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 β-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 (2 R,3 S)-β-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.