Chemical and mutational modification of histidines in violaxanthin de-epoxidase from Spinacia oleracea

The violaxanthin de-epoxidase (VDE) gene from spinach ( Spinacia oleracea ) was cloned, sequenced (GenBank AJ 250433), and expressed in Escherichia coli. The highest obtained conversion rate of violaxanthin was 86 nmol s⁻¹ per litre of growth medium, corresponding to an amount of active enzyme of 0.4 mg l⁻¹. Sequence comparison between VDE from different species were made and particular interest was focused on four highly conserved histidines (H121,124,167,173) and their possible involvement in enzymatic activity. Chemical modification of the histidines using DEPC or by site-directed mutations resulted in partial or total inactivation of the enzyme. The chemical modification could be reversed by hydroxylamine treatment, regenerating a large percentage of the original activity. The histidine residues, which are located in pairs close to each other, were pairwise substituted for either alanine or arginine. This resulted in one inactive mutant (H121,124R) and three mutants with very different activities and decreased binding of ascorbic acid, as reflected by an up to four-fold increase in K _m. A substitution of all four histidines for either alanine or arginine resulted in inactive enzymes. Based on these results it is suggested that the histidine residues are important for the activity of VDE.