Purification and characterization of acetyl coenzyme A: 10-hydroxytaxane O-acetyltransferase from cell suspension cultures of Taxus chinensis.
An O-acetyltransferase that catalyzes the regiospecific acetylation of a range of taxanes possessing an unsubstituted 10-hydroxyl group was detected and purified to apparent electrophoretic homogeneity from a cytosolic fraction of Taxus chinensis cell cultures. The purification involved negative calcium phosphate adsorption, sephadex desalting, DEAE, AcA44 chromatography, HighQ, CHT II, HiTrap Blue, Phenylsepharose and Mimetic Green purification steps. The purified acetyltransferase was found to be a monomeric protein of 71 +/- 1.5 kDa that is highly regio- and stereospecific towards the 10 beta-hydroxyl group of the taxane molecule and is also active towards 10-desacetylbaccatine III. The acetyltransferase reaction had a pH optimum of 9.0 with halfmaximal activities at pH 6.8 and 10.8, respectively. The temperature optimum was at 35 degrees C and the isoelectric point at 5.6. The apparent K(m) values for 10-desacetyltaxuyunnanine C and acetyl CoA were 23 and 61 microM, respectively. The turnover rate for the enzyme using both substrates was 0.2 mol mol-1 of enzyme. The kinetic optimum was determined to be Kcat/K(m) = 8.7 s-1 L M-1.
Menhard
B
B
Lehrstuhl für Pharmazeutische Biologie, Universität München, Germany.
Zenk
M H
MH
D000123:Acetyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D007202:Indicators and Reagents; D007700:Kinetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D017239:Paclitaxel / Q000031:analogs & derivatives* / Q000378:metabolism*; D013379:Substrate Specificity; D014197:Trees / Q000201:enzymology*
D007202:Indicators and Reagents; D000123:Acetyltransferases; C118550:acetyl coenzyme A - 10-hydroxytaxane O-acetyltransferase; D017239:Paclitaxel