The Family Keeps on Growing: Four Novel Fungal OYEs Characterized.

Aiming at expanding the portfolio of Old Yellow Enzymes (OYEs), which have been systematically studied to be employed in the chemical and pharmaceutical industries as useful biocatalysts, we decided to explore the immense reservoir of filamentous fungi. We drew from the genome of the two Ascomycetes Aspergillus niger and Botryotinia fuckeliana four new members of the OYE superfamily belonging to the classical and thermophilic-like subfamilies. The two BfOYEs show wider substrate spectra than the AnOYE homologues, which appear as more specialized biocatalysts. According to their mesophilic origins, the new enzymes neither show high thermostability nor extreme pH optimums. The crystal structures of BfOYE4 and AnOYE8 have been determined, revealing the conserved features of the thermophilic-like subclass as well as unique properties, such as a peculiar N-terminal loop involved in dimer surface interactions. For the classical representatives BfOYE1 and AnOYE2, model structures were built and analyzed, showing surprisingly wide open access to the active site cavities due to a shorter ß6-loop and a disordered capping subdomain.