Illuminating the mechanism and allosteric behavior of NanoLuc luciferase

We investigated the complex mechanism and allosteric behavior of NanoLuc luciferase, combining experimental and computational techniques. Our findings reveal that imidazopyrazinone luciferins bind not only to an intra-barrel catalytic site but also to an allosteric site on the enzyme's surface. Binding at one site prevents simultaneous binding at the other, due to a homotropic negative allostery mechanism. We discovered that restructuring the allosteric site enhances luminescence in the active site, providing insights critical for developing next-generation bioluminescent reporters. Our work elucidates the catalytic mechanism of NanoLuc, involving a radical charge-transfer mechanism, and demonstrates the potential for engineering superior in vivo reporters through allosteric site modification.