title: Purification and characterization of acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus.
abstract: The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyltransferase (EC 2.3.1.-) (DAT), which catalyzes the final step in vindoline biosynthesis in Catharanthus roseus, was purified 3300-fold using ammonium sulfate precipitation followed by gel filtration, anion exchange, hydroxyapatite, and affinity chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified DAT showed the presence of two major proteins having Mr values of 33,000 and 21,000, whereas native PAGE showed three protein bands, and isoelectric focusing-PAGE one diffuse protein band (pI = 4.7-5.3) plus two minor protein bands (pI = 5.7 and 6.1). Purified DAT possessed Km values of 6.5 microM and 1.3 microM for acetylcoenzyme A and deacetylvindoline, respectively, and Vmax values of 12.6 pkat/microgram protein (acetylcoenzyme A) and 10.1 pkat/micrograms protein (deacetylvindoline). Inhibition of DAT by tabersonine, coenzyme A, and cations (K+, Mg2+, and Mn2+) was observed, while the pH optimum of this enzyme was determined to be 7.5 to 9.
authors.affiliation: Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan.
authors.forename: R
authors.initials: R
authors.lastname: Power
authors.forename: W G
authors.initials: WG
authors.lastname: Kurz
authors.forename: V
authors.initials: V
authors.lastname: De Luca
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000123:Acetyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D002412:Cations / Q000494:pharmacology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D014747:Vinblastine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D002412:Cations; C009667:vindoline; D014747:Vinblastine; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C064442:acetylcoenzyme A-deacetylvindoline 4-O-acetyltransferase