Mechanistic Characterization of GedF Reveals a Ser–Tyr Catalytic Core with a Flexible Basic Residue in a Filamentous Fungal Anthraquinone SDR

Abstract

The cleavage of the C10–C4a bond in anthraquinones is a key step in generating ring-opened quinone derivatives in filamentous fungi. GedF, a short-chain dehydrogenase/reductase (SDR) from Aspergillus terreus, together with the dioxygenase GedK, mediates this transformation, yet the enzymatic mechanism of GedF remains unclear. Here, isotope labeling experiments confirm that reduction of Questin-to-Questin hydroquinone incorporates one proton from NADPH and one from water. Structural modeling, molecular docking, and site-directed mutagenesis reveal that GedF employs a noncanonical catalytic architecture featuring a conserved Ser–Tyr catalytic core instead of the classical Asn-Ser-Tyr-Lys tetrad typical of SDRs. Notably, mutagenesis and comparative analysis indicate that a positively charged residue is required for catalysis but is not strictly position-conserved, consistent with a role in maintaining the catalytic microenvironment and facilitating proton transfer. Phylogenetic and sequence analyses show that GedF belongs to the NAD(P)H-dependent SDR clade, and that variation in the positioning of basic residues occurs among homologues while preserving the conserved Ser–Tyr catalytic core. These findings elucidate the catalytic mechanism of GedF and uncover an alternative SDR catalytic strategy involved in anthraquinone ring-opening biosynthesis in filamentous fungi.