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Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase
Luciferases from copepods Metridia longa and Gaussia princeps are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encodi...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10220987/ https://www.ncbi.nlm.nih.gov/pubmed/37240867 http://dx.doi.org/10.3390/life13051222 |
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author | Markova, Svetlana V. Larionova, Marina D. Korotov, Igor A. Vysotski, Eugene S. |
author_facet | Markova, Svetlana V. Larionova, Marina D. Korotov, Igor A. Vysotski, Eugene S. |
author_sort | Markova, Svetlana V. |
collection | PubMed |
description | Luciferases from copepods Metridia longa and Gaussia princeps are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encoding the smallest MLuc7 isoform of M. longa luciferase. The single catalytic domain is shown to reside within the G32-A149 MLuc7 sequence and to be formed by both non-identical repeats, including 10 conserved Cys residues. Because this part of MLuc7 displays high homology with those of other copepod luciferases, our suggestion is that the determined boundaries of the catalytic domain are the same for all known copepod luciferases. The involvement of the flexible C-terminus in the retention of the bioluminescent reaction product in the substrate-binding cavity was confirmed by structural modeling and kinetics study. We also demonstrate that the ML7-N10 mutant (15.4 kDa) with deletion of ten amino acid residues at the N-terminus can be successfully used as a miniature bioluminescent reporter in living cells. Application of a shortened reporter may surely reduce the metabolic load on the host cells and decrease steric and functional interference at its use as a part of hybrid proteins. |
format | Online Article Text |
id | pubmed-10220987 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-102209872023-05-28 Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase Markova, Svetlana V. Larionova, Marina D. Korotov, Igor A. Vysotski, Eugene S. Life (Basel) Article Luciferases from copepods Metridia longa and Gaussia princeps are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encoding the smallest MLuc7 isoform of M. longa luciferase. The single catalytic domain is shown to reside within the G32-A149 MLuc7 sequence and to be formed by both non-identical repeats, including 10 conserved Cys residues. Because this part of MLuc7 displays high homology with those of other copepod luciferases, our suggestion is that the determined boundaries of the catalytic domain are the same for all known copepod luciferases. The involvement of the flexible C-terminus in the retention of the bioluminescent reaction product in the substrate-binding cavity was confirmed by structural modeling and kinetics study. We also demonstrate that the ML7-N10 mutant (15.4 kDa) with deletion of ten amino acid residues at the N-terminus can be successfully used as a miniature bioluminescent reporter in living cells. Application of a shortened reporter may surely reduce the metabolic load on the host cells and decrease steric and functional interference at its use as a part of hybrid proteins. MDPI 2023-05-21 /pmc/articles/PMC10220987/ /pubmed/37240867 http://dx.doi.org/10.3390/life13051222 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Markova, Svetlana V. Larionova, Marina D. Korotov, Igor A. Vysotski, Eugene S. Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase |
title | Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase |
title_full | Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase |
title_fullStr | Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase |
title_full_unstemmed | Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase |
title_short | Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Metridia longa Luciferase |
title_sort | localization of the catalytic domain of copepod luciferases: analysis of truncated mutants of the metridia longa luciferase |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10220987/ https://www.ncbi.nlm.nih.gov/pubmed/37240867 http://dx.doi.org/10.3390/life13051222 |
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