title: Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus.
abstract: 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.
authors.affiliation: Département de Sciences Biologiques, Université de Montréal, Québec, Canada.
authors.forename: E
authors.initials: E
authors.lastname: De Carolis
authors.forename: V
authors.initials: V
authors.lastname: De Luca
mesh_terms: 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
chemical_list: D010940:Plant Proteins; C009667:vindoline; D014747:Vinblastine; D007501:Iron; D006899:Mixed Function Oxygenases; C080259:CRSD4H protein, Catharanthus roseus