Saturation mutagenesis on Tyr205 of the cyclic dipeptide C2-prenyltransferase FtmPT1 results in mutants with strongly increased C3-prenylating activity.

Abstract

The fungal indole prenyltransferase FtmPT1 is involved in the biosynthesis of fumitremorgins and catalyzes, in the presence of dimethylallyl diphosphate, a predominant regular prenylation of cyclo-L-Trp-L-Pro (brevianamide F) at position C-2 of the indole nucleus. Analysis of the substrate-bound structure of FtmPT1 revealed that brevianamide F forms a hydrogen bond via its carbonyl oxygen in the diketopiperazine moiety with the hydroxyl group of Tyr205 near the center of the prenyltransferase (PT) barrel. In this study, Tyr205 was mutated to 19 other proteinogenic amino acids by one-step site-directed mutagenesis. The obtained mutants were assayed in the presence of dimethylallyl diphosphate with brevianamide F. The enzyme products were isolated on HPLC and their structures were elucidated by NMR and MS analyses. Mutation of Tyr205 to Phe or Met did not change the behavior of FtmPT1 significantly, with regularly C2-prenylated brevianamide F as the predominant product. Interestingly, 15 of the obtained mutants also produced regularly C3-prenylated brevianamide F, with relative yields between 33 and 110% of those of the regularly C2-prenylated derivatives. Among them, Y205C, Y205L, Y205N, Y205I, and Y205S showed similar brevianamide F consumption. Y205H, Y205Q, Y205V, Y205G, and Y205E showed activities between 47 and 77% of that of the wild type. These results provide a solid basis for the construction of a brevianamide F regular C3-prenyltransferase by site-directed mutagenesis. Assaying stereoisomers of brevianamide F, cyclo-D-Trp-D-Pro, cyclo-L-Trp-D-Pro, and cyclo-D-Trp-L-Pro, with two selected mutants Y205N and Y205L resulted in the formation of reversely C3-prenylated derivatives as predominant products, being in sharp contrast to their regularly C2- and C3-prenylated derivatives with cyclo-L-Trp-L-Pro.