MOLECULAR-CLONING AND CHARACTERIZATION OF 12-OXOPHYTODIENOATE REDUCTASE, AN ENZYME OF THE OCTADECANOID SIGNALING PATHWAY FROM ARABIDOPSIS-THALIANA - STRUCTURAL AND FUNCTIONAL-RELATIONSHIP TO YEAST OLD YELLOW ENZYME

Using partial amino acid sequence information for 12-oxophytodienoate- 10,11-reductase obtained from Corydalis sempervirens we have cloned th e homologous enzyme from Arabidopsis thaliana. The open reading frame of the cDNA encodes a polypeptide of 372 amino acids (M-r = 41,165) wi th significant similarity to the sequence of Old Yellow Enzyme from Sa ccharomyces carlsbergensis (Saito, K., Thiele, D. J., Davio, M., Lockr idge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724), a fl avin (FMN)-protein catalyzing the NADPH-dependent reduction of the ole finic bond of alpha,beta-unsaturated carbonyls. Specifically, all resi dues required for binding of FMN in Old Yellow Enzyme are conserved in the A. thaliana sequence, as are all residues associated with catalyt ic activity. The enzyme was functionally expressed from its cDNA in Es cherichia coli and thus proven to encode OPDA reductase. Further simil arities of OPDA reductase and yeast Old Yellow Enzyme include their bi nding to and elution by reductant from N-(4-hydroxybenzoyl)aminohexyl- Sepharose the immunoreactivity of yeast Old Yellow Enzyme with an anti serum raised against plant OPDA reductase and the demonstration that O ld Yellow Enzyme is an active OPDA reductase. It is thus conceivable t hat the physiological role of Old Yellow Enzymes now known from bacter ia, yeasts, and higher plants, is in oxylipin metabolism.