Dimethylallyltryptophan Synthase Superfamily Research Articles

Diprenylated cyclodipeptide production by changing the prenylation sequence of the nature’s synthetic machinery

Ascomycetous fungi are often found in agricultural products and foods as contaminants. They produce hazardous mycotoxins for human and animals. On the other hand, the fungal metabolites including mycotoxins are important drug candidates and the enzymes involved in the biosynthesis of these compounds are valuable biocatalysts for production of designed compounds. One of the enzyme groups are members of the dimethylallyl tryptophan synthase superfamily, which mainly catalyze prenylations of tryptophan and tryptophan-containing cyclodipeptides (CDPs). Decoration of CDPs in the biosynthesis of multiple prenylated metabolites in nature is usually initiated by regiospecific C2-prenylation at the indole ring, followed by second and third ones as well as by other modifications. However, the strict substrate specificity can prohibit the further prenylation of unnatural C2-prenylated compounds. To overcome this, we firstly obtained C4-, C5-, C6-, and C7-prenylated cyclo-l-Trp-l-Pro. These products were then used as substrates for the promiscuous C2-prenyltransferase EchPT1, which normally uses the unprenylated CDPs as substrates. Four unnatural diprenylated cyclo-l-Trp-l-Pro including the unique unexpected N1,C6-diprenylated derivative with significant yields were obtained in this way. Our study provides an excellent example for increasing structural diversity by reprogramming the reaction orders of natural biosynthetic pathways. Furthermore, this is the first report that EchPT1 can also catalyze N1-prenylation at the indole ring.Key points• Prenyltransferases as biocatalysts for unnatural substrates.• Chemoenzymatic synthesis of designed molecules.• A cyclodipeptide prenyltransferase as prenylating enzyme of already prenylated products.Graphical

C7-Prenylation of Tryptophan-Containing Cyclic Dipeptides by 7-Dimethylallyl Tryptophan Synthase Significantly Increases the Anticancer and Antimicrobial Activities.

Prenylated natural products have interesting pharmacological properties and prenylation reactions play crucial roles in controlling the activities of biomolecules. They are difficult to synthesize chemically, but enzymatic synthesis production is a desirable pathway. Cyclic dipeptide prenyltransferase catalyzes the regioselective Friedel–Crafts alkylation of tryptophan-containing cyclic dipeptides. This class of enzymes, which belongs to the dimethylallyl tryptophan synthase superfamily, is known to be flexible to aromatic prenyl receptors, while mostly retaining its typical regioselectivity. In this study, seven tryptophan-containing cyclic dipeptides 1a–7a were converted to their C7-regularly prenylated derivatives 1b–7b in the presence of dimethylallyl diphosphate (DMAPP) by using the purified 7-dimethylallyl tryptophan synthase (7-DMATS) as catalyst. The HPLC analysis of the incubation mixture and the NMR analysis of the separated products showed that the stereochemical structure of the substrate had a great influence on their acceptance by 7-DMATS. Determination of the kinetic parameters proved that cyclo-l-Trp–Gly (1a) consisting of a tryptophanyl and glycine was accepted as the best substrate with a KM value of 169.7 μM and a turnover number of 0.1307 s−1. Furthermore, docking studies simulated the prenyl transfer reaction of 7-DMATS and it could be concluded that the highest affinity between 7-DMATS and 1a. Preliminary results have been clearly shown that prenylation at C7 led to a significant increase of the anticancer and antimicrobial activities of the prenylated derivatives 1b–7b in all the activity test experiment, especially the prenylated product 4b.

Different behaviors of cyclic dipeptide prenyltransferases toward the tripeptide derivative ardeemin fumiquinazoline and its enantiomer.

In nature, cyclic dipeptide prenyltransferases catalyze regioselective Friedel-Crafts alkylations of tryptophan-containing cyclic dipeptides. This enzyme class, belonging to the dimethylallyl tryptophan synthase superfamily, is known to be flexible toward aromatic prenyl acceptors, while mostly retaining its typical regioselectivity. Ardeemin fumiquinazoline (FQ) (1), a tryptophan-containing cyclic tripeptide derivative, is assembled in Aspergillus fischeri by the non-ribosomal peptide synthetase ArdA and modified by the prenyltransferase ArdB, leading to the pharmaceutically active hexacyclic ardeemin. Therefore, 1 and its enantiomer ent-ardeemin FQ (2) constitute potential substrates for aromatic prenyltransferases. In this study, we investigated the acceptance of both enantiomers by two cyclic dipeptide C2-prenyltransferases BrePT and FtmPT1 and three C3-prenyltransferases CdpNPT, CdpC3PT, and AnaPT. LC-MS analysis of the incubation mixtures and NMR analysis of the isolated products revealed that the stereochemistry at C11 and C14 in 1 and 2 has a strong influence on their acceptance by these enzymes and the regioselectivity of the prenylation reactions. 1 was very well accepted by BrePT, FtmPT1, and CdpNPT, with C2- or C3-prenylated derivatives as predominant products, which fills the prenylation gaps by tryptophan prenyltransferases reported in a previous study. 2 was a poor substrate for all the enzymes and converted with low regioselectivity and mainly prenylated at C6 and C7 of the indole moiety.

Gem-Diprenylation of Acylphloroglucinols by a Fungal Prenyltransferase of the Dimethylallyltryptophan Synthase Superfamily.

