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@article{1409089,
author = {Marques, Sérgio Manuel and Dunajová, Zuzana and Prokop, Zbyněk and Chaloupková, Radka and Brezovský, Jan and Damborský, Jiří},
article_location = {DC USA},
article_number = {8},
doi = {http://dx.doi.org/10.1021/acs.jcim.7b00070},
keywords = {MOLECULAR-DYNAMICS SIMULATIONS; FORCE-FIELD; DIRECTED EVOLUTION; SYNTHETIC PATHWAY; PRODUCT RELEASE; MECHANISM; KINETICS; LINB; BIODEGRADATION; PARAMETERS},
language = {eng},
issn = {1549-9596},
journal = {JOURNAL OF CHEMICAL INFORMATION AND MODELING},
title = {Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling},
url = {https://loschmidt.chemi.muni.cz/peg/publications/catalytic-cycle-of-haloalkane-dehalogenases-toward-unnatural-substrates-explored-by-computational-modeling/},
volume = {57},
year = {2017}
}
TY - JOUR
ID - 1409089
AU - Marques, Sérgio Manuel - Dunajová, Zuzana - Prokop, Zbyněk - Chaloupková, Radka - Brezovský, Jan - Damborský, Jiří
PY - 2017
TI - Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling
JF - JOURNAL OF CHEMICAL INFORMATION AND MODELING
VL - 57
IS - 8
SP - 1970-1989
EP - 1970-1989
PB - AMER CHEMICAL SOC
SN - 15499596
KW - MOLECULAR-DYNAMICS SIMULATIONS
KW - FORCE-FIELD
KW - DIRECTED EVOLUTION
KW - SYNTHETIC PATHWAY
KW - PRODUCT RELEASE
KW - MECHANISM
KW - KINETICS
KW - LINB
KW - BIODEGRADATION
KW - PARAMETERS
UR - https://loschmidt.chemi.muni.cz/peg/publications/catalytic-cycle-of-haloalkane-dehalogenases-toward-unnatural-substrates-explored-by-computational-modeling/
N2 - The anthropogenic toxic compound 1,2,3-trichloropropane is poorly degradable by natural enzymes. We have previously constructed the haloalkane dehalogenase DhaA31 by focused directed evolution (Pavlova, M. et al. Nat. Chem. Biol. 2009, 5, 727-733), which is 32 times more active than the wild-type enzyme and is currently the most active variant known against that substrate. Recent evidence has shown that the structural basis responsible for the higher activity of DhaA31 was poorly understood. Here we have undertaken a comprehensive computational study of the main steps involved in the biocatalytic hydrolysis of 1,2,3-trichloropropane to decipher the structural basis for such enhancements. Using molecular dynamics and quantum mechanics approaches we have surveyed (i) the substrate binding, (ii) the formation of the reactive complex, (iii) the chemical step, and (iv) the release of the products. We showed that the binding of the substrate and its transport through the molecular tunnel to the active site is a relatively fast process. The cleavage of the carbon halogen bond was previously identified as the rate-limiting step in the wild-type. Here we demonstrate that this step was enhanced in DhaA31 due to a significantly higher number of reactive configurations of the substrate and a decrease of the energy barrier to the S(N)2 reaction. C176Y and V245F were identified as the key mutations responsible for most of those improvements. The release of the alcohol product was found to be the rate-limiting step in DhaA31 primarily due to the C176Y mutation. Mutational dissection of DhaA31 and kinetic analysis of the intermediate mutants confirmed the theoretical observations. Overall, our comprehensive computational approach has unveiled mechanistic details of the catalytic cycle which will enable a balanced design of more efficient enzymes. This approach is applicable to deepen the biochemical knowledge of a large number of other systems and may contribute to robust strategies in the development of new biocatalysts.
ER -
MARQUES, Sérgio Manuel, Zuzana DUNAJOVÁ, Zbyněk PROKOP, Radka CHALOUPKOVÁ, Jan BREZOVSKÝ and Jiří DAMBORSKÝ. Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling. \textit{JOURNAL OF CHEMICAL INFORMATION AND MODELING}. DC USA: AMER CHEMICAL SOC, 2017, vol.~57, No~8, p.~1970-1989. ISSN~1549-9596. Available from: https://dx.doi.org/10.1021/acs.jcim.7b00070.
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