Biochemical and Mechanistic Characterization of the Fungal Reverse N-1-Dimethylallyltryptophan Synthase DMATS1_Ff

Dimethylallyltryptophan synthases catalyze the regiospecific transfer of (oligo)prenylpyrophosphates to aromatic substrates like tryptophan derivatives. These reactions are key steps in many biosynthetic pathways of fungal and bacterial secondary metabolites. In vitro investigations on recombinant DMATS1_Ff from Fusarium fujikuroi identified the enzyme as the first selective reverse tryptophan-N-1 prenyltransferase of fungal origin. The enzyme was also able to catalyze the reverse N-geranylation of tryptophan. DMATS1_Ff was shown to be phylogenetically related to fungal tyrosine O-prenyltransferases and fungal 7-DMATS. Like these enzymes, DMATS1_Ff was able to convert tyrosine into its regularly O-prenylated derivative. Investigation of the binding sites of DMATS1_Ff by homology modeling and comparison to the crystal structure of 4-DMATS FgaPT2 showed an almost identical site for DMAPP binding but different residues for tryptophan coordination. Several putative active site residues were verified by site directed mutagenesis of DMATS1_Ff. Homology models of the phylogenetically related enzymes showed similar tryptophan binding residues, pointing to a unified substrate binding orientation of tryptophan and DMAPP, which is distinct from that in FgaPT2. Isotopic labeling experiments showed the reaction catalyzed by DMATS1_Ff to be nonstereospecific. Based on these data, a detailed mechanism for DMATS1_Ff catalysis is proposed.