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.
De Carolis
E
E
Département de Sciences Biologiques, Université de Montréal, Québec, Canada.
De Luca
V
V
D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins; D010946:Plants, Medicinal / Q000201:enzymology* / Q000378:metabolism; D014747:Vinblastine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism
D010940:Plant Proteins; C009667:vindoline; D014747:Vinblastine; D007501:Iron; D006899:Mixed Function Oxygenases; C080259:CRSD4H protein, Catharanthus roseus