Stereoisomer-specific allosteric activation of xanthine oxidoreductase by shikonin and alkannin: mechanistic insights and hyperuricemia risk assessment.

Xanthine oxidoreductase (XOR), the rate-limiting enzyme in uric acid metabolism, reversibly interconverts between dehydrogenase (XDH) and oxidase (XO) forms. Our previous study revealed allosteric activation of XOR by 1,4-naphthoquinones, highlighting their potential risk in food industry. Herein, we employed the isomeric 1,4-naphthoquinone probes shikonin (SHI) and alkannin (ALK) to elucidate the underlying mechanism. Site-directed mutagenesis demonstrated that SHI and ALK activate XOR by binding a Trp336-centered allosteric pocket, independent of classic Cys536-Cys993 bond. This binding destabilizes the relay cluster, promoting the XDH-to-XO transition. ALK showed stronger species-biased activation of XOR (human > rat > mouse ≈ microorganism). Both ligands enhanced catalytic efficiency and antagonized allopurinol's urate-lowering action. Molecular dynamics and MM/GBSA analyses indicated that ALK formed more stable hydrogen bonds and bound more strongly. These mechanistic insights were corroborated in vivo in rats, revealing a novel allosteric activation pathway with implications for XOR-targeted therapies and food safety risk assessment.