The final acylation step in taxol biosynthesis: cloning of the taxoid C13-side-chain N-benzoyltransferase from Taxus.
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,(1)H-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 k(cat) approximately 1.5 +/- 0.3 s(-1) and K(m) 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.
Walker
Kevin
K
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA. toyota@mail.wsu.edu
Long
Robert
R
Croteau
Rodney
R
D019880:Aldehyde-Ketone Transferases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017239:Paclitaxel / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020946:Taxus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*
D018744:DNA, Plant; D011994:Recombinant Proteins; D019880:Aldehyde-Ketone Transferases; C461940:taxoid C13-side-chain N-benzoyltransferase; D017239:Paclitaxel