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Effect of pH on the secondary structure and thermostability of beetle luciferases: structural origin of pH-insensitivity

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Abstract

Beetle luciferases were classified into three functional groups: (1) pH-sensitive yellow–green-emitting (fireflies) which change the bioluminescence color to red at acidic pH, high temperatures and presence of heavy metals; (2) the pH-insensitive green–yellow-emitting (click beetles, railroad worms and firefly isozymes) which are not affected by these factors, and (3) pH-insensitive red-emitting. Although the pH-sensing site in firefly luciferases was recently identified, it is unclear why some luciferases are pH-insensitive despite the presence of some conserved pH-sensing residues. Through circular dichroism, we compared the secondary structural changes and unfolding temperature of luciferases of representatives of these three groups: (1) pH-sensitive green–yellow-emitting Macrolampis sp2 (Mac) and Amydetes vivianii (Amy) firefly luciferases; (2) the pH-insensitive green-emitting Pyrearinus termitilluminans larval click beetle (Pte) and Aspisoma lineatum (Al2) larval firefly luciferases, and (3) the pH-insensitive red-emitting Phrixotrix hirtus railroadworm (PxRE) luciferase. The most blue-shifted luciferases, independently of pH sensitivity, are thermally more stable at different pHs than the red-shifted ones. The pH-sensitive luciferases undergo increases of α-helices and thermal stability above pH 6. The pH-insensitive Pte luciferase secondary structure remains stable between pH 6 and 8, whereas the Al2 luciferase displays an increase of the β-sheet at pH 8. The PxRE luciferase also displays an increase of α-helices at pH 8. The results indicate that green–yellow emission in beetle luciferases can be attained by: (1) a structurally rigid scaffold which stabilizes a single closed active site conformation in the pH-insensitive luciferases, and (2) active site compaction above pH 7.0 in the more flexible pH-sensitive luciferases.

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Funding

Fundação de Amparo à Pesquisa do Estado de São Paulo, 2010/05426-8, Vadim R. Viviani, 2018/02538-1, Atílio Tomazini, Conselho Nacional de Desenvolvimento Científico e Tecnológico, 405060/2021-1, Vadim R. Viviani.

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Authors and Affiliations

  1. Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar), Sorocaba, Brazil

    Atílio Tomazini & Vadim R. Viviani

  2. Graduate Program of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), São Carlos, Brazil

    Mariele Carvalho & Vadim R. Viviani

  3. Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, SP, Brazil

    Atílio Tomazini & Mario T. Murakami

  4. Department of Physics, Chemistry and Mathematics, Center for Sustainable Sciences and Technologies (CCTS), Federal University of São Carlos, Rod. João Leme dos Santos, SP 264, Km 110, Itinga, Sorocaba, SP, Brazil

    Vadim R. Viviani

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  1. Atílio Tomazini
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Tomazini, A., Carvalho, M., Murakami, M.T. et al. Effect of pH on the secondary structure and thermostability of beetle luciferases: structural origin of pH-insensitivity. Photochem Photobiol Sci 22, 893–904 (2023). https://doi.org/10.1007/s43630-022-00360-7

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