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bioRxiv · 10.1101/2025.06.16.659893

Novel mechanistic insights for catalytic bioluminescence of mammalian Gaussia Luciferase through mutant and ancestral analysis

Borum, R. M.·Lanzillotti, M.·Sahasrabuddhe, A.·Hui, J.·Xie, V.·Ferbas, J.
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Abstract

A mechanistic basis for luciferase bioluminescence provides a glimpse into its evolutionary role for organism survival, as it provides a blueprint to engineer luciferase enzymes for advanced technological applications. Gaussia Luciferase is among the brightest natural luciferases, but (1) the evolutionary development of its luminescence behavior remains unclear, (2) recent fundamental studies utilized E. Coli expression systems instead of eukaryotic expression systems, and (3) notable mutants have been discovered but not integrated into comprehensive mechanistic analysis. We describe new mechanistic observations from GLuc by addressing these gaps. We monitored fluorescent coelenerazine-to-coelenteramide conversion to study turnover kinetics of mammalian-derived GLuc; this assay characterized the positive cooperativity kinetics of GLuc. The non-luminescent mutants, R76A and R147A, still turn over the substrate with high efficiency, each demonstrating sustained positive cooperativity. Through mass spectrometry, mutational analysis, and analytical liquid chromatography, we demonstrate that GLuc undergoes methionine oxidation during substrate turnover, and that this impacts the flash-type luminescence of the luciferase; we did not observe indications of covalent attachment with the substrate, product, or their intermediates. Chromatography on luciferases derived from ancestral sequence reconstruction highlighted that the extent of methionine-induced surface changes was greater for earlier ancestral luciferases. Ancestral sequence reconstruction also revealed that earlier ancestral copepod luciferases produced less light when compared to GLuc.

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Borum, R. M., Lanzillotti, M., Sahasrabuddhe, A., Hui, J., Xie, V., Ferbas, J.. 2025-06-20. Novel mechanistic insights for catalytic bioluminescence of mammalian Gaussia Luciferase through mutant and ancestral analysis. https://doi.org/10.1101/2025.06.16.659893

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