An N-Aroyltransferase of the BAHD Superfamily Has Broad Aroyl CoA Specificity in Vitro with Analogues of N-Dearoylpaclitaxel

Abstract

The native N-debenzoyl-2'-deoxypaclitaxel:N-benzoyltransferase (NDTBT), from Taxus plants, transfers a benzoyl group from the corresponding CoA thioester to the amino group of the beta-phenylalanine side chain of N-debenzoyl-2'-deoxypaclitaxel, which is purportedly on the paclitaxel (Taxol) biosynthetic pathway. To elucidate the substrate specificity of NDTBT overexpressed in Escherichia coli, the purified enzyme was incubated with semisynthetically derived N-debenzoyltaxoid substrates and aroyl CoA donors (benzoyl; ortho-, meta-, and para-substituted benzoyls; various heterole carbonyls; alkanoyls; and butenoyl), which were obtained from commercial sources or synthesized via a mixed anhydride method. Several unnatural N-aroyl-N-debenzoyl-2'-deoxypaclitaxel analogues were biocatalytically assembled with catalytic efficiencies (V(max)/K(M)) ranging between 0.15 and 1.74 nmol.min(-1).mM(-1). In addition, several N-acyl-N-debenzoylpaclitaxel variants were biosynthesized when N-debenzoylpaclitaxel and N-de(tert-butoxycarbonyl)docetaxel (i.e., 10-deacetyl-N-debenzoylpaclitaxel) were used as substrates. The relative velocity (v(rel)) for NDTBT with the latter two N-debenzoyl taxane substrates ranged between approximately 1% and 200% for the array of aroyl CoAs compared to benzoyl CoA. Interestingly, NDTBT transferred hexanoyl, acetyl, and butyryl more rapidly than butenoyl or benzoyl from the CoA donor to taxanes with isoserinoyl side chains, whereas N-debenzoyl-2'-deoxypaclitaxel was more rapidly converted to its N-benzoyl derivative than to its N-alkanoyl or N-butenoyl congeners. Biocatalytic N-acyl transfer of novel acyl groups to the amino functional group of N-debenzoylpaclitaxel and its 2'-deoxy precursor reveal the surprisingly indiscriminate specificity of this transferase. This feature of NDTBT potentially provides a tool for alternative biocatalytic N-aroylation/alkanoylation to construct next generation taxanes or other novel bioactive diterpene compounds.