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 O2 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.

Center for Interdisciplinary Biosciences Technology and Innovation Park P J Safarik University in Kosice Trieda SNP 1 04011 Kosice Slovakia

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

International Clinical Research Center St Anne's University Hospital Brno Pekarska 53 656 91 Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Kamenice 5 Bld C13 625 00 Brno Czech Republic

Structure et Instabilité des Génomes Muséum National d'Histoire Naturelle INSERM CNRS Alliance Sorbonne Université 75005 Paris France

Unité de Chimie et Biocatalyse Institut Pasteur UMR 3523 CNRS 28 rue du Dr Roux 75724 Paris Cedex 15 Paris France

Université de Paris 12 rue de l'école de Médecine 75006 Paris France

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PDB
8AQH, 8AQI, 8AQ6, 8BO9, 7MJB, 7VSX, 5B0U, 7SNT