FgaPT2, a biocatalytic tool for alkyl-diversification of indole natural products

Aromatic prenyltransferases from natural product biosynthetic pathways display relaxed specificity for their aromatic substrates. While a growing body of evidence suggests aromatic prenyltransferases to be more tolerant towards their alkyl-donor substrates, most studies aimed at probing their donor-substrate specificity are limited to only a small set of alkyl pyrophosphate donors, restricting their broader utility as biocatalysts for synthetic applications. Here, we assess the donor substrate specificity of an l-tryptophan C4-prenyltransferase, also known as C4-dimethylallyltryptophan synthase, FgaPT2 from Aspergillus fumigatus, using an array of 34 synthetic unnatural alkyl-pyrophosphate analogues, and demonstrate FgaPT2 can catalyze the transfer of 25 of the 34 non-native alkyl groups from their corresponding synthetic alkyl-pyrophosphate analogues at N1, C3, C4 and C5 position of tryptophan in a normal and reverse manner. The kinetic studies and regio-chemical analysis of the alkyl-l-tryptophan products suggest that the alkyl-donor transfer by FgaPT2 is a function of the stability of the carbocation and the steric factors in the active site of the enzyme. Further, to demonstrate the biocatalytic utility of FgaPT2, this study also highlights the FgaPT2-catalyzed synthesis of a small set of alkyl-diversified indolocarbazole analogues. These results reveal FgaPT2 to be more tolerant to diverse non-native alkyl-donor substrates beyond their known acceptor substrate promiscuity and set the stage for its development as a novel biocatalytic tool for the differential alkylation of natural products for drug discovery and other synthetic applications.