Detailed Information on Publication Record
2017
Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling
MARQUES, Sérgio Manuel, Zuzana DUNAJOVÁ, Zbyněk PROKOP, Radka CHALOUPKOVÁ, Jan BREZOVSKÝ et. al.Basic information
Original name
Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling
Authors
MARQUES, Sérgio Manuel (620 Portugal, belonging to the institution), Zuzana DUNAJOVÁ (703 Slovakia, belonging to the institution), Zbyněk PROKOP (203 Czech Republic, belonging to the institution), Radka CHALOUPKOVÁ (203 Czech Republic, belonging to the institution), Jan BREZOVSKÝ (203 Czech Republic, belonging to the institution) and Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution)
Edition
JOURNAL OF CHEMICAL INFORMATION AND MODELING, DC USA, AMER CHEMICAL SOC, 2017, 1549-9596
Other information
Language
English
Type of outcome
Článek v odborném periodiku
Field of Study
10201 Computer sciences, information science, bioinformatics
Country of publisher
United States of America
Confidentiality degree
není předmětem státního či obchodního tajemství
References:
Impact factor
Impact factor: 3.804
RIV identification code
RIV/00216224:14310/17:00095517
Organization unit
Faculty of Science
UT WoS
000408790100022
Keywords in English
MOLECULAR-DYNAMICS SIMULATIONS; FORCE-FIELD; DIRECTED EVOLUTION; SYNTHETIC PATHWAY; PRODUCT RELEASE; MECHANISM; KINETICS; LINB; BIODEGRADATION; PARAMETERS
Změněno: 29/3/2018 23:17, Ing. Nicole Zrilić
Abstract
V originále
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.
Links
| GAP503/12/0572, research and development project |
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| LH14027, research and development project |
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| LM2015047, research and development project |
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| LM2015051, research and development project |
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| LM2015055, research and development project |
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| LO1214, research and development project |
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| MUNI/M/1888/2014, interní kód MU |
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| 4SGA8519, interní kód MU |
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