Abstract
The cDNA clone for a 10-deacetylbaccatin III-10-O-acetyl transferase, which catalyzes formation of the last diterpene intermediate in the Taxol biosynthetic pathway, has been isolated fromTaxus 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 fromTaxus. This amplicon was used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-inducedTaxuscells, from which the full-length 10-deacetylbaccatin III-10-O-transacetylase sequence was obtained. Expression of the ORF from pCWori+inEscherichia coliJM109 afforded a functional enzyme, as determined by1H-NMR and MS verification of the product baccatin III derived from 10-deacetylbaccatin III 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, hasKmvalues of 10 μM and 8 μM for 10-deacetylbaccatin III and acetyl CoA, respectively, and is apparently regiospecific toward the 10-hydroxyl group of the taxane ring. Amino acid sequence comparison of 10-deacetylbaccatin III-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% and 64–67%, respectively).
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Dates
| Type | When |
|---|---|
| Created | 23 years, 1 month ago (July 26, 2002, 10:31 a.m.) |
| Deposited | 2 years, 4 months ago (April 22, 2023, 8:05 a.m.) |
| Indexed | 1 month ago (Aug. 2, 2025, 1:22 a.m.) |
| Issued | 25 years, 7 months ago (Jan. 18, 2000) |
| Published | 25 years, 7 months ago (Jan. 18, 2000) |
| Published Online | 25 years, 7 months ago (Jan. 18, 2000) |
| Published Print | 25 years, 7 months ago (Jan. 18, 2000) |
@article{Walker_2000, title={Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA fromTaxusand functional expression inEscherichia coli}, volume={97}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.97.2.583}, DOI={10.1073/pnas.97.2.583}, number={2}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Walker, Kevin and Croteau, Rodney}, year={2000}, month=jan, pages={583–587} }