Influence of the C-terminal domain on the bioluminescence activity and color determination in green and red emitting beetle luciferases and luciferase-like enzyme


Beetle luciferases catalyze the bioluminescent oxidation of D-luciferin, producing bioluminescence colors ranging from green to red, using two catalytic steps: adenylation of D-luciferin to produce D-luciferyl-adenylate and PPi, and oxidation of D-luciferyl-adenylate, yielding AMP, CO2, and excited oxyluciferin, the emitter. Luciferases and CoA-ligases display a similar fold, with a large N-terminal domain, and a small C-terminal domain which undergoes rotation, closing the active site and promoting both adenylation and oxidative reactions. The effect of C-terminal domain deletion was already investigated for Photinus pyralis firefly luciferase, resulting in a red-emitting mutant with severely impacted luminescence activity. However, the contribution of C-terminal in the bioluminescence activities and colors of other beetle luciferases and related ancestral luciferases were not investigated yet. Here we compared the effects of the C-terminal domain deletion on green-emitting luciferases of Pyrearinus termitilluminans (Pte) click beetle and Phrixothrix vivianii railroadworm, and on the red-emitting luciferase of Phrixothrix hirtus railroadworm and luciferase-like enzyme of Zophobas morio. In all cases, the domain deletion severely impacted the overall bioluminescence activities and, slightly less, the oxidative activities, and usually red-shifted the bioluminescence colors. The results support the involvement of the C-terminal in shielding the active site from the solvent during the light emitting step. However, in Pte luciferase, the deletion caused only a 10 nm red-shift, indicating a distinctive active site which remains more shielded, independently of the C′-terminal. Altogether, the results confirm the main contribution of the C-terminal for the catalysis of the adenylation reaction and for active site shielding during the light emitting step.

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FAPESP 2010/05426-8, CNPq 401867/2016-1.

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VB produced Re and Pte deletion mutants, MC produced and characterized Zop deletion mutant, GFP characterized Pte and RE deletion mutants, AT made and discussed the three-dimensional models, VV idealized the work, prepared the manuscript and produced pxGR deletion mutant.

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Correspondence to Vadim R. Viviani.

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Bevilaqua, V.R., Carvalho, M.C., Pelentir, G.F. et al. Influence of the C-terminal domain on the bioluminescence activity and color determination in green and red emitting beetle luciferases and luciferase-like enzyme. Photochem Photobiol Sci 20, 113–122 (2021).

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  • Luciferases
  • CoA-ligases
  • C-terminal
  • Bioluminescence