title: Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis.
abstract: The last few steps in the biosynthesis of the anticancer drug Taxol in yew (Taxus) species are thought to involve the attachment of beta-phenylalanine to the C13-O-position of the advanced taxane diterpenoid intermediate baccatin III to yield N-debenzoyl-2'-deoxytaxol, followed by hydroxylation on the side chain at the C2'-position to afford N-debenzoyltaxol, and finally N-benzoylation to complete the pathway. A cDNA encoding the N-benzoyl transferase that catalyzes the terminal step of the reaction sequence was previously isolated from a family of transferase clones (derived from an induced Taxus cell cDNA library) by functional characterization of the corresponding recombinant enzyme using the available surrogate substrate N-debenzoyl-2'-deoxytaxol [K. Walker, R. Long, R. Croteau, Proc. Nat. Acad. Sci. USA 99 (2002) 9166-9171]. Semi-synthetic N-debenzoyltaxol was prepared by coupling of 7-triethylsilybaccatin III and (2R,3S)-beta-phenylisoserine protected as the N-Boc N,O-isopropylidene derivative by means of carbodiimide activation and formic acid deprotections. The selectivity of the recombinant N-transferase for N-debenzoyltaxol was evaluated, and the enzyme was shown to prefer, by a catalytic efficiency factor of two, N-debenzoyltaxol over N-debenzoyl-2'-deoxytaxol as the taxoid co-substrate in the benzoyl transfer reaction, consistent with the assembly sequence involving 2'-hydroxylation prior to N-benzoylation. Selectivity for the acyl/aroyl-CoA co-substrate was also examined, and the enzyme was shown to prefer benzoyl-CoA. Transfer from tigloyl-CoA to N-debenzoyltaxol to afford cephalomannine (Taxol B) was not observed, nor was transfer observed from hexanoyl-CoA or butanoyl-CoA to yield Taxol C or Taxol D, respectively. These results support the proposed sequence of reactions for C13-O-side chain assembly in Taxol biosynthesis, and suggest that other N-transferases are responsible for the formation of related, late pathway, N-acylated taxoids.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Clark Hall, Pullman, WA 99164-6340, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Long
authors.forename: Chandraiah
authors.initials: C
authors.lastname: Lagisetti
authors.forename: Robert M
authors.initials: RM
authors.lastname: Coates
authors.forename: Rodney B
authors.initials: RB
authors.lastname: Croteau
mesh_terms: D004789:Enzyme Activation; D017239:Paclitaxel / Q000737:chemistry*; D013379:Substrate Specificity; D020946:Taxus / Q000201:enzymology*; D014166:Transferases / Q000737:chemistry*
chemical_list: D014166:Transferases; D017239:Paclitaxel