Indole Prenyltransferases from Fungi: A New Enzyme Group with High Potential for the Production of Prenylated Indole Derivatives

Abstract

Prenylated indole derivatives are hybrid natural products containing both aromatic and isoprenoid moieties and are widely spread in plants, fungi and bacteria. Some of these complex natural products, e.g. the ergot alkaloids ergotamine and fumigaclavine C as well as the diketopiperazine derivative fumitremorgin C and its biosynthetic precursors tryprostatin A and B, show a wide range of biological and pharmacological activities. Prenyl transfer reactions catalysed by prenyltransferases represent key steps in the biosynthesis of these compounds and often result in formation of products which possess biological activities distinct from their non-prenylated precursors. Recently, a series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from fungi. We have cloned and overexpressed six of these genes, fgaPT1, fgaPT2, ftmPT1, ftmPT2, 7-dmats and cdpNPT from A. fumigatus in E. coli and S. cerevisiae. The overproduced enzymes were characterised biochemically. Three additional prenyltransferases, DmaW-Cs, TdiB and MaPT were identified and characterised in a Clavicipitalean fungus, Aspergillus nidulans and Malbranchea aurantiaca, respectively. Sequence analysis and alignments with known aromatic prenyltransferases as well as phylogenetic analysis revealed that these enzymes belong to a new group of "aromatic prenyltransferases". They differ clearly from membrane-bound aromatic prenyltransferases from different sources and soluble prenyltransferases from bacteria. The characterised enzymes are soluble proteins, catalyse different prenyl transfer reactions on indole moieties of various substrates and do not require divalent metal ions for their enzymatic reactions. All of the enzymes accepted only dimethylallyl diphosphate as prenyl donor. On the other hand, they showed broad substrate specificity towards their aromatic substrates. Diverse tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by these enzymes, providing a new strategy for convenient production of biologically active substances, e.g. by chemoenzymatic synthesis.

The structure of dimethylallyl tryptophan synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria

“…FgaPT2 prenylates the indole moiety with strict regiospecificity for C-4, whereas other fungal PTs show regiospecificity for positions 1, 2, 3, or 7 of the indole moiety (16,27). In 1976, Lee et al (9) pointed out that based on their chemical reactivity, positions 2, 5, and 7 of the indole nucleus would be the preferred sites for electrophilic substitution.…”

“…Several enzymes with sequence similarity to DMATS that prenylate tryptophan (or derivatives thereof) with different regiospecificities have been identified and, in some cases, catalyze ''reverse'' prenylations (i.e., attach the C-3 of the dimethylallyl moiety to the aromatic nucleus). These enzymes include 7-DMATS (catalyzing a regular prenylation at C-7 of the indole moiety), FtmPT1 (regular prenylation at C-2), FtmPT2 (regular prenylation at the ring nitrogen), CdpNPT (reverse prenylation at the ring nitrogen), FgaPT1 (reverse prenylation at C-2), and AnaPT (reverse prenylation at C-3), (16,27). We have generated structurebased alignments of each of these enzymes with FgaPT2.…”

“…Recently, several enzymes with sequence similarity to DMATS have been cloned from fungal genomes and were expressed, purified, and biochemically characterized, as reviewed by Steffan et al (16). These enzymes show different substrate specificities, catalyzing the prenylation of tryptophan or derivatives thereof.…”

“…Currently, BLAST searches reveal more than 100 entries with sequence similarity to DMATS in the database. The fungal indole PTs that have been biochemically investigated show remarkable flexibility for their aromatic substrates, and these enzymes may be of considerable interest for chemoenzymatic synthesis of bioactive compounds (16).…”

Genome mining and biosynthesis of fumitremorgin-type alkaloids in ascomycetes

“…27 Later on, orthologous clusters were also identified in A. fumigatus A1163 and N. fischeri NRRL181. 29,30 Comparison of these clusters revealed the presence of all of the 10 genes in both strains. Remarkably, the orthologous gene of orf17 27 in NRRL181, termed ftmP in the PhD thesis of A Grundmann, 30 is separated from ftmO by a segment of 24 kbp and is likely not a member of the gene cluster of fumitremorgintype alkaloids.…”