A NOVEL 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE INVOLVED IN VINDOLINE BIOSYNTHESIS - CHARACTERIZATION, PURIFICATION AND KINETIC-PROPERTIES

The enzyme, desacetoxyvindoline 4-hydroxylase, was purified to apparent homogeneity from Catharanthus roseus by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, green 19-agarose, hydroxylapatite, alpha-kg sepharose and Mono Q. The 4-hydroxylase was characterized by its strict specificity for position 4 of desacetoxyvindoline suggesting it to catalyze the second to last step in vindoline biosynthesis. The molecular mass of the native and denatured 4-hydroxylase was 45 kDa and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pIs 4.6, 4.7 and 4.8. The purified 4-hydroxylase exhibited no requirement for divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The enzyme was not inhibited by EDTA or SH-group reagents at concentrations up to 10 mM. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where alpha-kg is the first substrate to bind followed by the binding of O-2 and desacetoxyvindoline. Their K-m values for alpha-kg, O-2 and desacetoxyvindoline are 45 mu M, 45 mu M and 0.03 mu M, respectively. The first product to be released was deacetylvindoline followed by CO? and succinate, respectively.