Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 97, Issue 25, Pages 13591-13596Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.250491997
Keywords
paclitaxel; 2-debenzoyl-7,13-diacetylbaccatin III
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Funding
- NCI NIH HHS [CA-55254, R37 CA055254, R01 CA055254] Funding Source: Medline
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A cDNA clone encoding a taxane 2 alpha -O-benzoyltransferase has been isolated from Taxus cuspidata. The recombinant enzyme catalyzes the conversion of 2-debenzoyl-7,13-diacetylbaccatin III, a semisynthetic substrate, to 7,13-diacetylbaccatin III, and thus appears to function in a late-stage acylation step of the Taxol biosynthetic pathway. By employing a homology-based PCR cloning strategy for generating acyltransferase oligodeoxynucleotide probes, several gene fragments were amplified and used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which several full-length acyltransferases were obtained and individually expressed in Escherichia coli. The functionally expressed benzoyltransferase was confirmed by radio-HPLC,H-1-NMR, and combined HPLC-MS verification of the product, 7,13-diacetylbaccatin III, derived from 2-debenzoyl-7,13-diacetylbaccatin III and benzoyl-CoA as cosubstrates in the corresponding cell-free extract. The full-length cDNA has an open reading frame of 1,320 base pairs and encodes a protein of 440 residues with a molecular weight of 50,089. The recombinant benzoyltransferase has a pH optimum of 8.0. K-m values of 0.64 mM and 0.30 mM for the taxoid substrate and benzoyl-CoA, respectively, and is apparently regiospecific for acylation of the 2 alpha -hydroxyl group of the functionalized taxane nucleus. This enzyme may be used to improve the production yields of Taxol and for the semisynthesis of drug analogs bearing modified aroyl groups at the C2 position.
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