Detailed Information on Publication Record
2016
Engineering a de Novo Transport Tunnel
BREZOVSKÝ, Jan, Petra BABKOVÁ, Oksana DEGTJARIK, Andrea FOŘTOVÁ, Artur Wiktor GÓRA et. al.Basic information
Original name
Engineering a de Novo Transport Tunnel
Authors
BREZOVSKÝ, Jan (203 Czech Republic, belonging to the institution), Petra BABKOVÁ (203 Czech Republic, belonging to the institution), Oksana DEGTJARIK (112 Belarus), Andrea FOŘTOVÁ (203 Czech Republic, belonging to the institution), Artur Wiktor GÓRA (616 Poland, belonging to the institution), L. IERMAK (804 Ukraine), Petra ŘEZÁČOVÁ (203 Czech Republic), Pavel DVOŘÁK (203 Czech Republic, belonging to the institution), Ivana KUTÁ-SMATANOVÁ (203 Czech Republic), Zbyněk PROKOP (203 Czech Republic, belonging to the institution), Radka CHALOUPKOVÁ (203 Czech Republic, belonging to the institution) and Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution)
Edition
ACS Catalysis, WASHINGTON, AMER CHEMICAL SOC, 2016, 2155-5435
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: 10.614
RIV identification code
RIV/00216224:14310/16:00088545
Organization unit
Faculty of Science
UT WoS
000387306100036
Keywords in English
transport tunnel; protein engineering; protein design; activity; specificity; substrate inhibition; stability; substrate binding; product release; water dynamics
Změněno: 5/4/2017 13:35, Ing. Andrea Mikešková
Abstract
V originále
Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. The absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional “hole” into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members: up to 32 times increased substrate inhibition and 17 times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to those of the native enzyme and the most proficient haloalkane dehalogenase reported to date (kcat = 57 s–1 with 1,2-dibromoethane at 37 °C and pH 8.6). Crystallographic analysis and molecular dynamics simulations confirmed the successful introduction of a structurally defined and functional transport tunnel. Our study demonstrates that, whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels, and we anticipate that this engineering strategy will facilitate the creation of a wide range of useful biocatalysts.
Links
| GAP207/12/0775, research and development project |
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| GAP503/12/0572, research and development project |
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| GA16-06096S, research and development project |
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| LH14027, research and development project |
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| LM2011028, 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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