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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Viviani, V. R. (2002). The origin, diversity, and structure function relationships of insect Luciferases. Cellular and Molecular Life Sciences: CMLS, 59(11), 1833–1850.
Day, J. C., Tisi, L. C., & Bailey, M. J. (2004). Evolution of beetle bioluminescence: the origin of beetle luciferin. Luminescence, 19(1), 8–20.
A. M. Gulick, V. J. Starai, A. R. Horswill, K. M. Homick, and J. C. Escalante- Semerena. The 1.75 A Crystal Structure of acetyl-CoA Synthetase Bound to adenosine-5'-propylphosphate and Coenzyme A. Biochemistry, 2003, 42, n. 10, 2866–2873.
Wood, K. V. (1995). The chemical mechanism and evolutionary development of beetle bioluminescence. Photochemistry and Photobiology, 62, 662–673.
Babbitt, P. C., Kenyon, G. L., Martin, B. M., Charest, H., Slyvestre, M., Scholten, J. D., et al. (1992). Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases. Biochemistry, 31(24), 5594–5604.
Conti, E., Franks, N. P., & Brick, P. (1996). Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure (London, England), 4(3), 287–298.
Nakatsu, T., Ichiyama, S., Hiratake, J., Saldanha, A., Kobashi, N., Sakata, K., & Kato, H. (2006). Structural basis for the spectral difference in luciferase bioluminescence. Nature, 440(7082), 372–376.
Gulick, A. M. (2009). Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chemical Biology, 4(10), 811–827.
Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Magyar, R. A., Gonzalez, S. A., Ruggiero, M. C., & Stroh, J. G. (2004). An alternative mechanism of bioluminescence color determination in firefly luciferase. Biochemistry, 43(23), 7255–7262.
May, J. J., Kessler, N., Marahiel, M. A., & Stubbs, M. T. (2002). Crystal Structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases. Proceedings of the National Academy of Sciences of the United States of America, 99(19), 12120–12125.
Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Wilkinson, S. R., Khattak, N. F., Rosenberg, J. C., & Zimmer, M. (2005). Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain. Biochemistry, 44(5), 1385–1393.
Zako, T., Ayabe, K., Aburatani, T., Kamiya, N., Kitayama, A., Ueda, H., & Nagamune, T. (2003). Luminescent and substrate binding activities of firefly luciferase N-terminal domain. Biochimica et Biophysica Acta, 1649(2), 183–189.
Ayabe, K., Zako, T., & Ueda, H. (2005). The role of firefly luciferase N-terminal domain in efficient coupling of adenylation and oxidative steps. FEBS Letters, 579, 4389–4394.
V.R. Viviani, F.G.C. Arnoldi, B. Venkatesh, A.J.S. Neto, F.G.T. Ogawa, A.T.L. Oehlmeyer, Y. Ohmiya, (2006) Active-site properties of phrixotrix railroad worm green and red bioluminescence-eliciting luciferases. The Journal of Biochemistry 140(4), :467–474
I. Sánchez-linares, H. Pérez-Sánchez, J. M. Cecilia, and J. M. García, High-Throughput Parallel Blind Virtual Screening Using BINDSURF. BMC bioinformatics, 2012, 13 Suppl 14, n. Suppl 14, 1471–2105.
N. N. Ugarova, and L. Y. Brovko, Protein structure and bioluminescent spectra for firefly bioluminescence. Luminescence: the journal of biological and chemical luminescence, 2002, 17, n. 5, 321–330.
Hirano, T., Hasumi, Y., Ohtsuka, K., Maki, S., Niwa, H., Yamaji, M., & Hashizume, D. (2009). Spectroscopic studies of the light-color modulation mechanism of firefly (beetle) bioluminescence. Journal of the American Chemical Society, 131(6), 2385–2396.
D. Kato, Firefly Luciferase as Biocatalysts. In: MATSUDA, T. (Ed.). Future Directions in Biocatalysis. 2 ed.: Elsevier, 2017. p. 460.
Viviani, V. R., Silva, A. C., Perez, G. L., Santelli, R. V., Bechara, E. J., & Reinach, F. C. (1999). Cloning and molecular characterization of the cDNA for the Brazilian larval click-beetle Pyrearinus termitilluminans luciferase. Photochemistry and Photobiology, 70(2), 254–260.
Viviani, V. R., Prado, R. A., Neves, D. R., Kato, D., & Barbosa, J. A. R. G. (2013). A route from darkness to light: emergence and evolution of luciferase activity in AMP-CoA-ligases inferred from a mealworm luciferase-like enzyme. Biochemistry, 52(23), 3963–3973.
Viviani, V. R., Scorsato, V., Prado, R. A., Pereira, J. G., Niwa, K., Ohmiya, Y., & Barbosa, J. A. R. G. (2010). The origin of luciferase activity in zophobas mealworm AMP/CoA-ligase (Protoluciferase): luciferin stereoselectivity as a switch for the oxygenase activity. Photochemical Photobiological Sciences, 9(8), 1111–1119.
Viviani, V. R., Arnoldi, F. G., Neto, A. J., Oehlmeyer, T. L., Bechara, E. J. H., & Ohmiya, Y. (2008). The structural origin and biological function of pH-sensitivity in firefly luciferases. Photochemical Photobiological Sciences, 7(2), 59–169.
V. R. Viviani, A. J. Silva Neto, F. G. Arnoldi, J. A.R.G. Barbosa, and Y. Ohmiya. The influence of the loop between residues 223–235 in beetle luciferase bioluminescence spectra: a solvent gate for the active site of pH-sensitive luciferases. Photochemistry and photobiology, 2008, 84, n. 1, 138–144.
Viviani, V. R., & Ohmiya, Y. (2006). Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: insights on the origin of protoluciferase activity and bioluminescence colours. Luminescence, 21(4), 262–267.
Roy, A., Kucukural, A., & Zhang, Y. (2010). I-TASSER: a unified platform for automated protein structure and function prediction. Nature protocols, 5(4), 725–738.
Rodrigues, C. H., Pires, D. E., & Ascher, D. B. (2018). DynaMut: predicting the impact of mutations on protein conformation, flexibility and stability. Nucleic Acids Research, 46(W1), 350–355.
Delano, W. L, The PyMOL Molecular Graphics System. 2008. Disponível em: http://www.pymol.org.
FAPESP 2010/05426-8, CNPq 401867/2016-1.
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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). https://doi.org/10.1007/s43630-020-00007-5