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Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site

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Abstract

The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

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Authors and Affiliations

  1. Laboratorio de Bioqu´ımica e Biotecnologia de Sistemas Bioluminescentes, Universidade Federal de Sao Carlos (UFSCAR), Campus de Sorocaba, Sorocaba, SP, Brazil

    R. A. Prado & V. R. Viviani

  2. Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, 70790-160, Brasília, DF, Brazil

    J. A. Barbosa

  3. National Institute of Advanced Science and Technology (AIST), Osaka, Japan

    Y. Ohmiya

Authors
  1. R. A. Prado
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  2. J. A. Barbosa
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  3. Y. Ohmiya
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  4. V. R. Viviani
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Correspondence to V. R. Viviani.

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Prado, R.A., Barbosa, J.A., Ohmiya, Y. et al. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site. Photochem Photobiol Sci 10, 1226–1232 (2011). https://doi.org/10.1039/c0pp00392a

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  • DOI: https://doi.org/10.1039/c0pp00392a

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