Cloning, expression and functional studies of violaxanthin de-epoxidase from Spinacia oleracea

Abstract

Violaxanthin de-epoxidase (VDE) is a key enzyme of the xanthophyll cycle of all higher plants. It catalyzes the conversion of violaxanthin to zeaxanthin upon high light, resulting in photoprotection of the plant by energy dissipation. VDE has been purified from spinach thylakoids and the amount of the enzyme was estimated to be as low as 1 VDE per 20-100 electron transport chains. To obtain recombinant VDE, the gene was cloned by screening a spinach cDNA library (GenBank AJ 250433). Overexpression of the spinach VDE gene in E. coli resulted in highly active recombinant protein. Four histidine residues are conserved in spinach, lettuce and Arabidopsis. In tobacco one of the histidines is replaced by a leucine. When release of VDE from thylakoids was measured by sonication at different pH, the release was found to be strongly pH dependent with an inflexion point at pH 6,6 and a cooperativity of 4 for protons. Since there are 4 histidines with a pK value of the side group around 6,0, we were interested in investigating the role of the histidine for VDE activity further. By incubating VDE with DEPC, the histidines were chemically modified by carbethoxylation and VDE activity was fully inhibited. The modification of histidines can be reversed, and after incubating the chemically modified VDE with hydroxylamine, most of the catalytic activity was regained. The histidine residues clearly play a role in VDE?s catalytic activity, and we propose that they are important for the binding of VDE to the thylakoid membrane at low pH.