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 dramatically boost 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, as well as it protonates the excited amide product to secure high emission intensity. Concomitantly, an aspartate, supported by two tyrosines, fine-tune the electronic state of amide product, promoting the formation of the blue color emitter. This information is critical to engineering the next-generation of light-producing biosystems.