期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2002
卷号：99
期号：20
页码：12715-12720
DOI：10.1073/pnas.192463699
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The structural pharmacophore of Taxol, responsible for binding the N terminus of the {beta}-subunit of tubulin to arrest cell proliferation, comprises, in part, the 13-O-(N-benzoyl-3-phenylisoserinoyl) side chain. To identify the side chain transferase of Taxol biosynthesis, a set of transacylases obtained from an enriched cDNA library (constructed from mRNA isolated from Taxus cuspidata cells induced with methyl jasmonate for Taxol production) was screened. A cDNA clone (designated TAX7) encoding a taxoid C-13 O-phenylpropanoyltransferase was isolated which yielded a recombinant enzyme that catalyzes the selective 13-O-acylation of baccatin III with {beta}-phenylalanoyl CoA as the acyl donor to form N-debenzoyl-2'-deoxytaxol. This enzymatic product was converted to 2'-deoxytaxol by chemical N-benzoylation, and the identity of this derivative was confirmed by spectrometric analyses. The full-length cDNA has an ORF of 1,335 bases and encodes a 445-aa protein with a calculated molecular weight of 50,546. Evaluation of kinetic parameters revealed Km values of 2.4 {+/-} 0.5 {micro}M and 4.9 {+/-} 0.3 {micro}M for baccatin III and {beta}-phenylalanoyl-CoA, respectively. The pH optimum for the recombinant O-(3-amino-3-phenylpropanoyl)transferase is at 6.8. Identification of this clone completes acquisition of the five aroyl/acyltransferases involved in the biosynthesis of Taxol. Application of these transacylase genes in suitable host cells can improve the production yields of Taxol and could enable the preparation of second-generation Taxol analogs possessing greater bioactivity and improved water solubility.
关键词：side chain attachment‖C-13 O -acyltransferase‖paclitaxel‖ baccatin III‖amino acid-CoA esters
