Simultaneous C7- and N1-prenylation of cyclo-l-Trp-l-Trp catalyzed by a prenyltransferase from Aspergillus oryzae

Abstract

A putative prenyltransferase gene cTrpPT was amplified from Aspergillus oryzae DSM1147, cloned into pQE70 and overexpressed in Escherichia coli. The overproduced His₆-CTrpPT was purified to near homogeneity and incubated with L-tryptophan or tryptophan-containing cyclic dipeptides in the presence of dimethylallyl diphosphate. The formation of the enzyme products was monitored with HPLC. It was shown that CTrpPT differed clearly from other known indole prenyltransferases in several aspects. This enzyme showed higher substrate specificity towards aromatic substrates, but lower regioselectivity regarding the prenylation position than other indole prenyltransferases. Cyclo-L-Trp-L-Trp was much better accepted than other cyclic dipeptides tested in this study. In comparison to other indole prenyltransferases with one dominant enzyme product, at least two product peaks were detected in the reaction mixtures of CTrpPT. ¹H- and ¹³C-NMR analyses, including long-range ¹H–¹³C connectivities in Heteronuclear Multiple-Bond Correlation (HMBC) and Nuclear Overhauser Effect Spectroscopy (NOESY), proved the structures of the enzyme products as C7- and N1-prenylated derivatives with a ratio of 1 : 1.2 using cyclo-L-Trp-L-Trp as substrate. The K_M values were determined at about 2.5 mM for dimethylallyl diphosphate and 0.3 mM for cyclo-L-Trp-L-Trp with a turnover number of 0.33 s⁻¹.