Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00159816%3A_____%2F17%3A00067124" target="_blank" >RIV/00159816:_____/17:00067124 - isvavai.cz</a>

Alternative codes found

RIV/00216224:14310/17:00095517

Result on the web

<a href="http://dx.doi.org/10.1021/acs.jcim.7b00070" target="_blank" >http://dx.doi.org/10.1021/acs.jcim.7b00070</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/acs.jcim.7b00070" target="_blank" >10.1021/acs.jcim.7b00070</a>

Result language

angličtina

Original language name

Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling

Original language description

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, 727MINUS SIGN 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 SN2 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.

Czech name

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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

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OECD FORD branch

30104 - Pharmacology and pharmacy

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)

Publication year

2017

Confidentiality

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

Name of the periodical

Journal of Chemical Information and Modeling

ISSN

1549-9596

e-ISSN

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Volume of the periodical

57

Issue of the periodical within the volume

8

Country of publishing house

US - UNITED STATES

Number of pages

21

Pages from-to

1970-1989

UT code for WoS article

000408790100022

EID of the result in the Scopus database

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