Browsing by Author "Saxena, Ajay Kumar"

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    Crystal structure of ArOYE6 reveals a novel C-terminal helical extension and mechanistic insights into the distinct class III OYEs from pathogenic fungi
    (John Wiley & Sons, 2022) Singh, Yeshveer; Sharma, Ruby; Mishra, Manasi; Verma, Praveen K.; Saxena, Ajay Kumar
    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.