Enantioselective Phenol Coupling by Laccases in the Biosynthesis of Fungal Dimeric Naphthopyrones.

Biaryl compounds are ubiquitous metabolites that are often formed by dimerization through oxidative phenol coupling. Hindered rotation around the biaryl bond can cause axial chirality. In nature, dimerizations are catalyzed by oxidative enzymes such as laccases. This class of enzymes is known for non-specific oxidase reactions while inherent enantioselectivity is hitherto unknown. Here, we describe four related fungal laccases that catalyze γ-naphthopyrone dimerization in a regio- and atropselective manner. In vitro assays revealed that three enzymes were highly P-selective (ee >95 %), while one enzyme showed remarkable flexibility. Its selectivity for M- or P-configured dimers varied depending on the reaction conditions. For example, a lower enzyme concentration yielded primarily (P)-ustilaginoidin A, whereas the M atropisomer was favored at higher concentration. These results demonstrate inherent enantioselectivity in an enzyme class that was previously thought to comprise only non-selective oxidases.

Diversity in Fungal Intermolecular Phenol Coupling of Polyketides: Regioselective Laccase-Based Systems.

Polyketides form a structurally diverse and pharmaceutically important class of secondary metabolites. Both diversity and biological activity are largely facilitated by post-polyketide synthase tailoring including methylation, oxidation, reduction, glycosylation, and dimerization. Cytochrome P450 enzymes (CYPs), flavin-dependent monooxygenases (FMOs), and laccases are known to catalyze phenol coupling in the biosynthesis of polyketide dimers. Polyketide homodimers resulting from enzyme catalysis are often formed in a highly regio- and stereoselective manner, in contrast to analogous nonenzymatic dimerization. Although it is known that CYPs and FMOs are capable of selectively generating one of several putative isomers, hitherto described laccases depend on auxiliary proteins to achieve similar selectivity. Herein, regioselective phenol coupling catalyzed by a fungal laccase is demonstrated. The heterologously produced Av-VirL from Aspergillus viridinutans selectively generated the 6,6'-homodimer of (R)-semivioxanthin. Genome analysis is used to show that laccase-based phenol-coupling systems are widespread in fungi. Homologues of Av-VirL were identified in the putative biosynthetic gene clusters of vioxanthin, xanthomegnin, and xanthoepocin, and of the perylenequinones hypocrellin A, elsinochrome A, and cercosporin. These findings show that laccases are capable of selective phenol coupling in the absence of auxiliary proteins.

Phylogenetic Studies, Gene Cluster Analysis, and Enzymatic Reaction Support Anthrahydroquinone Reduction as the Physiological Function of Fungal 17ß-Hydroxysteroid Dehydrogenase.

17ß-Hydroxysteroid dehydrogenase (17ß-HSDcl) from the filamentous fungus Curvularia lunata (teleomorph Cochliobolus lunatus) catalyzes NADP(H)-dependent oxidoreductions of androgens and estrogens. Despite detailed biochemical and structural characterization of 17ß-HSDcl, its physiological function remains unknown. On the basis of amino acid sequence alignment, phylogenetic studies, and the recent identification of the physiological substrates of the homologous MdpC from Aspergillus nidulans and AflM from Aspergillus parasiticus, we propose an anthrahydroquinone as the physiological substrate of 17ß-HSDcl. This is also supported by our analysis of a secondary metabolite biosynthetic gene cluster in C. lunata m118, containing 17ß-HSDcl and ten other genes, including a polyketide synthase probably involved in emodin formation. Chemoenzymatic reduction of emodin by 17ß-HSDcl in the presence of sodium dithionite verified this hypothesis. On the basis of these results, the involvement of a 17ß-HSDcl in the biosynthesis of other anthrahydroquinone-derived natural products is proposed; hence, 17ß-HSDcl should be more appropriately referred to as a polyhydroxyanthracene reductase (PHAR).