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@article{1503358,
author = {Vaňáček, Pavel and Šebestová, Eva and Babková, Petra and Nevolová, Šárka and Daniel, Lukáš and Dvořák, Pavel and Štěpánková, Veronika and Chaloupková, Radka and Brezovský, Jan and Prokop, Zbyněk and Damborský, Jiří},
article_location = {WASHINGTON},
article_number = {3},
doi = {http://dx.doi.org/10.1021/acscatal.7b03523},
keywords = {diversity; sequence space; bioinformatics; biocatalyst; biochemical characterization; activity; substrate specificity; haloalkane dehalogenases},
language = {eng},
issn = {2155-5435},
journal = {ACS Catalysis},
title = {Exploration of Enzyme Diversity by Integrating Bioinformatics with Expression Analysis and Biochemical Characterization},
url = {http://dx.doi.org/10.1021/acscatal.7b03523},
volume = {8},
year = {2018}
}
TY - JOUR
ID - 1503358
AU - Vaňáček, Pavel - Šebestová, Eva - Babková, Petra - Nevolová, Šárka - Daniel, Lukáš - Dvořák, Pavel - Štěpánková, Veronika - Chaloupková, Radka - Brezovský, Jan - Prokop, Zbyněk - Damborský, Jiří
PY - 2018
TI - Exploration of Enzyme Diversity by Integrating Bioinformatics with Expression Analysis and Biochemical Characterization
JF - ACS Catalysis
VL - 8
IS - 3
SP - 2402-2412
EP - 2402-2412
PB - AMER CHEMICAL SOC
SN - 21555435
KW - diversity
KW - sequence space
KW - bioinformatics
KW - biocatalyst
KW - biochemical characterization
KW - activity
KW - substrate specificity
KW - haloalkane dehalogenases
UR - http://dx.doi.org/10.1021/acscatal.7b03523
N2 - Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Here, we describe an integrated system for automated in silico screening and systematic characterization of diverse family members. The workflow consists of (i) identification and computational characterization of relevant genes by sequence/structural bioinformatics, (ii) expression analysis and activity screening of selected proteins, and (iii) complete biochemical/biophysical characterization and was validated against the haloalkane dehalogenase family. The sequence-based search identified 658 potential dehalogenases. The subsequent structural bioinformatics prioritized and selected 20 candidates for exploration of protein functional diversity. Out of these 20, the expression analysis and the robotic screening of enzymatic activity provided 8 soluble proteins with dehalogenase activity. The enzymes discovered originated from genetically unrelated Bacteria, Eukaryota, and also Archaea. Overall, the integrated system provided biocatalysts with broad catalytic diversity showing unique substrate specificity profiles, covering a wide range of optimal operational temperature from 20 to 70 degrees C and an unusually broad pH range from 5.7 to 10. We obtained the most catalytically proficient native haloalkane dehalogenase enzyme to date (k(cat)/K-0.5 = 96.8 mM(-1) s(-1) the most thermostable enzyme with melting temperature 71 degrees C, three different cold-adapted enzymes showing dehalogenase activity at near-to-zero temperatures, and a biocatalyst degrading the warfare chemical sulfur mustard. The established strategy can be adapted to other enzyme families for exploration of their biocatalytic diversity in a large sequence space continuously growing due to the use of next-generation sequencing technologies.
ER -
VAŇÁČEK, Pavel, Eva ŠEBESTOVÁ, Petra BABKOVÁ, Šárka NEVOLOVÁ, Lukáš DANIEL, Pavel DVOŘÁK, Veronika ŠTĚPÁNKOVÁ, Radka CHALOUPKOVÁ, Jan BREZOVSKÝ, Zbyněk PROKOP a Jiří DAMBORSKÝ. Exploration of Enzyme Diversity by Integrating Bioinformatics with Expression Analysis and Biochemical Characterization. \textit{ACS Catalysis}. WASHINGTON: AMER CHEMICAL SOC, 2018, roč.~8, č.~3, s.~2402-2412, 21 s. ISSN~2155-5435. Dostupné z: https://dx.doi.org/10.1021/acscatal.7b03523.
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