Publications by authors named "Elena O Smirnova"

6 Publications

  • Page 1 of 1

Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (, Chordata).

Int J Mol Sci 2021 Apr 29;22(9). Epub 2021 Apr 29.

Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, P.O. Box 30, 420111 Kazan, Russia.

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher's) lancelet (, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9,11,12,13,15)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and -12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, -12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo--12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name " EAS/AOS" (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22094737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124189PMC
April 2021

The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis.

Phytochemistry 2020 Nov 11;179:112512. Epub 2020 Sep 11.

Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center, Russian Academy of Sciences, P.O. Box 30, Kazan, 420111, Russia. Electronic address:

The CYP74 family of cytochromes P450 includes four enzymes of fatty acid hydroperoxide metabolism: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), and epoxyalcohol synthase (EAS). The present work is concerned with catalytic specificities of three recombinant DESs, namely, the 9-DES (LeDES, CYP74D1) of tomato (Solanum lycopersicum), 9-DES (NtDES, CYP74D3) of tobacco (Nicotiana tabacum), and 13-DES (LuDES, CYP74B16) of flax (Linum usitatissimum), as well as their alterations upon the site-directed mutagenesis. Both LeDES and NtDES converted 9-hydroperoxides of linoleic and α-linolenic acids to divinyl ethers colneleic and colnelenic acids (respectively) with only minorities of HPL and EAS products. In contrast, LeDES and NtDES showed low efficiency towards the linoleate 13-hydroperoxide, affording only the low yield of epoxyalcohols. LuDES exhibited mainly the DES activity towards α-linolenate 13-hydroperoxide (preferred substrate), and HPL activity towards linoleate 13-hydroperoxide, respectively. In contrast, LuDES converted 9-hydroperoxides primarily to the epoxyalcohols. The F291V and A287G mutations within the I-helix groove region (SRS-4) of LuDES resulted in the loss of DES activity and the acquirement of the epoxyalcohol synthase activity. Thus, the studied enzymes exhibited the versatility of catalysis and its qualitative alterations upon the site-directed mutagenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phytochem.2020.112512DOI Listing
November 2020

Catalysis by allene oxide synthases (CYP74A and CYP74C): Alterations by the Phe/Leu mutation at the SRS-1 region.

Phytochemistry 2020 Jan 10;169:112152. Epub 2019 Oct 10.

Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center, Russian Academy of Sciences, P.O. Box 30, Kazan, 420111, Russia. Electronic address:

The CYP74 family of cytochromes P450 includes four fatty acid hydroperoxide metabolizing enzymes: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase, and epoxyalcohol synthase (EAS). All P450s have six substrate recognition sites (SRSs) in their structures. Some CYP74 mutations in SRSs leading to their interconversions including substitutions in "F/L toggle" (SRS-1 region) were reported before. For further elucidation of the role of this site in CYP74 catalysis, the effect of Phe/Leu mutation on the specificity of selected AOSs was studied in the present work. Mutant forms of ZmAOS1 (CYP74A19, Zea mays), LeAOS3 (CYP74C3, Lycopersicon esculentum), and PpAOS2 (CYP74A8, Physcomitrella patens) acquired partial EAS activity. Mutant forms of ZmAOS1 and PpAOS2 possessed additional HPL activities. The results validate the significance of the "F/L toggle" as a catalytic determinant of CYP74s, as well as the importance of the conserved Phe at this site for the AOS catalysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phytochem.2019.112152DOI Listing
January 2020

Detection of the first higher plant epoxyalcohol synthase: Molecular cloning and characterisation of the CYP74M2 enzyme of spikemoss Selaginella moellendorffii.

Phytochemistry 2018 Dec 6;156:73-82. Epub 2018 Sep 6.

Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan, 420111, Russia. Electronic address:

The CYP74M2 gene of a model plant, the spikemoss Selaginella moellendorffii Hieron, was cloned and the catalytic properties of corresponding recombinant protein were studied. The recombinant CYP74M2 protein was active towards 13-hydroperoxides of linoleic and a-linolenic acids (13-HPOD and 13-HPOT, respectively). In contrast to previously studied CYP74M1 and CYP74M3, which possessed the divinyl ether synthase activity, CYP74M2 behaved as a dedicated epoxyalcohol synthase (EAS). For instance, the 13-HPOD was converted to three epimeric oxiranyl carbinols 1-3 (formed at a ratio ca. 4:2:1), namely the (11R,12S,13S), (11R,12R, 13S), and (11S,12S,13S) epimers of (9Z)-11-hydroxy-12,13-epoxy-9-octadecenoic acid. Besides these products, a minority of oxiranyl vinyl carbinols like (10E)-11-hydroxy-12,13-epoxy-9-octadecenoic acid was formed. The 13-HPOT conversion by CYP74M2 afforded two stereoisomers of 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the H-NMR, 2D-COSY, HSQC, and HMBC. Thus, the CYP74M2 is the dedicated epoxyalcohol synthase. To our knowledge, no enzymes of this type have been detected in higher plants yet.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phytochem.2018.08.010DOI Listing
December 2018

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 Apr 8;1863(4):369-378. Epub 2018 Jan 8.

Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbalip.2018.01.002DOI Listing
April 2018

Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Molecular cloning and identification of divinyl ether synthases CYP74M1 and CYP74M3.

Biochim Biophys Acta 2016 Apr 9;1861(4):301-9. Epub 2016 Jan 9.

Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia. Electronic address:

Nonclassical P450s of CYP74 family control the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. At least ten genes attributed to four novel CYP74 subfamilies have been revealed by the recent sequencing of the spikemoss Selaginella moellendorffii Hieron genome. Two of these genes CYP74M1 and CYP74M3 have been cloned in the present study. Both recombinant proteins CYP74M1 and CYP74M3 were active towards the 13(S)-hydroperoxides of α-linolenic and linoleic acids (13-HPOT and 13-HPOD, respectively) and exhibited the activity of divinyl ether synthase (DES). Products were analyzed by gas chromatography-mass spectrometry. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the (1)H NMR, 2D-COSY, HSQC and HMBC. CYP74M1 (SmDES1) specifically converted 13-HPOT to (11Z)-etherolenic acid and 13-HPOD to (11Z)-etheroleic acid. CYP74M3 (SmDES2) turned 13-HPOT and 13-HPOD mainly to etherolenic and etheroleic acids, respectively. CYP74M1 and CYP74M3 are the first DESs detected in non-flowering plants. The obtained results demonstrate the existence of the sophisticated oxylipin biosynthetic machinery in the oldest taxa of vascular plants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbalip.2016.01.001DOI Listing
April 2016