The final acylation step in Taxol biosynthesis: Cloning of the taxoid C13-side-chain N-benzoyltransferase from Taxus

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The National Academy of Sciences

RESUMO

The formation of several acyl groups and an amide group of Taxol is catalyzed by regioselective CoA thioester-dependent acyltransferases. Several full-length acyltransferase sequences, obtained from a cDNA library constructed from mRNA isolated from Taxus cuspidata cells induced for Taxol production with methyl jasmonate, were individually expressed in Escherichia coli, from which a cDNA clone encoding a 3′-N-debenzoyl- 2′-deoxytaxol N-benzoyltransferase was identified. This recombinant enzyme catalyzes the stereoselective coupling of the surrogate substrate N-debenzoyl-(3′RS)-2′-deoxytaxol with benzoyl-CoA to form predominantly one 3′-epimer of 2′-deoxytaxol. The product 2′-deoxytaxol was confirmed by radio-HPLC,1H-NMR, and chemical ionization-MS. This enzymatic reaction constitutes the final acylation in the Taxol biosynthetic pathway. The full-length cDNA coding for the N-benzoyltransferase has an ORF of 1,323 nucleotides and encodes a 441-residue protein with a calculated molecular weight of 49,040. The recombinant enzyme expressed in E. coli has a pH optimum at 8.0, a kcat ≈ 1.5 ± 0.3 s−1 and Km values of 0.42 mM and 0.40 mM for the N-deacylated taxoid and benzoyl-CoA, respectively. In addition to improving the production yields of Taxol in genetically engineered host systems, this enzyme provides a means of attaching modified aroyl groups to taxoid precursors for the purpose of improving drug efficacy.

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