Structure-function relationship in the CYP74 family: conversion of divinyl ether synthases into allene oxide synthases by site-directed mutagenesis

Abstract

Non-classical P450s of CYP74 family control several enzymatic conversions of fatty acid hydroperoxides to bioactive oxylipins in plants, some invertebrates and bacteria. The family includes two dehydrases, namely allene oxide synthase (AOS) and divinyl ether synthase (DES), and two isomerases, hydroperoxide lyase (HPL) and epoxyalcohol synthase. To study the interconversion of different CYP74 enzymes, we prepared the mutant forms V379F and E292G of tobacco (CYP74D3) and flax (CYP74B16) divinyl ether synthases (DESs), respectively. In contrast to the wild type (WT) enzymes, both mutant forms lacked DES activity. Instead, they produced the typical AOS products, α-ketols and (in the case of the flax DES mutant) 12-oxo-10,15-phytodienoic acid. This is the first demonstration of DES into AOS conversions caused by single point mutations.

Keywords: (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid; (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid; 13-HPOT; 9-HPOD; AOS; Allene oxide synthase; CYP74 family; Cytochrome P450; DES; Divinyl ether synthase; HPL; I-helix central domain; IHCD; IMAC; Linum usitatissimum DES (CYP74B16); LuDES; Me; Nicotiana tabacum DES (CYP74D3); NtDES; SRS; Site-directed mutagenesis; TMS; WT; allene oxide synthase; divinyl ether synthase; hydroperoxide lyase; immobilized metal affinity chromatography; methyl; substrate recognition site; trimethylsilyl; wild type.