Artur Góra, Ph.D.

Project: REDEHAL - Fine-tuning of haloalkane dehalogenases by access tunnels re-engineering

Person in Charge: doc. Mgr. Jiří Damborský, Dr.

Host institution: Loschmidt laboratories, Institute of Experimental Biology, Faculty of Science, Masaryk University

Country of Origin: Poland 

Country of scientific activity: Japan

Project duration: 36 months

Scientific panel: Life sciences

Abstract:

Methods for protein modification, redesign or de novo design are of the great importance in modern chemical and pharmacological industry. Recent project will explore possibility of the haloalkane dehalogenases tunnels re-engineering towards enzymes with higher selectivity and reactivity towards desired substrates. The novelty of the proposed project lies in engineering of catalytic properties of enzymes by modification of access tunnels. This is a new concept potentially applicable to other enzymes with buried active sites. Combination of computational and experimental methods will provide detailed description of the free energy profiles and barriers of the passage of water molecules and substrates/products through the access tunnels. This will provide new insights into the role of water solvent in the catalytic mechanism of enzymes and identification of bottlenecks for the process of enzymatic catalysis. Understanding of these mechanisms will broaden our basic knowledge of enzymatic catalysis, which has important implications in the basic research as well as in applications. Based on the analysis carried out, it will be possible to design new haloalkane dehalogenase mutants with improved catalytic properties. For challenging ligand access study, new computational methods will be developed to improve prediction of accurate tunnels re-engineering towards enzyme desired selectivity and activity.

The ongoing project summary:
REDEHAL project explores possibility of tunnels re-engineering in bacterial enzymes haloalkane dehalogenases. The novelty of the project lies in engineering of catalytic properties of enzymes by modification of their access tunnels. Combination of computational and experimental methods will provide detailed description of the barriers governing the passage of water molecules, substrates and products through the access tunnels. This will provide new insights into the role of water-tunnel interactions in the catalytic mechanism of enzymes and help identify additional bottlenecks for the process of biocatalysis. Understanding of these mechanisms will broaden our basic knowledge of enzymatic catalysis, which is important for basic research as well as for practical applications. Based on the analysis carried out, it will be possible to design new haloalkane dehalogenase with improved catalytic properties. New computational methods will be developed for study of ligand passage through the access tunnels to improve design of enzymes with desired selectivity and activity.

Overall objectives of the project are:

•  to develop fast and accurate in silico methods for design of mutants with desired activity or selectivity
• to propose structure of mutants with desired properties and their experimental validation

Activities implemented within the project (first year):

• training in molecular modeling, molecular visualization and bioinformatic tools (Amber, AutoDock, Pymol, FOLDX, HotSpot Wizard, CAVER),
• participation in software development (CAVER plug-in and CAVER 3.0),
• description of haloalkane dehalogenases and other enzymes structures with focus on characterization of their tunnels and gating mechanisms,
• co-operation with experimental groups (analysis and explanation of experimental results, providing input data for design of new enzymes).

Brief summary of the results achieved to-date:

• Prediction of tunnel opening in haloalkane dehalogenases DatA
• Proposal of two single-point mutants DatA F178A and DatA F247A with opened access tunnel. Proposed mutants were constructed and are currently under experimental testing.

• Prediction of tunnel closing in haloalkane dehalogenases LinB.

• Two single point mutations A247C and D147C were proposed to close access tunnel by creation of disulfide bond with previously designed L177C LinB mutant. Proposed mutants were designed and are currently under experimental verification.
• Detection of tunnel-forming residues in selected enzymes – input data for testing hypothesis on stabilizing effect of mutations of tunnel residues

Already achieved project outputs result in: 

•  acceleration of work over development of new (CAVER 3.0) and improvement of already existing and widely used software (CAVER plug-in for PyMol),
• improving the scientific potential of host group by transfer of knowledge,
• proposing of new possibly interesting mutants of haloalkane dehalogenases, enzymes with potential applicability in industry and environmental protection issues,
• participation in preparation of patent: “Improving stability of proteins at higher temperatures and in the presence of organic solvents by engineering of tunnels and channels”