Illuminating the mechanism and allosteric behavior of NanoLuc luciferase

NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis<br> remains puzzling. Here experimental and computational techniques were combined, revealing that<br> imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on<br> the enzyme surface. Binding to the allosteric site prevents simultaneous binding to the catalytic site, and<br> vice versa, through concerted conformational changes. We demonstrate that restructuration of the<br> allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel<br> arginine coordinates the imidazopyrazinone component of luciferin which then react with O 2 via a radical<br> charge-transfer mechanism, and it also protonates the resulting excited amide product to form a light-<br> emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, is fine-tuning the blue<br> color emitter to secure a high emission intensity. This information is critical to engineering the next-<br> generation of ultrasensitive bioluminescent reporters.