Highly stable single-strand-specific 3'-nuclease/nucleotidase from Legionella pneumophila

0495886 - BTÚ 2019 RIV NL eng J - Článek v odborném periodiku
Trundová, Mária - Koval, Tomáš - Owens, R. J. - Fejfarová, Karla - Dušková, Jarmila - Kolenko, Petr - Dohnálek, Jan
Highly stable single-strand-specific 3'-nuclease/nucleotidase from Legionella pneumophila.
International Journal of Biological Macromolecules. Roč. 114, JUL 15 2018 (2018), s. 776-787. ISSN 0141-8130. E-ISSN 1879-0003
Grant CEP: GA MŠMT LG14009; GA MŠMT(CZ) LM2015043; GA ČR GA15-05228S; GA MŠMT(CZ) ED2.1.00/19.0390; GA MŠMT(CZ) EF16_013/0001776
Institucionální podpora: RVO:86652036
Klíčová slova: S1-P1 nuclease * Legionella * Escherichia coli expression
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 4.784, rok: 2018

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 structurefunction 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. (C) 2018 Elsevier B.V. All rights reserved.
Trvalý link: http://hdl.handle.net/11104/0288768