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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F86652036%3A_____%2F18%3A00495886" target="_blank" >RIV/86652036:_____/18:00495886 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1016/j.ijbiomac2018.03.113" target="_blank" >http://dx.doi.org/10.1016/j.ijbiomac2018.03.113</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.ijbiomac2018.03.113" target="_blank" >10.1016/j.ijbiomac2018.03.113</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

International Journal of Biological Macromolecules

ISSN

0141-8130

e-ISSN

—

Svazek periodika

114

Číslo periodika v rámci svazku

JUL 15 2018

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

12

Strana od-do

776-787

Kód UT WoS článku

000435056900090

EID výsledku v databázi Scopus

2-s2.0-85052476283