| PAVLOVÁ, Martina, Martin KLVAŇA, Radka CHALOUPKOVÁ, Pavel BANÁŠ, Michal OTYEPKA, Rebecca WADE, Yuji NAGATA a Jiří DAMBORSKÝ. Design and Evolution of Haloalkane Dehalogenase with Enhanced Conversion of 1,2,3-Trichloropropane by Modification of Access Tunnels. Nature Chemical Biology. Nature Chemical Biology, 2009, roč. 5, xx, s. 727-733. ISSN 1552-4450. |
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@article{727119,
author = {Pavlová, Martina and Klvaňa, Martin and Chaloupková, Radka and Banáš, Pavel and Otyepka, Michal and Wade, Rebecca and Nagata, Yuji and Damborský, Jiří},
article_number = {xx},
keywords = {xxx},
language = {eng},
issn = {1552-4450},
journal = {Nature Chemical Biology},
title = {Design and Evolution of Haloalkane Dehalogenase with Enhanced Conversion of 1,2,3-Trichloropropane by Modification of Access Tunnels},
url = {http://loschmidt.chemi.muni.cz/peg/abstracts/nchemb09.html},
volume = {5},
year = {2009}
}
TY - JOUR
ID - 727119
AU - Pavlová, Martina - Klvaňa, Martin - Chaloupková, Radka - Banáš, Pavel - Otyepka, Michal - Wade, Rebecca - Nagata, Yuji - Damborský, Jiří
PY - 2009
TI - Design and Evolution of Haloalkane Dehalogenase with Enhanced Conversion of 1,2,3-Trichloropropane by Modification of Access Tunnels
JF - Nature Chemical Biology
VL - 5
IS - xx
SP - 727-733
EP - 727-733
PB - Nature Chemical Biology
SN - 15524450
KW - xxx
UR - http://loschmidt.chemi.muni.cz/peg/abstracts/nchemb09.html
N2 - Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.
ER -
PAVLOVÁ, Martina, Martin KLVAŇA, Radka CHALOUPKOVÁ, Pavel BANÁŠ, Michal OTYEPKA, Rebecca WADE, Yuji NAGATA a Jiří DAMBORSKÝ. Design and Evolution of Haloalkane Dehalogenase with Enhanced Conversion of 1,2,3-Trichloropropane by Modification of Access Tunnels. \textit{Nature Chemical Biology}. Nature Chemical Biology, 2009, roč.~5, xx, s.~727-733. ISSN~1552-4450.
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