Design and Evolution of Haloalkane Dehalogenase with Enhanced Conversion of 1,2,3-Trichloropropane by Modification of Access Tunnels

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F09%3A00032484" target="_blank" >RIV/00216224:14310/09:00032484 - isvavai.cz</a>

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Design and Evolution of Haloalkane Dehalogenase with Enhanced Conversion of 1,2,3-Trichloropropane by Modification of Access Tunnels

Popis výsledku v původním jazyce

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 compound1,2,3-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.

Název v anglickém jazyce

Design and Evolution of Haloalkane Dehalogenase with Enhanced Conversion of 1,2,3-Trichloropropane by Modification of Access Tunnels

Popis výsledku anglicky

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 compound1,2,3-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.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

CE - Biochemie

OECD FORD obor

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Projekt

<a href="/cs/project/LC06010" target="_blank" >LC06010: Centrum biokatalýzy a biotransformací</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>Z - Vyzkumny zamer (s odkazem do CEZ)

Rok uplatnění

2009

Kód důvěrnosti údajů

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

Název periodika

Nature Chemical Biology

ISSN

1552-4450

e-ISSN

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Svazek periodika

5

Číslo periodika v rámci svazku

xx

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

7

Strana od-do

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Kód UT WoS článku

000270039900010

EID výsledku v databázi Scopus

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