Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis
AUTOR(ES)
Walker, Kevin
FONTE
The National Academy of Sciences
RESUMO
The structural pharmacophore of Taxol, responsible for binding the N terminus of the β-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 β-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 μM and 4.9 ± 0.3 μM for baccatin III and β-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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=130526Documentos Relacionados
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