Discovery of a Fungal Multicopper Oxidase That Catalyzes the Regioselective Coupling of a Tricyclic Naphthopyranone To Produce Atropisomers

Abstract

Atropisomeric dinapinones A1 and A2 (DPA1 and DPA2) were isolated from aculture of Talaromyces pinophilus FKI-3864. Monapinone coupling enzyme (MCE), which dimerizesnaphthopyranone monapinone A(MPA), was purified from ac ell-free extract of T. pinophilus FKI-3864. MCE regioselectively dimerizes MPAatthe 8,8'-positions to synthesizet he atropisomers DPA1 and DPA2 in ar atio of approximately 1:2.5 without acofactor.The optimal pH value and temperature for MCE were 4.0 and 50 8 8C, and the apparent K m and V max values for MPAwere (72.7 AE 23.2) mm and (1.21 AE 0.170) mmol min À1 mg À1 protein. The MCE polypeptide is significantly homologous with multicopper oxidases.H eterologous expression of MCE and functional analysis confirmed that MCE catalyzest he regioselective coupling reaction of MPAt op roduce DPA. No fungal multicopper oxidase has previously been reported to catalyze regioselective intermolecular oxidative phenol coupling to produce naphthopyranone atropisomers.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

The fungal gene cluster for biosynthesis of the antibacterial agent viriditoxin

“…How this coupling occurs in fungi is beginning to be understood, as both P450 monooxygenases and laccases/multicopper oxidases are able to catalyze this coupling. While P450 monooxygenases have been shown to be capable of stereoselective coupling [14], none of the fungal laccase/multicopper oxidases enzymes that have been implicated in biaryl coupling have demonstrated stereospecificity [4,22,23]. In plants, laccases are able to catalyze stereoselective coupling in combination with dirigent proteins; however, no such proteins have been reported in fungi [24].…”

Heterologous biosynthesis of elsinochrome A sheds light on the formation of the photosensitive perylenequinone system

“…Stereo- and regio-selective oxidative coupling of polycyclic aromatic compounds have been shown to be catalysed by P450s in fungi 28–30. More recently, a LMCO from the fungus Talaromyces pinophilus that could catalyse the regioselective coupling of naphthopyranone was discovered 31. The involvement of LMCOs in regioselective oxidative coupling of naphthopyrones have also been reported 32,33.…”

“…Given that there are several precedents for LMCOs involved in intermolecular phenolic coupling,31–33 we propose that the ElcG LMCO catalyses the first intermolecular coupling of 10 in a regio- and stereo-selective manner via a phenol radical coupling mechanism (Fig. 6-i).…”

The fungal gene cluster for biosynthesis of the antibacterial agent viriditoxin

“…VdtB shows homology to laccase enzymes previously implicated in oxidative coupling, including GIP1 that is suggested to play a role in the dimerization of two rubrofusarin molecules to form aurofusarin in F. graminearum [4] and MCE that dimerizes monapinone A into dinapinone A in Talaromyces pinophilus [16]. Thus, we thus hypothesized that VdtB would be required for forming the biaryl bond between two semi-viriditoxin molecules to produce viriditoxin.…”

“…It is known that both P450 monooxygenases and laccases/multicopper oxidases are able to catalyze this coupling. While P450 monooxygenases have been shown to be capable of stereoselective coupling [14], none of the fungal laccase/multicopper oxidases enzymes which have been implicated in biaryl coupling have demonstrated stereospecificity [4,15,16]. In plants, laccases are able to catalyze stereoselective coupling in combination with dirigent proteins, however, no such proteins have been reported in fungi [17].…”