The Promiscuous Flavin-Dependent Monooxygenase PboD from Aspergillus ustus Increases the Structural Diversity of Hydroxylated Pyrroloindoline Diketopiperazines.

The potential of natural products as pharmaceutical and agricultural agents is based on their large structural diversity, resulting in part from modifications of the backbone structure by tailoring enzymes during biosynthesis. Flavin-dependent monooxygenases (FMOs), as one such group of enzymes, play an important role in the biosynthesis of diverse natural products, including cyclodipeptide (CDP) derivatives. The FMO PboD was shown to catalyze C-3 hydroxylation at the indole ring of cyclo-l-Trp-l-Leu in the biosynthesis of protubonines, accompanied by pyrrolidine ring formation. PboD substrate promiscuity was investigated in this study by testing its catalytic activity toward additional tryptophan-containing CDPs in vitro and biotransformation in Aspergillus nidulans transformants bearing a truncated protubonine gene cluster with pboD and two acetyltransferase genes. High acceptance of five CDPs was detected for PboD, especially of those with a second aromatic moiety. Isolation and structure elucidation of five pyrrolidine diketopiperazine products, with two new structures, proved the expected stereospecific hydroxylation and pyrrolidine ring formation. Determination of kinetic parameters revealed higher catalytic efficiency of PboD toward three CDPs consisting of aromatic amino acids than of its natural substrate cyclo-l-Trp-l-Leu. In the biotransformation experiments with the A. nidulans transformant, modest formation of hydroxylated and acetylated products was also detected.

A Flavin-Dependent Oxygenase Catalyzes Hydroxylation and Simultaneous Pyrrolidine Ring Formation in Protubonine Biosynthesis in Aspergillus ustus.

The hydroxylated and diacetylated cyclo-l-Trp-l-Leu derivative (-)-protubonine B was isolated from a culture of Aspergillus ustus 3.3904. Genome mining led to the identification of a putative biosynthetic gene cluster coding for a bimodular nonribosomal peptide synthetase, a flavin-dependent monooxygenase, and two acetyltransferases. Heterologous expression of the pbo cluster in Aspergillus nidulans showed that it is responsible for the formation of the isolated metabolite. Gene deletion experiments and structural elucidation of the isolated intermediates confirmed the biosynthetic steps. In vitro experiments with the recombinant protein proved that the flavin-dependent oxygenase is responsible for stereospecific hydroxylation at the indole ring accompanied by pyrrolidine ring formation.

Biosynthesis of p-Terphenyls in Aspergillus ustus Implies Enzymatic Reductive Dehydration and Spontaneous Dibenzofuran Formation.

p-Terphenyls contain a central benzene ring substituted with two phenyl residues at its para positions. Heterologous expression of a biosynthetic gene cluster from Aspergillus ustus led to the formation of four new p-terphenyl derivatives. Gene deletion experiments proved the formation and reductive dehydration of the terphenylquinone atromentin, followed by O-methylation and prenylation. Spontaneous dibenzofuran formation led to the final products. These results provide new insights into the biosynthesis of p-terphenyls in fungi and dibenzofuran formation in the biosynthesis of numerous natural products.