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

Result code in 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>

Alternative codes found

RIV/00159816:_____/20:00074102

Result on the web

<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>

Result language

angličtina

Original language name

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

Original language description

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.

Czech name

—

Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10608 - Biochemistry and molecular biology

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

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

Publication year

2020

Confidentiality

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

Name of the periodical

Computational and Structural Biotechnology Journal

ISSN

2001-0370

e-ISSN

—

Volume of the periodical

18

Issue of the periodical within the volume

2020

Country of publishing house

NL - THE KINGDOM OF THE NETHERLANDS

Number of pages

11

Pages from-to

922-932

UT code for WoS article

000607729500017

EID of the result in the Scopus database

2-s2.0-85082857838