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

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.

Methyl Jasmonate Induces Lauric Acid ω-Hydroxylase Activity and Accumulation of CYP94A1 Transcripts but Does Not Affect Epoxide Hydrolase Activities inVicia sativaSeedlings

“…This was confirmed by the recent cloning of distinct -hydroxylases from V. sativa (Tijet et al, 1998; R. Le Bouquin and I. Benveniste, unpublished data). One of these enzymes, CYP94A1, when expressed in yeast (Tijet et al, 1998), catalyzed the -hydroxylation of 18:1, 18:2, and 18:3 (oleic, linoleic, and linolenic acids, respectively) and of the model substrate 12:0 (lauric acid).Jasmonates, which derive from 18:3, are important regulatory molecules in plant defense (for review, see Sembner and Parthier 1993; Creelman and Mullet, 1997;Mueller, 1997). The proteinase inhibitor PI-2 was the first wellcharacterized defense-related protein induced by jasmonate (Farmer and Ryan, 1990).…”

“…This was confirmed by the recent cloning of distinct -hydroxylases from V. sativa (Tijet et al, 1998;R. Le Bouquin and I. Benveniste, unpublished data).…”

“…We recently cloned CYP94A1, a Cyt P450-dependent -hydroxylase from V. sativa that hydroxylates C12 to C18 fatty acids (Tijet et al, 1998). Here we studied the accumulation of CYP94A1 transcripts during induction of V. sativa seedlings in water or in water containing 1 mm MetJA.…”

“…Le Bouquin and I. Benveniste, unpublished data). One of these enzymes, CYP94A1, when expressed in yeast (Tijet et al, 1998), catalyzed the -hydroxylation of 18:1, 18:2, and 18:3 (oleic, linoleic, and linolenic acids, respectively) and of the model substrate 12:0 (lauric acid).…”

Cloning and Characterization of the WAX2 Gene of Arabidopsis Involved in Cuticle Membrane and Wax Production

“…The CR4 gene was cloned and shown to encode a receptor-like kinase involved in the regulation of global aspects of leaf development rather than strictly cuticle membrane metabolism. Other genes were cloned whose predicted protein sequences suggest functions in the synthesis of cutin (the primary component of cuticle membrane); these genes include CYP86A1 , CYP94A1 , CYP94A2 , and LCR Tijet et al, 1998;LeBouquin et al, 1999;Wellesen et al, 2001). However, these genes have yet to be linked with cutin synthesis in planta.…”