期刊
FEBS JOURNAL
卷 285, 期 8, 页码 1456-1476出版社
WILEY
DOI: 10.1111/febs.14418
关键词
de novo protein design; enzyme catalysis; enzyme tunnels engineering; haloalkane dehalogenases; protein engineering
资金
- Grant Agency of the Czech Republic [16-24223S, 17-24321S]
- Ministry of Education, Youth, and Sports of the Czech Republic [LO1214, LQ1605, LM2015047, LM2015051, LM2015055, LM2015063]
- Masaryk University [MUNI/M/1888/2014]
- 'Employment of Best Young Scientists for International Cooperation Empowerment' project [CZ.1.07/2.3.00/30.0037]
- European Social Fund
- State Budget of the Czech Republic
- SoMoPro II Programme (project BIOGATE) [4SGA8519]
- People Programme (Marie Curie action) of the EU's Seventh Framework Programme [291782]
- South Moravian Region
- RECETOX Research Infrastructure [LM2015051, CZ.02.1.01/0.0/0.0/16_013/0001761]
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.
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