Kinetic mechanism of Renilla luciferase guides induced-fit engineering for improved bioluminescence

Renilla luciferase (RLuc) remains one of the most popular bioluminescence reporters, but its molecular principle has yet to be fully understood. Here, we reveal a detailed kinetic mechanism of the RLuc catalytic cycle which uncovers multiple limiting factors: (i) an oxygen-induced irreversible inactivation, (ii) a low oxygen saturation, and (iii) rate-limiting induced-fit conformational dynamics coupled with the product release. Furthermore, we could determine the actual enzyme kcat value at all saturating substrates to be 22 s-1. This value is 5-fold higher than the previously reported apparent kcat values determined at physiological, non-saturating oxygen concentration. Our integrative analysis by transient kinetics, X-ray crystallography, and molecular dynamics linked the rate-limiting flexible enzyme opening to the dynamics of the loops surrounding the active site, which prompted targeted engineering of this limiting step by loop grafting. The resulting variant AncFT-L14 (AncFT7) showed a prolonged stable light emission thanks to the selectively improved induced-fit kinetics. Additional characterization of AncFT-L14 identified increased catalytic efficiency kcat/Km, product inhibition factor Kp/Km, and a glow-type signal characteristic. Our results provide mechanistic details of RLuc catalysis and will govern future enzyme engineering to design the next generations of bioluminescence-based tools.