Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis.
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 K(m) values of 2.4 +/- 0.5 microM and 4.9 +/- 0.3 microM 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.
Walker
Kevin
K
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA. toyota@mail.wsu.edu
Fujisaki
Shingo
S
Long
Robert
R
Croteau
Rodney
R
D000214:Acyl Coenzyme A / Q000737:chemistry*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017239:Paclitaxel / Q000378:metabolism; D013379:Substrate Specificity; D020946:Taxus / Q000378:metabolism; D013997:Time Factors
D000214:Acyl Coenzyme A; D018076:DNA, Complementary; C078024:phenylpropionyl-coenzyme A; C089123:C-13-deacylase; D002265:Carboxylic Ester Hydrolases; D017239:Paclitaxel