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

Eur J Biochem. 2001 May;268(10):3083-90. doi: 10.1046/j.1432-1327.2001.02207.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohols / metabolism
  • Amino Acid Sequence
  • Base Sequence
  • Catalysis
  • Chromatography, Thin Layer
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • DNA, Complementary / metabolism
  • Fatty Acids / metabolism*
  • Gas Chromatography-Mass Spectrometry
  • Gene Library
  • Kinetics
  • Microsomes / metabolism
  • Mixed Function Oxygenases / chemistry*
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Molecular Sequence Data
  • Oxygen / metabolism*
  • Plants, Toxic*
  • Saccharomyces cerevisiae / metabolism
  • Stearic Acids / metabolism
  • Substrate Specificity
  • Time Factors
  • Tobacco / enzymology*

Substances

  • Alcohols
  • DNA, Complementary
  • Fatty Acids
  • Stearic Acids
  • 9,10-epoxystearic acid
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • cytochrome P-450 CYP94A5 (Nicotiana tabacum)
  • Oxygen

Associated data

  • GENBANK/AF092916