Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F20%3A00114731" target="_blank" >RIV/00216224:14310/20:00114731 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00159816:_____/20:00074102

Výsledek na webu

<a href="https://doi.org/10.1016/j.csbj.2020.03.029" target="_blank" >https://doi.org/10.1016/j.csbj.2020.03.029</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.csbj.2020.03.029" target="_blank" >10.1016/j.csbj.2020.03.029</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

Popis výsledku v původním jazyce

Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure-function relationships of &gt;100.000 members of the alpha/beta-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14-12.6 mu M(-1)s(-1)) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005-0.014 mu M(-1)s(-1)). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii-iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

Název v anglickém jazyce

Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

Popis výsledku anglicky

Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure-function relationships of &gt;100.000 members of the alpha/beta-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14-12.6 mu M(-1)s(-1)) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005-0.014 mu M(-1)s(-1)). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii-iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

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)<br>S - Specificky vyzkum na vysokych skolach<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2020

Kód důvěrnosti údajů

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

Název periodika

Computational and Structural Biotechnology Journal

ISSN

2001-0370

e-ISSN

—

Svazek periodika

18

Číslo periodika v rámci svazku

2020

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

11

Strana od-do

922-932

Kód UT WoS článku

000607729500017

EID výsledku v databázi Scopus

2-s2.0-85082857838