Bioluminescence Profiling of NanoKAZ/NanoLuc Luciferase Using a Chemical Library of Coelenterazine Analogues |
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Authors: | Dr Eloi P Coutant Dr Glwadys Gagnot Vincent Hervin Racha Baatallah Prof?Dr Sophie Goyard Dr Yves Jacob Dr Thierry Rose Prof?Dr Yves L Janin |
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Institution: | 1. Institut Pasteur, UMR 3523, CNRS, Unité de Chimie et Biocatalyse, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France;2. Center for Innovation and Technological Research, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France;3. Unité de Génétique Moléculaire des Virus à ARN, Institut Pasteur, UMR 3569, CNRS, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France |
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Abstract: | We describe here an extensive structure-bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 O-acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo1,2-a]pyrazin-3(7H)-one provided a 1.5-fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal-to-background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate-dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer-dependent fraction of a photon). |
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Keywords: | enzyme catalysis heterocycles luciferins luminescence natural products |
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