Biocatalytic phenol coupling of [gamma]-naphthopyrones and biosynthesis of ibotenic acid

Abstract: The present work deals with the identification and investigation of enzymes involved in the biosynthesis of fungal natural products. It is composed of two parts. Part I covers the biosynthesis of dimeric γ-naphthopyrone metabolites in ascomycetes. Part II covers the biosynthesis of ibotenic acid in the mushroom Amanita muscaria.

I. Dimeric γ-naphthopyrones are produced by many strains of filamentous fungi. Their chemical diversity emerges from differing regiochemistries of the biaryl bond. Moreover, the hindered rotation around this bond gives rise to atropisomers. Typically, one fungal strain produces dimeric γ-naphthopyrones with one defined regio- and stereochemistry. Therefore, a highly selective enzymatic system responsible for the oxidative coupling of the monomeric γ-naphthopyrones was expected.
To identify the enzymes responsible for the biosynthesis of the γ-naphthopyrones, genomics, transcriptomics, and retrobiosynthesis were combined. Despite the high structural similarity of the various γ-naphthopyrone dimers, two unrelated groups of enzymes were shown to catalyze the oxidative phenol coupling reaction. Ustilaginoidin A is biosynthesized by laccase enzymes in fungi such as Ustilaginoidea virens. Bifonsecin-type products such as nigerone and bifonsecin B are formed by the action of an entirely new group of CYP enzymes in Aspergillus species. Representative enzymes of both groups were heterologously produced and experimentally investigated.
The ustilaginoidin laccases UstL, CheL, and MytL were shown to regio- and atropselectively catalyze the coupling of the achiral monomeric γ-naphthopyrone, nor-rubrofusarin. CheL and MytL were highly selective for production of (P)-ustilaginoidin A, with enantiomeric excesses of above 95%. UstL could even be tuned to either M- or P-atropselectivity by the reaction conditions. Overall, this is the first report of stereoselectivity in laccases.
Likewise, the bifonsecin CYP enzymes AunB and BfoB were shown to catalyze selective phenol coupling reactions. Their native substrates fonsecin B and rubrofusarin B were each homodimerized regioselectively; AunB yielded the 7,10′-dimers, whereas BfoB yielded the 10,10′-dimers. However, the degree of atropselectivity was dependent on the monomeric substrate. Both enzymes coupled rubrofusarin B with moderate atropselectivity, while fonsecin B was converted to nearly atropisomerically pure dimers.
The unexpected identification of two separate enzymatic systems for the dimerization of γ-naphthopyrones represents a notable case of convergent evolution, and highlights the importance of phenol coupling for the generation of chemical diversity in nature.

II. Ibotenic acid is the primary toxic metabolite of the mushroom Amanita muscaria, or fly agaric. Despite its interesting pharmacology and long history, the biosynthetic origin of ibotenic acid was hitherto uninvestigated.
Using retrobiosynthesis and transcriptomics, a candidate biosynthetic gene cluster for the formation of ibotenic acid was identified in A. muscaria. Deducing from the genes present in the gene cluster, probable biosynthetic pathways were inferred. The putative first biosynthetic step is the introduction of a hydroxyl group at position 3 of glutamic acid.
Accordingly, the enzyme proposed to catalyze this reaction, IboH, was heterologously produced and experimentally investigated. It was shown that it is a 2 oxoglutarate-dependent enzyme which catalyzes stereoselective hydroxylation of L glutamic acid to threo-3 hydroxyglutamic acid. This suggested that the ibotenic acid biosynthetic gene cluster was correctly identified. The results will enable the complete reconstruction of the biosynthetic pathway, eventually facilitating the exploitation of the involved enzymes for various possible applications, such as biotechnological production of ibotenic acid and its analogs

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Universität Freiburg, Dissertation, 2019

Klassifikation und Themen

• Schlagwort: Biosynthesis ; Phenol ; Sekundärmetabolit ; Biosynthese ; Enzymsystem

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2019
• Urheber: Obermaier, Sebastian
• Beteiligte Personen und Organisationen: Müller, Michael ; Albert-Ludwigs-Universität Freiburg. Fakultät für Chemie und Pharmazie

Weitere Informationen

• DOI: 10.6094/UNIFR/151278
• URN: urn:nbn:de:bsz:25-freidok-1512785
• Rechteinformation: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 25.03.2025, 13:55 MEZ