Increasing structural diversity by prenylation-based modifications
Fungi have the ability to generate tremendously complex and diverse natural products. Fungal secondary metabolites are highly relevant in mankind’s daily life by playing an important role in medicine, agriculture and manufacturing industries. Since the discovery of antibiotics in the first half of t...
prenylation-based modifications by enzymatic or nonenzymatic reactions ; Biosynthesis of flavoglaucin and congeners
Pilze, Sekundärmetabolite, biosynthetischen Genclustern, nicht-Häm-FeII / 2-Oxoglutarat (2-OG) abhängige Oxygenase, spontane Umlagerung, Polyketi
prenylierungsbasierte Modifikationen durch enzymatische oder nichtenzymatische Reaktionen; Biosynthese von Flavoglaucin und Analoga
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|Summary:||Fungi have the ability to generate tremendously complex and diverse natural products. Fungal secondary metabolites are highly relevant in mankind’s daily life by playing an important role in medicine, agriculture and manufacturing industries. Since the discovery of antibiotics in the first half of the last century, an enormous variety of natural products has been discovered in different fungi. With the advent of the genomics revolution, scientists have realized that the remarkable chemical space of fungal secondary metabolites has resulted from the diversification of biosynthetic gene clusters (BGC). Enzymes as efficient catalysts are the bridge between these biosynthetic genes and the resulting small molecules. The initial chemical scaffolds are assembled by backbone enzyme(s) and undergo decorations catalysed by a set of tailoring enzymes to mature the products. Prenyltransferases are one representative family of these tailoring enzymes. “Aromatic” prenyltransferases accept a broad spectrum of substrates including, but no limited to, indole derivatives, benzene carbaldehydes and naphthalenes. Prenylated metabolites can be further modified by enzymatic or nonenzymatic reactions to facilitate the functional group density. Thus, understanding the complexity and diversity of natural product scaffolds requires investigation of whole biosynthetic assembly lines in vivo as well as the participating enzymes and their mechanisms.
There are substantial studies demonstrating the diversification of enzymatic post-modifications on prenyl moieties. For example, the nonheme FeII/2-oxoglutarate (2-OG)-dependent oxygenase FtmOx1 from Aspergillus fumigatus is involved in the biosynthesis of fumitremorgin-type mycotoxins and catalyses an endoperoxide formation by insertion of an oxygen molecule into two prenyl moieties. Following this work, we cloned and overexpressed its homologous gene NFIA_045530 from Neosartorya fischeri. The recombinant protein EAW25734 encoded by NFIA_045530 was purified to apparent homogeneity and incubated with intermediates of the fumitremorgin biosynthetic pathway. LC-MS analysis revealed no consumption of fumitremorgin B, the natural substrate of FtmOx1, but good conversion with its biosynthetic precursor tryprostatin B in the presence of FeII and 2-OG. Structure elucidation confirmed the three products as 22-hydroxylisotryprostatin B, 14a-hydroxylisotryprostatin B and 14a, 22-dihydroxylisotryprostatin B. Further detailed biochemical characterization proved EAW25734 to be a nonheme FeII/2-OG-dependent oxygenase, which catalyses a double bond migration within the dimethylallyl moiety accompanied by hydroxylation. We proposed that the reaction mechanism for this transformation is a radical rearrangement prior to accepting a hydroxyl radical from FeIII. The major origin of the hydroxyl groups at C14a and C22 was confirmed to be O2 by labelling experiments. Solvent exchange was also observed for that at C22. LC-MS analysis of the fungal culture revealed the presence of 22-hydroxylisotryprostatin B, indicating the hijacking of tryprostatin B by EAW25734 from the fumitremorgin pathway. Our study demonstrates a notable oxidative modification of prenyl moieties.
In cooperation with Dr. Jinglin Wang, we investigated spontaneous rearrangements of 4-dimethylallyl-1,3-dihydroxynaphthalene to two tetrahydrobenzofuran and one bicyclo[3.3.1]nonane derivatives. Incubations of FgaPT2, 1,3-dihydroxynaphthalene and DMAPP under 18O2-enriched atmosphere and with 18O-enriched water confirmed that the two additional hydroxyl groups were originated from one molecule of O2. Thus, a radical-involved mechanism was proposed starting with a reactive C4-peroxyl intermediate, which led to radical shifts and the formation of tricyclic products. These results provide one additional example for the nonenzymatic oxidative cyclisation and give valuable insights into the structural diversification by spontaneous reactions.
In cooperation with Jonas Nies, a nine-gene fog cluster was identified in Aspergillus ruber. Genome mining revealed the presence of a prenyltransferase gene fogH in the fog cluster. The involvement of the fog cluster in the biosynthesis of the prenylated salicylaldehyde flavoglaucin and congeners was confirmed by heterologous expression of the whole cluster in Aspergillus nidulans. The highly-reducing polyketide synthase FogA, together with three additional enzymes, was proven to be responsible for the formation of the benzyl alcohol intermediates. Deletion of fogH led to the accumulation of C5-hydroxylated hydroquinones, which were unstable and partially oxidised to their benzoquinone forms. Biochemical characterization revealed that the prenyltransferase FogH can accept both hydroquinone and benzoquinone forms as substrates. Consecutively, the alcohols were oxidized to the final aldehyde products by an oxidase, which only accepts prenylated derivatives as substrates. Meanwhile, the spontaneous oxidoreduction from prenylated benzoquinone alcohols to final hydroquinone aldehydes was observed as a minor side reaction during isolation. Therefore, this study demonstrated a highly efficient and programmed biosynthetic machinery for the flavoglaucin formation and highlighted the importance of the prenyltransferase FogH in the assembly line.
In the review on fungal benzene carbaldehydes, we summarised their structural features, distribution, biological activities and biosynthesis with focus on alkylated derivatives and meroterpenoids. The first group carries different alkyl chains (C3, C5, C7, C9 or C11) at the ortho-position to the aldehyde group and the second group contains structural features derived from a C5, C10 or C15 prenyl moiety. In addition, simple benzaldehydes, benzophenones, spirocyclic and other benzene carbaldehydes were also included. Most of the reviewed compounds are salicylaldehyde derivatives, which are assembled by polyketide synthases from ascomycetes and released directly as aldehydes or afterwards oxidised/reduced by tailoring enzymes.|
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