J 2009

Redesigning Dehalogenase Access Tunnels as a Strategy for Degrading an Anthropogenic Substrate.

PAVLOVÁ, Martina, Martin KLVAŇA, Radka CHALOUPKOVÁ, Pavel BANÁŠ, Michal OTYEPKA et. al.

Basic information

Original name

Redesigning Dehalogenase Access Tunnels as a Strategy for Degrading an Anthropogenic Substrate.

Name in Czech

Zmena designu tunelů dehalogenáz jako strategie degradace antropogenních substrátů.

Authors

PAVLOVÁ, Martina (203 Czech Republic, belonging to the institution), Martin KLVAŇA (203 Czech Republic, belonging to the institution), Radka CHALOUPKOVÁ (203 Czech Republic, belonging to the institution), Pavel BANÁŠ (203 Czech Republic), Michal OTYEPKA (203 Czech Republic), R. WADE (276 Germany), Yuji NAGATA (392 Japan) and Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution)

Edition

NATURE CHEMICAL BIOLOGY, 2009, 1552-4450

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 16.058

RIV identification code

RIV/00216224:14310/09:00028563

Organization unit

Faculty of Science

UT WoS

000270039900010

Keywords in English

Dehalogenase; Access Tunnels; degradation

Tags

International impact, Reviewed
Změněno: 23/3/2012 11:05, prof. Mgr. Jiří Damborský, Dr.

Abstract

ORIG CZ

V originále

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.

In Czech

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Links

GA201/07/0927, research and development project
Name: Vizualizace proteinových struktur
Investor: Czech Science Foundation, Visualization of protein structures
GA203/08/0114, research and development project
Name: Specifické iontové efekty pro proteiny v roztocích a podobné biologicky relevantní systémy.
Investor: Czech Science Foundation, Specific ion effects for proteins in solutions and related biologically relevant systems
IAA401630901, research and development project
Name: Evoluce substrátové specifity u enzymů aktivních s xenobiotickými látkami
Investor: Academy of Sciences of the Czech Republic, Evolution of substrate specificity in enzymes acting on xenobiotic compounds
LC06010, research and development project
Name: Centrum biokatalýzy a biotransformací
Investor: Ministry of Education, Youth and Sports of the CR, Center of Biocatalysis and Biotransformation
MSM0021622412, plan (intention)
Name: Interakce mezi chemickými látkami, prostředím a biologickými systémy a jejich důsledky na globální, regionální a lokální úrovni (INCHEMBIOL) (Acronym: INCHEMBIOL)
Investor: Ministry of Education, Youth and Sports of the CR, Interactions among the chemicals, environment and biological systems and their consequences on the global, regional and local scales (INCHEMBIOL)