A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: characterization, purification and kinetic properties

Abstract

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, α-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 α-kg is the first substrate to bind followed by the binding of O2 and desacetoxyvindoline. Their K m values for α-kg, O2 and desacetoxyvindoline are 45 μM, 45 μM and 0.03 μM, respectively. The first product to be released was deacetylvindoline followed by CO2 and succinate, respectively.

DOI: 10.1007/BF00033888

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@article{Carolis2004AN2, title={A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: characterization, purification and kinetic properties}, author={Emidio De Carolis and Vincenzo De Luca}, journal={Plant Cell, Tissue and Organ Culture}, year={2004}, volume={38}, pages={281-287} }