Violaxanthin conversion by recombinant diatom and plant de-epoxidases, expressed in Escherichia coli – comparative analysis

Monika Olchawa-Pajor; Monika Bojko; Wojciech Strzałka; Kazimierz Strzałka; Dariusz Latowski

doi:10.18388/abp.2019_2831

Monika Olchawa-Pajor Department of Environmental Protection, Institute of Mathematical and Natural Sciences, State Higher Vocational School in Tarnow, Poland https://orcid.org/0000-0002-8263-6429
Monika Bojko Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland https://orcid.org/0000-0002-6806-0187
Wojciech Strzałka Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland https://orcid.org/0000-0002-4541-8545
Kazimierz Strzałka 1Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland https://orcid.org/0000-0002-8367-4865
Dariusz Latowski Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland https://orcid.org/0000-0001-9801-3990

DOI: https://doi.org/10.18388/abp.2019_2831

Abstract

The purpose of this research was to obtain recombinant violaxanthin de-epoxidases (VDEs) from two species. The first one was VDE of Arabidopsis thaliana (L.) Heynh. (WT Columbia strain) (AtVDE) which in vivo catalyzes conversion of violaxanthin (Vx) to zeaxanthin (Zx) via anteraxanthin (Ax). The second one was VDE of Phaeodactylum tricornutum Bohlin, 1897 (CCAP 1055/1 strain) (PtVDE) which is responsible for de-epoxidation of diadinoxanthin (Ddx) to diatoxanthin (Dtx). As the first step of our experiments, open reading frames coding for studied enzymes were amplified and subsequently cloned into pET-15b plasmid. For recombinant proteins production Escherichia coli Origami b strain was used. The molecular weight of the produced enzymes were estimated approximately at 45kDa and 50kDa for AtVDE and PtVDE, respectively. Both enzymes, purified under native conditions by immobilized metal affinity chromatography, displayed comparable activity in assay mixture and converted up to 90% Vx in 10 min in two steps enzymatic de-epoxidation, irrespective of enzyme origin. No statistically significant differences were observed when kinetics of the reactions catalyzed by these enzymes were compared. Putative role of selected amino-acid residues of AtVDE and PtVDE was also considered. The significance of the first time obtained recombinant PtVDE as a useful tool in various comparative investigations of de-epoxidation reactions in main types of xanthophyll cycles existing in nature are also indicated.