Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1) involved in cutin monomers synthesis.
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.
Tijet
N
N
Institut de Biologie Moléculaire des Plantes-CNRS, Département d'Enzymologie Cellulaire et Moléculaire, 28 rue Goethe, F-67083 Strasbourg Cedex, France.
Helvig
C
C
Pinot
F
F
Le Bouquin
R
R
Lesot
A
A
Durst
F
F
Salaün
J P
JP
Benveniste
I
I
D002994:Clofibrate / Q000494:pharmacology*; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology*; D005227:Fatty Acids / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D007850:Lauric Acids / Q000378:metabolism; D008563:Membrane Lipids / Q000096:biosynthesis*; D006899:Mixed Function Oxygenases / Q000502:physiology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry; D010940:Plant Proteins / Q000502:physiology; D010946:Plants, Medicinal; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013053:Spectrophotometry; D013379:Substrate Specificity
D018076:DNA, Complementary; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D007850:Lauric Acids; D008563:Membrane Lipids; D010446:Peptide Fragments; D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C040948:11-dodecynoic acid; C000521:cutin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D002994:Clofibrate