Illuminating the mechanism and allosteric behavior of NanoLuc luciferase

• Published in: Nature communications Vol. 14; no. 1; p. 7864
• Main Authors: Nemergut, Michal, Pluskal, Daniel, Horackova, Jana, Sustrova, Tereza, Tulis, Jan, Barta, Tomas, Baatallah, Racha, Gagnot, Glwadys, Novakova, Veronika, Majerova, Marika, Sedlackova, Karolina, Marques, Sérgio M., Toul, Martin, Damborsky, Jiri, Prokop, Zbynek, Bednar, David, Janin, Yves L., Marek, Martin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 29-11-2023; Nature Publishing Group; Nature Portfolio
• Subjects: 631/45/603; 631/535/1266; 631/92/607; 82/29; 82/80; 82/83; Allosteric properties; Binding; Bioluminescence; Catalysis; Charge transfer; Emitters; Enzymes; Humanities and Social Sciences; Luciferin; multidisciplinary; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-43403-y

NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, 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 the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O
2
via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.
NanoLuc luciferase is a popular bioluminescent enzyme, but the molecular details of its mechanism of action on luciferins such as coelenterazine remained elusive. Here the authors use, protein crystal structures and biochemical analyses to provide an atomistic description of its catalytic mechanism and allosteric behaviour.