Aspergillus terreus aromatic prenyltransferase (AtaPT) catalyzes predominantly C-monoprenylation of acylphloroglucinols in the presence of different prenyl diphosphates. With dimethylallyl diphosphate (DMAPP) as prenyl donor, gem-diprenylated products 1D3, 2D3, and 3D3 were also detected. High conversion of 1D1 to 1D3, 2D1 to 2D3, and 3D1 to 3D3 was demonstrated by incubation with AtaPT and DMAPP. The first example of gem-diprenylation by a member of the dimethylallyltryptophan synthase superfamily is provided.

Mutation on Gly115 and Tyr205 of the cyclic dipeptide C2-prenyltransferase FtmPT1 increases its catalytic activity toward hydroxynaphthalenes.

The fungal cyclic dipeptide prenyltransferase FtmPT1 from Aspergillus fumigatus catalyzes a regular C2-prenylation of brevianamide F (cyclo-L-Trp-L-Pro) and is involved in the biosynthesis of a number of biologically active natural products including tryprostatins, spirotryprostatins, verruculogen, and fumitremorgins. FtmPT1, like other members of the dimethylallyltryptophan synthase superfamily, was shown to have high substrate promiscuity for tryptophan-containing cyclic dipeptides and a few other aromatic substrates. A previous study demonstrated the acceptance of 1-naphthol by FtmPT1, but with very low product yield. In this study, we report the significantly increased acceptance of 1-naphthol and other hydroxynaphthalenes by FtmPT1_G115A and six FtmPT1_Y205X single mutants as well as FtmPT1_G115A_Y205C. These results provided an example for creation of biocatalysts with improved catalytic activity by site-directed mutagenesis.

One Substrate – Seven Products with Different Prenylation Positions in One‐Step Reactions: Prenyltransferases Make it Possible

AbstractPrenylated indole alkaloids derived from L‐tryptophan are widely distributed in nature and show diverse biological and pharmacological activities, usually distinct from their non‐prenylated precursors. Prenyltransferases catalyze the transfer reactions of prenyl moieties onto the indole nucleus and contribute largely to the structural diversity of these compounds. In this study, we demonstrate the acceptance of cyclo‐L‐homotryptophan‐D‐valine, an unnatural cyclic dipeptide, by eight prenyltransferases of the dimethylallyltryptophan synthase superfamily. Seven products with one prenyl moiety at each position of the indole nucleus and one diprenylated derivative were isolated from enzyme assays of cyclo‐L‐homotryptophan‐D‐valine with dimethylallyl diphosphate. To the best of our knowledge, this is the first report for production of seven monoprenylated products from one substrate by one‐step reactions.magnified image

Geranylation of Cyclic Dipeptides by the Dimethylallyl Transferase AnaPT Resulting in a Shift of Prenylation Position on the Indole Ring

The dimethylallyl transferase AnaPT from Neosartorya fischeri is involved in the biosynthesis of acetylaszonalenin and catalyses the regioselective and stereospecific C3α-prenylation of (R)-benzodiazepinedione in the presence of dimethylallyl diphosphate. This enzyme also converts several tryptophan-containing cyclic dipeptides to C3α-prenylated indolines. In this study, we demonstrate the geranylation of (R)-benzodiazepinedione and five other cyclic dipeptides by AnaPT in the presence of geranyl diphosphate (GPP). Interestingly, structure elucidation by NMR and MS analyses revealed that, with GPP, the geranyl moiety is attached to C-6 or C-7 rather than C-3 of the indole ring of the enzyme products. For (R)-benzodiazepinedione, one dominant C6-geranylated derivative was obtained, whereas the other five substrates yielded both C6- and C7-geranylated products. Neither acceptance of GPP by a dimethylallyl transferase from the dimethylallyltryptophan synthase superfamily, nor the alkylation shift from C-3 to the benzene ring of the indole nucleus has been reported previously.

Biochemical Characterization of Indole Prenyltransferases: FILLING THE LAST GAP OF PRENYLATION POSITIONS BY A 5-DIMETHYLALLYLTRYPTOPHAN SYNTHASE FROM ASPERGILLUS CLAVATUS

The putative prenyltransferase gene ACLA_031240 belonging to the dimethylallyltryptophan synthase superfamily was identified in the genome sequence of Aspergillus clavatus and overexpressed in Escherichia coli. The soluble His-tagged protein EAW08391 was purified to near homogeneity and used for biochemical investigation with diverse aromatic substrates in the presence of different prenyl diphosphates. It has shown that in the presence of dimethylallyl diphosphate (DMAPP), the recombinant enzyme accepted very well simple indole derivatives with L-tryptophan as the best substrate. Product formation was also observed for tryptophan-containing cyclic dipeptides but with much lower conversion yields. In contrast, no product formation was detected in the reaction mixtures of L-tryptophan with geranyl or farnesyl diphosphate. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific regular prenylation at C-5 of the indole nucleus of the simple indole derivatives. EAW08391 was therefore termed 5-dimethylallyltryptophan synthase, and it filled the last gap in the toolbox of indole prenyltransferases regarding their prenylation positions. K(m) values of 5-dimethylallyltryptophan synthase were determined for L-tryptophan and DMAPP at 34 and 76 μM, respectively. Average turnover number (k(cat)) at 1.1 s(-1) was calculated from kinetic data of L-tryptophan and DMAPP. Catalytic efficiencies of 5-dimethylallyltryptophan synthase for L-tryptophan at 25,588 s(-1)·M(-1) and for other 11 simple indole derivatives up to 1538 s(-1)·M(-1) provided evidence for its potential usage as a catalyst for chemoenzymatic synthesis.