Acetyl CoA:10-deacetylbaccatin-III-10-O-acetyltransferase activity in leaves and cell suspension cultures of Taxus cuspidata

Abstract

Abstract Partially purified acetyl CoA:10-deacetylbaccatin-III-10-orthoxy-acetyltransferase from leaves and 3-year-old cell suspension cultures of Taxus cuspidata yields baccatin-III from 10-deacetyl-baccatin-III in the presence of acetyl-CoA. The enzyme is substrate selective and does not significantly catalyze the conversion of 10-deacetyltaxol to paclitaxel (Taxol®). Ammonium sulfate precipitations and anion exchange column chromatography yielded partially purified enzyme from Taxus cuspidata leaves and cell suspensions. The 0–40% ammonium sulfate-precipitated protein fraction showed consistent and significant enzyme activity. Cell culture-protein extracts yielded higher activities of the enzyme than did leaf-protein extracts. 1 ml aliquots of enzyme preparation with 150 nmol of 10-deacetylbaccatin-III and 4.4 nmol of [1- 14 C]acetyl CoA for 1 h at 30°C produce approximately 1.1 pmol baccatin-III per hour per mg of protein. The products of the reactions were eluted using photodiode array HPLC and fractions containing baccatin-III or paclitaxel were collected and scintillation counted to determine the amount of radiolabeled product formed. Identity of product peaks was confirmed by retention time, photodiode array UV spectrophotometry and co-chromatography with authentic standards.