Partial purification and characterization of acetyl coenzyme A: Taxa-4(20),11(12)-dien-5 alpha-ol O-acetyl transferase that catalyzes the first acylation step of Taxol biosynthesis

The acetylation of taxa-4(20),11 (12)-dien-5 alpha-ol is considered to be t he third specific step of Taxol biosynthesis that precedes further hydroxyl ation of the taxane nucleus. An operationally soluble acetyl CoA:taxadienol -O-acetyl transferase was demonstrated in extracts of Taxus canadensis and Taxus cuspidata cells induced with methyl jasmonate to produce Taxol. The r eaction was dependent on both cosubstrates and active enzyme, and the produ ct of this acetyl transferase was identified by radiochromatographic and GC -MS analysis. Following determination of the time course of acetyl transfer ase appearance in induced cell cultures, the operationally soluble enzyme w as partially purified by a combination of anion exchange, hydrophobic inter action, and affinity chromatography on immobilized coenzyme A resin. This a cetyl transferase has a pI and pH optimum of 4.7 and 9.0, respectively, and a molecular weight of about 50,000 as determined by gel permeation chromat ography. The enzyme shows high selectivity and high affinity for both cosub strates, with K-m values of 4.2 and 5.5 mu M for taxadienol and acetyl CoA, respectively. The enzyme does not acetylate the more advanced Taxol precur sors, 10-deacetylbaccatin III or baccatin III. This acetyl transferase is i nsensitive to monovalent and divalent metal ions, is only weakly inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and coenzyme A, and resemble s in general properties the few other O-acetyl transferases of higher plant origin that have been examined. (C) 1999 Academic Press.