Green leaf divinyl ether synthase: Gene detection, molecular cloning and identification of a unique CYP74B subfamily member

Abstract

Enzymes of the CYP74 family (P450 superfamily) play a key role in the plant lipoxygenase signalling cascade. Recently we detected a pathogen inducible divinyl ether synthase (DES) in flax leaves [Chechetkin, Blufard, Hamberg, Grechkin, 2008]. This prompted us to examine the CYP74 genes in the flax leaf transcriptome. Since the flax genome is not sequenced, we used the PCR approach with degenerate primers related to the conserved domains of selected CYP74 genes; this revealed several CYP74 transcripts in flax leaves. One transcript belongs to the previously described allene oxide synthase ( LuAOS, CYP74A, GenBank ID: U00428.1). Another one contains the ORF (1473 bp) of an unknown CYP74B16 gene. Three more nearly identical sequences, including one expressed pseudogene, were also identified. The recombinant CYP74B16 protein expressed in Escherichia coli had 491 amino acid residues and MW of 56 kDa. The preferred substrate of this enzyme is the 13-hydroperoxide of α-linolenic acid, and the reaction product was identified by mass spectrometry, NMR and UV spectroscopy as the divinyl ether (9 Z,11 E)-12-[(1′ Z,3′ Z)-hexadienyloxy]-9,11-dodecadienoic acid, (ω5 Z)-etherolenic acid. All previously known CYP74B subfamily enzymes are hydroperoxide lyases. The novel flax enzyme CYP74B16 (LuDES) is an unprecedented DES member of the CYP74B subfamily.