Catalytic mechanism for Renilla-type luciferases
Authors/Creators
- 1. Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic, International Clinical Research Center, St. Anne's University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic
- 2. Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
- 3. Structure et Instabilité des Génomes (StrInG), Muséum National d'Histoire Naturelle, INSERM, CNRS, Alliance Sorbonne Université, 75005 Paris, France
- 4. Central European Institute of Technology, CEITEC BUT, Purkynova 656/123, 61200 Brno, Czech Republic
- 5. School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), 555 Moo 1 Payupnai, Wangchan Valley, Rayong, 21210 Thailand
- 6. Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic, International Clinical Research Center, St. Anne's University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic, Enantis, INBIT, Kamenice 771/34, 625 00 Brno, Czech Republic
Description
The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type catalysis through crystallographic, spectroscopic, and computational experiments. Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing unprecedented snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O2 via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue light-emitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase‒luciferin pairs for ultrasensitive optical bioassays.
Files
CatMechRen-TypeLuc.zip
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