Crystal structure of ArOYE6 reveals a novel C-terminal helical extension and mechanistic insights into the distinct class III OYEs from pathogenic fungi

Abstract

Old Yellow Enzymes (OYEs) play critical role in antioxidation, detoxification and ergot alkaloid biosynthesis processes in various organisms. The yeast- and bacteria-like OYEs have been structurally characterized earlier, however, filamentous fungal pathogens possess a novel OYE class i.e. class III, whose biochemical and structural intricacies remain unexplored to date. Here, we present the 1.6 Å X-ray structure of a class III member, old yellow enzyme 6 from necrotrophic fungus Ascochyta rabiei (ArOYE6), in FMN-bound form (PDB ID-7FEV) and provide mechanistic insights into their catalytic capability. We demonstrate that ArOYE6 exists as a monomer in solution, forms (β/α)8 barrel structure harboring non-covalently bound FMN at cofactor binding site, and utilizes reduced nicotinamide adenine dinucleotide phosphate as its preferred reductant. The large hydrophobic cavity situated above FMN, specifically accommodates 12-oxo-phytodienoic acid and N-ethylmaleimide substrates as observed in ArOYE6-FMN-substrate ternary complex models. Site-directed mutations in the conserved catalytic (His196, His199, Tyr201) and FMN-binding (Lys249, Arg348) residues render the enzyme inactive. Intriguingly, ArOYE6 structure contains a novel C-terminus (369-445 residues) made of three α-helices, which stabilizes the FMN binding pocket as its mutation/truncation lead to complete loss of FMN binding. Moreover, the loss of extended C-terminus does not alter the monomeric nature of ArOYE6. In this study, for the first time, we provide the structural and biochemical insights for a fungi-specific class III OYE homolog and dissect the oxidoreductase mechanism. Our findings hold broad biological significance during host-fungus interactions owing to conservation of this class among pathogenic fungi, and would have potential implications in the pharmacochemical industry.