Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus.
A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the hydroxylation at position 4 of the indole alkaloid, desacetoxyvindoline has been purified to near homogeneity from Catharanthus roseus. The purification procedure combined conventional chromatographic methods and cosubstrate affinity chromatography on alpha-ketoglutarate-Sepharose. The specific activity of the 4-hydroxylase was enriched over 2000-fold compared to the crude homogenate with a recovery of 1.6%. The molecular mass of the native and denatured 4-hydroxylase was found to be 45 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 pI values 4.6, 4.7, and 4. 8. The enzyme did not require most divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. 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 2-oxoglutarate is the first substrate to bind followed by the binding of O2 and desacetoxyvindoline. The first product to be released was deacetylvindoline followed by CO2 and succinate, respectively.[1]References
- Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus. De Carolis, E., De Luca, V. J. Biol. Chem. (1993) [Pubmed]
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