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

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.