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

Abstract

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 f ujikuroi 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.

Structural Basis of Substrate Promiscuity and Catalysis by the Reverse Prenyltransferase N-Dimethylallyl-l-tryptophan Synthase from Fusarium fujikuroi

“…18,19 Burkhardt and colleagues recently reported the biochemical characterization of dimethylallyl-L-tryptophan synthase 1 (DMATS1) from the fungus Fusarium f ujikuroi. 23 DMATS1 catalyzes the reverse N-prenylation of L-Trp with DMAPP, a reaction previously observed for bacterial enzymes but not fungal enzymes (Figure 1). A dissociative mechanism 24 is proposed in which the ionization of DMAPP yields an allylic cation that alkylates the indole nitrogen of L-Trp, with deprotonation mediated by a conserved glutamate, E90 (Figure 2).…”

“…To confirm the importance of R257, Y389, and E90 for catalytic function, we prepared, purified, and analyzed the R257L, Y389A, and E90A mutants of DMATS1. Because the substrate scope of DMATS1 had already been determined previously, 23 we assayed each mutant using all three catalytically competent prenyl donor−acceptor pairs. Enzyme activity was measured using a coupled assay to detect pyrophosphate release during catalysis.…”

“…26,27 Consistent with this evolutionary relationship, DMATS1 also catalyzes the forward Oprenylation of L-Tyr with DMAPP (Figures 1 and 2), although the catalytic efficiency is 400-fold lower with L-Tyr than with L-Trp. 23 Product O-dimethylallyl-L-tyrosine is an intermediate in the biosynthesis of sirodesmin antibiotics in other fungal species. 28,29 With regard to multiple prenyl donors and aromatic amino acid acceptors, the catalytic versatility of DMATS1 is unusual among the greater family of native aromatic prenyltransferases.…”

“…The structure of the ternary complex reveals substrate orientation during catalysis and is consistent with the proposed mechanism. 23 We additionally report crystal structures of unliganded DMATS1, DMATS1 complexed with L-Trp, DMATS1 complexed with L-Trp and geranyl S-thiolodiphosphate (GSPP), and DMATS1 complexed with L-tyrosine (L-Tyr) and DMSPP. These structures illuminate the molecular basis of substrate specificity as well as key features of catalysis confirmed through functional analysis of site-specific mutants.…”

Cp*Co(III)‐Catalyzed Dehydrative C2‐Prenylation of Pyrrole and Indole with Allyl Alcohols

“…In nature, the addition of a prenyl (C 5 ) or its higher homologs (n × C 5 ) such as geranyl unit generally occurs via either 'normal' mode or 'reverse' mode and is controlled by the active sites of various soluble prenyltransferase enzymes having common "ABBA" type structural motif under metal-independent fashion (such as Mg 2 + ). [2] Generally, dimethylallyl di-phosphate (DMAPP) is used to transfer the prenyl moiety (Scheme 1a). Despite their high level of efficiency, these enzymatic prenylations are mostly limited to narrow substrate scope.…”