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

Abstract

The purpose of study presented here 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 catalyzes conversion of violaxanthin (Vx) to zeaxanthin (Zx) via anteraxanthin (Ax) in vivo. 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 the studied enzymes were amplified and subsequently cloned into the pET-15b plasmid. For recombinant protein production, the Escherichia coli Origami b strain was used. The molecular weight of the produced enzymes was approximately estimated to be 45kDa and 50kDa for AtVDE and PtVDE, respectively. Both enzymes, purified under native conditions by immobilized metal affinity chromatography, displayed comparable activity in an assay mixture and converted up to 90% of Vx in 10 min in a two step enzymatic de-epoxidation, irrespective of the enzyme's origin. No statistically significant differences were observed when kinetics of the reactions catalyzed by these enzymes were compared. A putative role of the selected amino-acid residues of AtVDE and PtVDE was also considered. Significance of recombinant PtVDE (purified here for the first time ever) is also indicated as a useful tool in various comparative investigations of deepoxidation reactions in main types of xanthophyll cycles existing in nature.