Highly stable single-strand-specific 3′-nuclease/nucleotidase from Legionella pneumophila
The Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1–P1 type nuclease. Bacterial zinc-dependent 3′-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1...
| Main Authors: | , , , , , , |
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| Format: | Journal article |
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Elsevier
2018
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| _version_ | 1797074250322935808 |
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| author | Trundová, M Kovaľ, T Owens, R Fejfarová, K Dušková, J Kolenko, P Dohnálek, J |
| author_facet | Trundová, M Kovaľ, T Owens, R Fejfarová, K Dušková, J Kolenko, P Dohnálek, J |
| author_sort | Trundová, M |
| collection | OXFORD |
| description | The Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1–P1 type nuclease. Bacterial zinc-dependent 3′-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1 (Lpn1), in contrast to many eukaryotic representatives with in-depth studies available. To help explain the principle properties and role of these enzymes in intracellular prokaryotic pathogens we have designed and optimized a heterologous expression protocol utilizing E. coli together with an efficient purification procedure, and performed detailed characterization of the enzyme. Replacement of Ni2+ ions by Zn2+ ions in affinity purification proved to be a crucial step in the production of pure and stable protein. The production protocol provides protein with high yield, purity, stability, and solubility for structure-function studies. We show that highly thermostable Lpn1 is active mainly towards RNA and ssDNA, with pH optima 7.0 and 6.0, respectively, with low activity towards dsDNA; the enzyme features pronounced substrate inhibition. Bioinformatic and experimental analysis, together with computer modeling and electrostatics calculations point to an unusually high positive charge on the enzyme surface under optimal conditions for catalysis. The results help explain the catalytic properties of Lpn1 and its substrate inhibition. |
| first_indexed | 2024-03-06T23:33:25Z |
| format | Journal article |
| id | oxford-uuid:6cd2bb34-bc15-410d-84f7-c2997d1fe00a |
| institution | University of Oxford |
| last_indexed | 2024-03-06T23:33:25Z |
| publishDate | 2018 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | oxford-uuid:6cd2bb34-bc15-410d-84f7-c2997d1fe00a2022-03-26T19:13:45ZHighly stable single-strand-specific 3′-nuclease/nucleotidase from Legionella pneumophilaJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:6cd2bb34-bc15-410d-84f7-c2997d1fe00aSymplectic Elements at OxfordElsevier2018Trundová, MKovaľ, TOwens, RFejfarová, KDušková, JKolenko, PDohnálek, JThe Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1–P1 type nuclease. Bacterial zinc-dependent 3′-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1 (Lpn1), in contrast to many eukaryotic representatives with in-depth studies available. To help explain the principle properties and role of these enzymes in intracellular prokaryotic pathogens we have designed and optimized a heterologous expression protocol utilizing E. coli together with an efficient purification procedure, and performed detailed characterization of the enzyme. Replacement of Ni2+ ions by Zn2+ ions in affinity purification proved to be a crucial step in the production of pure and stable protein. The production protocol provides protein with high yield, purity, stability, and solubility for structure-function studies. We show that highly thermostable Lpn1 is active mainly towards RNA and ssDNA, with pH optima 7.0 and 6.0, respectively, with low activity towards dsDNA; the enzyme features pronounced substrate inhibition. Bioinformatic and experimental analysis, together with computer modeling and electrostatics calculations point to an unusually high positive charge on the enzyme surface under optimal conditions for catalysis. The results help explain the catalytic properties of Lpn1 and its substrate inhibition. |
| spellingShingle | Trundová, M Kovaľ, T Owens, R Fejfarová, K Dušková, J Kolenko, P Dohnálek, J Highly stable single-strand-specific 3′-nuclease/nucleotidase from Legionella pneumophila |
| title | Highly stable single-strand-specific 3′-nuclease/nucleotidase from Legionella pneumophila |
| title_full | Highly stable single-strand-specific 3′-nuclease/nucleotidase from Legionella pneumophila |
| title_fullStr | Highly stable single-strand-specific 3′-nuclease/nucleotidase from Legionella pneumophila |
| title_full_unstemmed | Highly stable single-strand-specific 3′-nuclease/nucleotidase from Legionella pneumophila |
| title_short | Highly stable single-strand-specific 3′-nuclease/nucleotidase from Legionella pneumophila |
| title_sort | highly stable single strand specific 3 nuclease nucleotidase from legionella pneumophila |
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