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

Partially purified acetyl CoA: 10-deacetylbaccatin-III-10-orthoxy-acetyltra nsferase from leaves and 3-year-old cell suspension cultures of Taxus cuspi data yields baccatin-III from 10-deacetyl-baccatin-III in the presence of a cetyl-CoA. The enzyme is substrate selective and does not significantly cat alyze the conversion of 10-deacetyltaxol to paclitaxel (Taxol(R)). Ammonium sulfate precipitations and anion exchange column chromatography yielded pa rtially purified enzyme from Taxus cuspidata leaves and cell suspensions. T he 0-40% ammonium sulfate-precipitated protein fraction showed consistent a nd significant enzyme activity. Cell culture-protein extracts yielded highe r 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 o f [1-C-14]acetyl CoA for 1 h at 30 degrees C produce approximately 1.1 pmol baccatin-III per hour per mg of protein. The products of the reactions wer e eluted using photodiode array HPLC and fractions containing baccatin-III or paclitaxel were collected and scintillation counted to determine the amo unt of radiolabeled product formed. Identity of product peaks was confirmed by retention time, photodiode array UV spectrophotometry and co-chromatogr aphy with authentic standards. (C) 1998 Elsevier Science Ltd. All rights re served.