with 12-hydroxylauric acid. These compounds were not produced by incubating microsomes from yeast transformed with a void plasmid, demonstrating the involvement of CYP94C1. This new enzyme also metabolized fatty acids of varying aliphatic chain lengths (C12 to C18) and in-chain modifications, for example, degree of unsaturation or the presence of an epoxide as an additional polar functional group. Transcription of the gene encoding CYP94C1 is enhanced by stress, treatment with the hormone methyl jasmonate and wounding. Treatment with methyl jasmonate also induced lauric acid metabolism in microsomes prepared from Arabidopsis. The induction of hydroxylase activity was dose dependent and increased with exposure time, reaching 16x higher in microsomes from 24-h treated Arabidopsis compared with control plants. Analysis of the metabolites showed a mixture of 12-, 11- and 10-hydroxylauric acids, revealing for the first time the presence of fatty acid in-chain hydroxylase in Arabidopsis.
authors.affiliation: Institut de Biologie Moléculaire des Plantes, CNRS-Université Louis Pasteur, Strasbourg, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Kandel
authors.forename: Vincent
authors.initials: V
authors.lastname: Sauveplane
authors.forename: Vincent
authors.initials: V
authors.lastname: Compagnon
authors.forename: Rochus
authors.initials: R
authors.lastname: Franke
authors.forename: Yves
authors.initials: Y
authors.lastname: Millet
authors.forename: Lukas
authors.initials: L
authors.lastname: Schreiber
authors.forename: Danièle
authors.initials: D
authors.lastname: Werck-Reichhart
authors.forename: Franck
authors.initials: F
authors.lastname: Pinot
mesh_terms: D000085:Acetates / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015152:Blotting, Northern; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D003998:Dicarboxylic Acids / Q000378:metabolism*; D006900:Hydroxylation; D008861:Microsomes / Q000378:metabolism; D054883:Oxylipins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
chemical_list: D000085:Acetates; D029681:Arabidopsis Proteins; D003517:Cyclopentanes; D017931:DNA Primers; D003998:Dicarboxylic Acids; D054883:Oxylipins; C072239:methyl jasmonate; D003577:Cytochrome P-450 Enzyme System; C571537:cytochrome P-450 94C1, Arabidopsis