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

• Published in: FEBS letters 2013-08, Vol.587 (16), p.2552-2558
• Main Authors: Toporkova, Yana Y., Ermilova, Valeria S., Gorina, Svetlana S., Mukhtarova, Lucia S., Osipova, Elena V., Gogolev, Yuri V., Grechkin, Alexander N.
• Format: Article
• Language: English
• Subjects: (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid; (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid; 13-HPOT; 9-HPOD; Allene oxide synthase; Amino Acid Sequence; AOS; Computational Biology; CYP74 family; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P450; DES; Divinyl ether synthase; Flax - enzymology; Gas Chromatography-Mass Spectrometry; HPL; hydroperoxide lyase; I-helix central domain; IHCD; IMAC; immobilized metal affinity chromatography; Intramolecular Oxidoreductases - chemistry; Linum usitatissimum DES (CYP74B16); LuDES; methyl; Molecular Sequence Data; Mutagenesis, Site-Directed; Nicotiana - enzymology; Nicotiana tabacum DES (CYP74D3); NtDES; Plant Proteins - chemistry; Point Mutation; Protein Conformation; Recombinant Proteins - chemistry; Sequence Homology, Amino Acid; Site-directed mutagenesis; SRS; Structure-Activity Relationship; substrate recognition site; TMS; trimethylsilyl; wild type
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2013.06.030

•The interrelation of CYP74 enzymes was tested by single point mutations.•The mutant forms of tobacco and flax divinyl ether synthases (DESs) were prepared.•Mutation sites V379F and E292G belong to the ERR-triad and I-helix domains.•Both mutations converted DESs into allene oxide synthases (AOSs).•DES to AOS conversions caused by single point mutations are described for the first time. 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.