Analysis of Rhizonin Biosynthesis Reveals Origin of Pharmacophoric Furylalanine Moieties in Diverse Cyclopeptides

Abstract

Rhizonin A and B are hepatotoxic cyclopeptides produced by bacterial endosymbionts (Mycetohabitans endofungorum) of the fungus Rhizopus microsporus. Their toxicity critically depends on the presence of 3‐furylalanine (Fua) residues, which also occur in pharmaceutically relevant cyclopeptides of the endolide and bingchamide families. The biosynthesis and incorporation of Fua by non‐ribosomal peptide synthetases (NRPS), however, has remained elusive. By genome sequencing and gene inactivation we elucidated the gene cluster responsible for rhizonin biosynthesis. A suite of isotope labeling experiments identified tyrosine and l‐DOPA as Fua precursors and provided the first mechanistic insights. Bioinformatics, mutational analysis and heterologous reconstitution identified dioxygenase RhzB as necessary and sufficient for Fua formation. RhzB is a novel type of heme‐dependent aromatic oxygenases (HDAO) that enabled the discovery of the bingchamide biosynthesis gene cluster through genome mining.

A MassQL-Integrated Molecular Networking Approach for the Discovery and Substructure Annotation of Bioactive Cyclic Peptides

“…At present, only two other NMefAla-containing peptides, bingchamide B and rhizonin A, are known, , both originating from bacteria. Only recently did Ehinger et al prove the production of furylalanine moieties in rhizonin A by the endofungal symbiont Mycetohabitans endofungorum . Thus, it is likely that the endolides are also of bacterial origin, but to date we have not been able to cultivate the endosymbiont in axenic cultures (a commonly observed phenomenon) .…”

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“…Genome sequencing and gene inactivation experiments have identified the gene cluster responsible for the biosynthesis of rhizonin A 37 and B, hepatotoxic cyclopeptides produced by bacterial endosymbionts of the fungus Rhizopus microspores . 28 Feeding studies to elucidate the biosynthesis of the 3-furylalanine residues, which are critical for toxicity, revealed that these are derived from tyrosine and l -DOPA. The mechanism of the C–C bond forming reaction by heterodimeric hinokiresinol synthase (HRS) from Asparagus officinalis that forms ( Z )-hinokiresinol 38 from 4-coumaryl 4-coumarate (Scheme 2) has been elucidated.…”