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Double Function Hydroperoxide lyases/epoxyalcohol Synthases (CYP74C) of Higher Plants: Identification and Conversion Into Allene Oxide Synthases by Site-Directed Mutagenesis

Double Function Hydroperoxide lyases/epoxyalcohol Synthases (CYP74C) of Higher Plants: Identification and Conversion Into Allene Oxide Synthases by Site-Directed Mutagenesis

Yana Y Toporkova, Svetlana S Gorina, Elena K Bessolitsyna, Elena O Smirnova, Valeria S Fatykhova, Fredi Brühlmann, Tatiana M Ilyina, Lucia S Mukhtarova, Alexander N Grechkin

Biochim Biophys Acta Mol Cell Biol Lipids. 2018 Apr;1863(4):369-378.

PMID: 29325723

Abstract:

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the "hydroperoxide-binding domain" (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP87864208	trans-5-Phenyl-4-pentenyl hydroperoxide		87864-20-8	Price

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