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 M r values of 33,000 and 21,000, whereas native PAGE showed three protein bands, and isoelectric focusing-PAGE one diffuse protein band (p I = 4.7−5.3) plus two minor protein bands (p I = 5.7 and 6.1). Purified DAT possessed K m values of 6.5 μ m and 1.3μ m for acetylcoenzyme A and deacetylvindoline, respectively, and V max values of 12.6 pkat/μg protein (acetylcoenzyme A) and 10.1 pkat/μg protein (deacetylvindoline). Inhibition of DAT by tabersonine, coenzyme A, and cations (K + , Mg 2+ , and Mn 2+ ) was observed, while the pH optimum of this enzyme was determined to be 7.5 to 9.