Illuminating the mechanism of NanoLuc luciferase action

M. Marek^1,2, M. Nemergut^1,2, D. Pluskal¹, J. Horackova¹, T. Sustrova¹, T. Barta³, J. Tulis¹, V. Novakova^1,2, M. Majerova^1,2, M. Toul^1,2, S. M. Marques^1,2, Yves Janin⁴, J. Damborsky^1,2, D. Bednar^1,2, Z. Prokop^1,2

¹Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

²International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 65691 Brno, Czech Republic

³Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic

⁴Structure et Instabilité des Génomes (StrInG), Muséum National d'Histoire Naturelle, INSERM, CNRS, Alliance Sorbonne Université, 75005 Paris, France

martin.marek@recetox.muni.cz

NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational structural techniques were combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface [1]. Binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost dramatically the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin to attack O₂ via a radical charge-transfer mechanism, while it protonates the excited amide product to secure high emission intensity. Concomitantly, an aspartate, supported by two tyrosines, fine-tune the electronic state of the amide product, promoting the formation of the blue colored emission. Thus, we show that NanoLuc, despite its structural dissimilarity, employs analogous tricks to secure a blue light-emitting phenolate anion, as we recently revealed for Renilla-type luciferase [2]. Such information should be critical to engineer the next-generation of light-producing biosystems.

1. Nemergut M., Pluskal D., Horackova J, Sustrova T., Tulis J., Barta T., Baatallah R., Gagnot G., Novakova V., Majerova M., Marques S. M., Toul M., Damborsky J., Bednar D., Prokop Z., Janin Y. L., Marek M. (2022). Illuminating the mechanism and allosteric behavior of NanoLuc luciferase. bioRxiv (2022), https://doi.org/10.1101/2022.12.05.519101

2. Schenkmayerova A., Toul, M., Pluskal D., Baatallah R., Gagnot G., Pinto G. P., Santana V. T., Stuchla M., Neugebauer P., Chaiyen P., Damborsky J., Bednar D., Janin Y. L., Prokop Z., Marek M. (2023). Catalytic mechanism for Renilla-type luciferases. Nature Catalysis (2023), https://doi.org/10.1038/s41929-022-00895-z

This work was supported by the Czech Science Foundation (22-09853S).