Molecular cloning of a 10-deacetylbaccatin Ill-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli

摘要

The cDNA clone for a 10-deacetylbaccatin lIl-10-O-acetyl trans- ferase, which catalyzes formation of the last diterpene intermedi- ate in the Taxol biosynthetic pathway, has been isolated from Taxus cuspidata. By using consensus sequences from an assembly of transacylases of plant origin and from many deduced proteins of unknown function, a homology-based PCR cloning strategy was employed to amplify initially a 911-bp gene fragment of the putative taxane C-10 hydroxyl acetyl transferase from Taxus. This amplicon was used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which the full-length 10-deacetylbaccatin lll-10-O-transacetylase sequence was obtained. Expression of the ORF from pewori~+ in Escherichia coli JM109 afforded a functional enzyme, as deter- mined by ~1H-NMR and MS verification of the product baccatin lll derived from 10-deacetylbaccatin lll and acetyl CoA. The full-length cDNA has an ORF of 1,320 bp corresponding to a deduced protein of 440 residues with a calculated molecular weight of 49,052, consistent with the size of the operationally soluble, monomeric, native acetyl transferase. The recombinant acetyl transferase has a pH optimum of 7.5, has Km values of 10 pM and 8 UM for 10-deacetylbaccatin lll and acetyl CoA. respectively, and is appar- ently regiospecific toward the 10-hydroxyl group of the taxane ring. Amino acid sequence comparison of 10-deacetylbaccatin lll-10-O-acetyl transferase with taxadienol-5-O-acetyl transferase and with other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (80/100 and 64-67/100, respectively).