Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis
| Autor: | V.S. Fatykhova, Fredi Brühlmann, Lucia S. Mukhtarova, Svetlana S. Gorina, Yana Y. Toporkova, Elena K. Bessolitsyna, Tatiana Ilyina, Alexander N. Grechkin, Elena O. Smirnova |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Lipid Peroxides Linolenic Acids Stereochemistry Linoleic acid Mutant Gas Chromatography-Mass Spectrometry Substrate Specificity 03 medical and health sciences chemistry.chemical_compound Cytochrome P-450 Enzyme System Amino Acid Sequence Site-directed mutagenesis Molecular Biology Phylogeny Aldehyde-Lyases Plant Proteins chemistry.chemical_classification biology ATP synthase Fatty acid Cytochrome P450 Cell Biology Plants Recombinant Proteins Intramolecular Oxidoreductases Kinetics 030104 developmental biology Enzyme Linoleic Acids chemistry Glycine Biocatalysis Mutagenesis Site-Directed biology.protein Mutant Proteins Sequence Alignment |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1863:369-378 |
| ISSN: | 1388-1981 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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