A putative brevianamide F reverse prenyltransferase gene brePT was amplified from Aspergillus versicolor NRRL573 by using primers deduced from its orthologue notF in Aspergillus sp. MF297-2 and overexpressed in Escherichia coli. The soluble His-tagged protein BrePT was purified to near homogeneity and assayed with tryptophan-containing cyclic dipeptides in the presence of dimethylallyl diphosphate. BrePT showed much higher flexibility towards its aromatic substrates than NotF and accepted all of the 14 tested tryptophan-containing cyclic dipeptides. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific reverse prenylation at C2 of the indole nucleus. K (M) values of BrePT were determined for its putative substrates brevianamide F and DMAPP at 32 and 98 mu M, respectively. Average turnover number (k (cat)) at 0.4 s(-1) was calculated from kinetic data of brevianamide F and DMAPP. K (M) values in the range of 0.082-2.9 mM and k (cat) values from 0.003 to 0.15 s(-1) were determined for other 11 cyclic dipeptides. Similar to known fungal indole prenyltransferases, BrePT did not accept geranyl or farnesyl diphosphate as prenyl donor for its prenylation.