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@article{1415292,
author = {Kaushik, Shubhangi and Marques, Sérgio Manuel and Khirsariya, Prashant Kumar and Paruch, Kamil and Libichová, Lenka and Brezovský, Jan and Prokop, Zbyněk and Chaloupková, Radka and Damborský, Jiří},
article_number = {8},
doi = {http://dx.doi.org/10.1111/febs.14418},
keywords = {de novo protein design; enzyme catalysis;enzyme tunnels engineering; haloalkanedehalogenases; protein engineering},
language = {eng},
issn = {1742-464X},
journal = {the FEBS Journal},
title = {Impact of the access tunnel engineering on catalysis is strictly ligand-specific},
url = {https://doi.org/10.1111/febs.14418},
volume = {285/2018},
year = {2018}
}
TY - JOUR
ID - 1415292
AU - Kaushik, Shubhangi - Marques, Sérgio Manuel - Khirsariya, Prashant Kumar - Paruch, Kamil - Libichová, Lenka - Brezovský, Jan - Prokop, Zbyněk - Chaloupková, Radka - Damborský, Jiří
PY - 2018
TI - Impact of the access tunnel engineering on catalysis is strictly ligand-specific
JF - the FEBS Journal
VL - 285/2018
IS - 8
SP - 1456-1476
EP - 1456-1476
SN - 1742464X
KW - de novo protein design
KW - enzyme catalysis;enzyme tunnels engineering
KW - haloalkanedehalogenases
KW - protein engineering
UR - https://doi.org/10.1111/febs.14418
L2 - https://doi.org/10.1111/febs.14418
N2 - The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes’ catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products. Thus, we have systematically studied the effects of single-point mutations in a tunnel-lining residue of a haloalkane dehalogenase on the binding kinetics and catalytic conversion of both linear and branched haloalkanes. The hotspot residue Y176 was identified using computer simulations and randomized through saturation mutagenesis, and the resulting variants were screened for shifts in binding rates. Strikingly, opposite effects of the substituted residues on the catalytic efficiency toward linear and branched substrates were observed, which was found to be due to substrate-specific requirements in the critical steps of the respective catalytic cycles. We conclude that not only the catalytic sites, but also the access pathways must be tailored specifically for each individual ligand, which is a new paradigm in protein engineering and de novo protein design. A rational approach is proposed here to address more effectively the task of designing ligand-specific tunnels using computational tools.
ER -
KAUSHIK, Shubhangi, Sérgio Manuel MARQUES, Prashant Kumar KHIRSARIYA, Kamil PARUCH, Lenka LIBICHOVÁ, Jan BREZOVSKÝ, Zbyněk PROKOP, Radka CHALOUPKOVÁ a Jiří DAMBORSKÝ. Impact of the access tunnel engineering on catalysis is strictly ligand-specific. \textit{the FEBS Journal}. 2018, roč.~285/2018, č.~8, s.~1456-1476. ISSN~1742-464X. Dostupné z: https://dx.doi.org/10.1111/febs.14418.
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