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

Toul, M.; Horackova, J.; Schenkmayerova, A.; Planas-Iglesias, J.; Landolt, T.; Sucharitakul, J.; Janin, Y.; Prakinee, K.; Chaiyen, P.; Stavrakis, S.; deMello, A.; Johnson, K. A.; Damborsky, J.; Marek, M.; Bedar, D.; Prokop, Z.

doi:10.1101/2025.09.16.675553

bioRxiv · 10.1101/2025.09.16.675553

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

Toul, M.·Horackova, J.·Schenkmayerova, A.·Planas-Iglesias, J.·Landolt, T.·Sucharitakul, J.·Janin, Y.·Prakinee, K.·Chaiyen, P.·Stavrakis, S.·deMello, A.·Johnson, K. A.·Damborsky, J.·Marek, M.·Bedar, D.·Prokop, Z.

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Abstract

Luciferases are widely used bioluminescent reporters, yet the molecular determinants of their catalytic efficiency and light-emission stability remain incompletely understood. Here, we reconstruct the complete catalytic pathway of Renilla luciferase by combining steady-state, transient, and temperature-dependent kinetics with crystallography and molecular simulations. We show that the enzyme is substantially undersaturated with oxygen (Km,O2 = 719 M), causing its true turnover number (kcat = 21.9 s-1) to be systematically underestimated. Concurrently, elevated oxygen drives irreversible enzyme inactivation after ~1,500 turnovers, revealing a fundamental trade-off that limits oxygen-affinity engineering. Instead, the genuine bottleneck of the catalytic cycle is the induced-fit conformational opening of the product-bound enzyme. Selective engineering of this transition step through rational loop grafting yielded a variant AncFT-L14 with enhanced catalytic efficiency and glow-type bioluminescence with substantially slower signal decay in cell lysates. Collectively, our results identify conformational dynamics as a primary tunable determinant of luciferase function. More broadly, this work establishes a mechanistically grounded framework for the development of next-generation bioluminescent tools and for engineering enzymes controlled by dynamically gated ligand exchange.

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Toul, M., Horackova, J., Schenkmayerova, A., Planas-Iglesias, J., Landolt, T., Sucharitakul, J., Janin, Y., Prakinee, K., Chaiyen, P., Stavrakis, S., deMello, A., Johnson, K. A., Damborsky, J., Marek, M., Bedar, D., Prokop, Z.. 2025-09-18. Kinetic mechanism of Renilla luciferase guides induced-fit engineering for improved bioluminescence. https://doi.org/10.1101/2025.09.16.675553

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