Diprenylated cyclodipeptide production by changing the prenylation sequence of the nature's synthetic machinery

Abstract

Ascomycetous fungi are often found in agricultural products and foods as contaminants. They produce hazardous mycotoxins for human and animals. On the other hand, the fungal metabolites including mycotoxins are important drug candidates and the enzymes involved in the biosynthesis of these compounds are valuable biocatalysts for production of designed compounds. One of the enzyme groups are members of the dimethylallyl tryptophan synthase superfamily, which mainly catalyze prenylations of tryptophan and tryptophan-containing cyclodipeptides (CDPs). Decoration of CDPs in the biosynthesis of multiple prenylated metabolites in nature is usually initiated by regiospecific C2-prenylation at the indole ring, followed by second and third ones as well as by other modifications. However, the strict substrate specificity can prohibit the further prenylation of unnatural C2-prenylated compounds. To overcome this, we firstly obtained C4-, C5-, C6-, and C7-prenylated cyclo-L-Trp-L-Pro. These products were then used as substrates for the promiscuous C2-prenyltransferase EchPT1, which normally uses the unprenylated CDPs as substrates. Four unnatural diprenylated cyclo-L-Trp-L-Pro including the unique unexpected N1,C6-diprenylated derivative with significant yields were obtained in this way. Our study provides an excellent example for increasing structural diversity by reprogramming the reaction orders of natural biosynthetic pathways. Furthermore, this is the first report that EchPT1 can also catalyze N1-prenylation at the indole ring. KEY POINTS: • Prenyltransferases as biocatalysts for unnatural substrates. • Chemoenzymatic synthesis of designed molecules. • A cyclodipeptide prenyltransferase as prenylating enzyme of already prenylated products.

Keywords: Chemoenzymatic synthesis; Cyclodipeptides; EchPT1; Indole prenyltransferases; Multiple prenylation.

Fig. 1

Examples of biosynthetic pathways for multiprenylated CDP derivatives with C2-prenylation as the first decoration step

Fig. 2

SDS-PAGE analysis of the purified prenyltransferases. The proteins were separated on a 12% polyacrylamide gel and stained with Coomassie brilliant blue R-250. Lanes from left to right: a, EchPT1; b, FgaPT2_R244L; c, FgaPT2_Y398F; d, 6-DMATS_Sa; e, 7-DMATS; M, protein marker

Fig. 3

a LCMS analysis of the acceptance of cyclo-l-Trp-l-Pro (1) by EchPT1. b–e LCMS analysis of the acceptance of deoxybrevianamide E (2) by FgaPT2_R244L, FgaPT2_Y398F, 6-DMATS_Sa, and 7-DMATS. UV absorptions at 280 nm are illustrated. All the assays were performed in duplicates. The conversion yield with EchPT1 is given as the mean value

Fig. 4

LCMS analysis of cyclo-l-Trp-l-Pro (1) prenylation by a FgaPT2_R244L, b FgaPT2_Y398F, c 6-DMATS_Sa, and d 7-DMATS. Only absorptions at 280 nm are illustrated. All the assays were performed in duplicates. The conversion yields are given as mean values

Fig. 5

LCMS analysis of acceptance of monoprenylated derivatives (3, 4, 5, and 6) by EchPT1. Products 9 and 10 with a total conversion yield of 23.6 ± 1.4% were isolated from the incubation mixture of 6 with 5. Absorptions at 280 nm are illustrated. All the assays were performed in duplicates. The conversion yields are given as mean values

Fig. 6

Determination of kinetic parameters of EchPT1 toward 3–5 and DMAPP with 3