Impact of the access tunnel engineering on catalysis is strictly ligand-specific

The FEBS Journal
Shubhangi KaushikJirí Damborský

Abstract

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 si...Continue Reading

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Citations

Jun 16, 2018·PloS One·Kalyanasundaram SubramanianPeter J Schaap
Sep 16, 2018·Applied and Environmental Microbiology·Shalev GihazAyelet Fishman
Jul 11, 2019·Journal of the Royal Society, Interface·Sophie Sacquin-Mora
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Apr 17, 2020·International Journal of Molecular Sciences·Bartłomiej SurpetaJan Brezovsky
Apr 21, 2020·Computational and Structural Biotechnology Journal·Piia KokkonenDavid Bednar
Jul 3, 2020·Computational and Structural Biotechnology Journal·Martin MarekJiri Damborsky
May 26, 2021·Scientific Reports·Yael Baruch-Shpigler, David Avnir
Aug 31, 2021·Frontiers in Molecular Biosciences·Edward KingRay Luo

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