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Catalytic mechanism for Renilla-type luciferases

Nature Catalysis volume 6, pages 23–38 (2023)Cite this article

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Abstract

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 molecular 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.

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Fig. 1: Inhibitory effects of azaCTZ on AncFT and RLuc8 bioluminescence.
Fig. 2: Structures of azaCTZ- and CEI-bound AncFT luciferase.
Fig. 3: Structures of azaCTZ-, CEI- and CNM-bound RLuc8 variants.
Fig. 4: Structural and functional determinants of Renilla-type bioluminescence.
Fig. 5: Spectroscopic dissection of the mechanism of Renilla-type bioluminescence.
Fig. 6: Emission spectra of RLuc8 and AncFT luciferases with native CTZ and its derivatives.
Fig. 7: A proposed catalytic mechanism of CTZ-powered Renilla-type bioluminescence.

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Data availability

Atomic coordinates and structural factors have been deposited in the PDB (www.wwpdb.org)70 under accession codes 7QXR, 7QXQ, 7OMD, 7OMR and 7OMO. We will release the atomic coordinates and experimental data on article publication. The primary data from molecular dynamics simulations are available in Zenodo repository with the identifier https://doi.org/10.5281/zenodo.7241544. Source data are provided with this paper.

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Acknowledgements

We thank the Czech Science Foundation (grant no. GA22-09853S) and the Czech Ministry of Education (INBIO grant nos. CZ.02.1.01/0.0/0.0/16_026/0008451, RECETOX RI LM2018121 and e-INFRA LM2018140). This project has received funding from the European Union’s Horizon 2020 research and innovation programme (grant nos. TEAMING 857560 and Sinfonia 814418) and the Marie Sklodowska-Curie Action (grant no. 792772). M.M. acknowledges financial support from GAMU of the Masaryk University (grant no. MUNI/H/1561/2018) and M.T. is a Brno PhD Talent Scholarship holder funded by the Brno City Municipality. CIISB research infrastructure project (grant no. LM2018127) is acknowledged for financial support of the measurements at Biomolecular Interactions and Crystallization Core Facility. P.C. acknowledges research support from VISTEC, TSRI and the NSRF via the Programme Management Unit for Human Resources & Institutional Development, Research and Innovation grant no. B05F640089 and Kasikorn Bank. P.N. acknowledges European Research Council grant (no. 714850) under the European Union´s Horizon 2020 research and innovation programme. G.G. acknowledges a PhD fellowship from the University of Paris Descartes, Sorbonne Paris City. The design and synthesis of azaCTZ also benefited from the Valoexpress funding calls of the Institut Pasteur. The crystallographic experiments were performed using the PXIII beamline at the Swiss Light Source (SLS) in Villigen (Switzerland). We are grateful to the members of the SLS synchrotron for the use of their beamline and help during data collection.

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  1. These authors contributed equally: Andrea Schenkmayerova, Martin Toul.

Authors and Affiliations

  1. Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic

    Andrea Schenkmayerova, Martin Toul, Daniel Pluskal, Gaspar P. Pinto, Marketa Stuchla, Jiri Damborsky, David Bednar, Zbynek Prokop & Martin Marek

  2. International Clinical Research Center, St Anne’s University Hospital Brno, Brno, Czech Republic

    Andrea Schenkmayerova, Martin Toul, Gaspar P. Pinto, Jiri Damborsky, David Bednar, Zbynek Prokop & Martin Marek

  3. Unité de Chimie et Biocatalyse, Institut Pasteur, UMR 3523, CNRS, Paris, France

    Racha Baatallah & Glwadys Gagnot

  4. Université de Paris, Paris, France

    Glwadys Gagnot

  5. Central European Institute of Technology, CEITEC BUT, Brno, Czech Republic

    Vinicius T. Santana & Petr Neugebauer

  6. School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand

    Pimchai Chaiyen

  7. Enantis, INBIT, Brno, Czech Republic

    Jiri Damborsky

  8. Structure and Instability of Genomes (StrInG), Museum of National Natural History, INSERM, CNRS, Alliance Sorbonne University, Paris, France

    Yves L. Janin

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Contributions

R.B., G.G. and Y.L.J. designed, synthesized and characterized azaCTZ. A.S., M.S. and M.M. constructed and cloned DNA mutants and produced all recombinant proteins. D.P., A.S. and Z.P. performed luciferase and steady-state kinetics assays. A.S. and M.M. performed cocrystallization experiments, collected X-ray data and solved protein–ligand structures. M.T. and Z.P. carried out high-performance liquid chromatography experiments. V.T.S., A.S., M.T., M.M. and P.N. conducted EPR experiments. M.T. and Z.P. performed oxygen-free assays and spectral experiments. G.P.P., J.D. and D.B. performed and analysed molecular dynamics simulations. P.C. contributed to experimental design and data interpretation. M.M., Z.P. and J.D. designed the study and contributed to data interpretation. All authors contributed to the writing of the manuscript and preparation of tables and figures.

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Correspondence to Zbynek Prokop or Martin Marek.

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Nature Catalysis thanks Naohiro Kato, Per-Olof Syren, Roberto Steiner, Anderson Garbuglio Oliveira and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Schenkmayerova, A., Toul, M., Pluskal, D. et al. Catalytic mechanism for Renilla-type luciferases. Nat Catal 6, 23–38 (2023). https://doi.org/10.1038/s41929-022-00895-z

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