Theoretical study on the catalytic mechanism of oxyluciferin hydrolysis by luciferin‐regenerating enzyme in firefly bioluminescence substrate regeneration

Abstract Firefly bioluminescence (BL) is widely utilized in bioimaging due to its high quantum yield; however, its performance in long‐term monitoring is limited by rapid signal decay resulting from product inhibition by oxyluciferin ( oLu ). In nature, fireflies employ the luciferin‐regenerating enzyme (LRE) to catalyze oLu hydrolysis, thereby removing inhibition and initiating substrate regeneration for subsequent emission. While previous studies have established the three‐step regeneration pathway—LRE‐catalyzed oLu hydrolysis, CHBT condensation with L‐Cys, and L/D‐luciferin isomerization—the initiating step lacks molecular‐level clarity. Specifically, how the active site environment of LRE facilitates this reaction remains unknown, which hinders a comprehensive understanding of the recycling process and the rational optimization of sustained bioluminescent systems. To address this, we investigate its microscopic mechanism using molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations. The results reveal that LRE provides a critical residue, Asp111, which acts as a general base to facilitate nucleophilic addition. The subsequent ring opening follows a stepwise mechanism, characterized by sequential C4‐N3 bond cleavage and proton transfer‐coupled C2‐S1 bond breaking as the rate‐determining step. Overall, this work elucidates the molecular role of LRE in substrate regeneration and provides a theoretical foundation for understanding how fireflies restart their bioluminescent cycles.