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
K. Walker et al., 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, ARCH BIOCH, 364(2), 1999, pp. 273-279
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.