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
remains puzzling. Here experimental and computational 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. 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 then react with O 2 via a radical
charge-transfer mechanism, and it also protonates the resulting excited amide product to form a light-
emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, is fine-tuning the blue
color emitter to secure a high emission intensity. This information is critical to engineering the next-
generation of ultrasensitive bioluminescent reporters.