CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the omega-alcohol and to the corresponding diacid.
A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10-epoxystearic acid was converted into one major metabolite identified by GC/MS as 18-hydroxy-9,10-epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 +/- 0.2 microM and Vmax,app = 27 +/- 1 nmol x min(-1) x nmol(-1) P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10-epoxy-octadecan-1,18-dioic acid. The diacid was also produced in microsomal incubations of 18-hydroxy-9,10-epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the omega-hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.
Le Bouquin
R
R
Institut de Biologie Moléculaire des Plantes-CNRS UPR2357, Département: Réponses au Stress, Strasbourg, France.
Skrabs
M
M
Kahn
R
R
Benveniste
I
I
Salaün
J P
JP
Schreiber
L
L
Durst
F
F
Pinot
F
F
D000438:Alcohols / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D015723:Gene Library; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D010947:Plants, Toxic; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013229:Stearic Acids / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014026:Nicotiana / Q000201:enzymology*
D000438:Alcohols; D018076:DNA, Complementary; D005227:Fatty Acids; D013229:Stearic Acids; C013519:9,10-epoxystearic acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C431568:cytochrome P-450 CYP94A5 (Nicotiana tabacum); D010100:Oxygen