A putative prenyltransferase gene of the dimethylallyltryptophan synthase (DMATS) family, An13g01840, was identified in the genome sequence of Aspergillus niger. The deduced polypeptide CAK41583 consists of 465 amino acids with a calculated molecular mass of 52.7 kDa. To evaluate gene function, the coding sequence was cloned into pET28a and overexpressed in Escherichia coli. The soluble His(6)-fusion protein was purified to near homogeneity on Ni-NTA agarose and used for enzyme assays with diverse aromatic substrates in the presence of dimethylallyl diphosphate. HPLC analysis revealed product formation in the incubation mixtures with l-tyrosine and five derivatives thereof. Structure elucidation of the enzyme products by NMR and MS analyses confirmed O-prenylations and proved the identification of a tyrosine O-prenyltransferase (TyrPT). As in the case of SirD from Leptosphaeria maculans, TyrPT also accepted 4-amino-l-phenylalanine for an N-prenylation and l-tryptophan for a C7-prenylation. The K (M) values of TyrPT for l-tyrosine, l-tryptophan, and dimethylallyl diphosphate (DMAPP) were found to be 0.24, 0.19, and 0.71 mM, respectively. The k (cat) of l-tyrosine and l-tryptophan reactions were determined at 0.58 and 0.0053 s(-1), respectively. The results presented in this study enhance the relationship of tyrosine O- and tryptophan C7-prenyltranferases and provide meanwhile a new enzyme for production of prenylated derivatives. In comparison to the known tyrosine prenyltransferase SirD, TyrPT showed significantly higher catalytic activity for several substrates, e.g., 4-amino-l-phenylalanine as well as 4- and 5-methyl-DL-tryptophan.