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Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity
Petra Babkova1,2, Eva Sebestova1, Jan Brezovsky1,2
1Loschmidt Laboratories, Department of Experimental Biology and, Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00, Brno, Czech Republic.
Chembiochem : a European Journal of Chemical Biology
|April 19, 2017
Summary
Ancestral sequence reconstruction resurrected five ancestral haloalkane dehalogenases (HLDs) with enhanced stability and activity. This protein engineering approach efficiently develops novel biocatalysts and robust directed evolution templates.
Area of Science:
- Protein engineering
- Biocatalysis
- Molecular evolution
Background:
- Ancestral sequence reconstruction (ASR) is a valuable method for studying protein structure-function relationships.
- Haloalkane dehalogenases (HLDs) are enzymes with significant biotechnological applications.
- The HLD-II subfamily presents an opportunity for exploring enzyme evolution and engineering.
Purpose of the Study:
- To reconstruct and characterize ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily using ASR.
- To evaluate the thermodynamic stability, specific activity, and substrate specificity of resurrected ancestral enzymes.
- To assess the potential of ASR for developing novel biocatalysts and improving enzyme properties.
Main Methods:
- Ancestral sequence reconstruction (ASR) to predict five ancestral HLD sequences.
- Gene synthesis, expression in Escherichia coli, and purification of ancestral enzymes (AncHLD1-5).
- Experimental characterization including thermodynamic stability (Tm), specific activity assays, and substrate specificity profiling.
Main Results:
- Resurrected ancestral HLDs displayed significantly enhanced thermodynamic stability (up to 24°C increase in Tm) compared to extant enzymes.
- Ancestral HLDs exhibited higher specific activities, particularly for short, multi-substituted halogenated substrates.
- Multivariate statistical analysis indicated a shift in substrate specificity for AncHLD1 and AncHLD2, demonstrating successful engineering of substrate preference.
Conclusions:
- ASR is an efficient strategy for resurrecting ancestral proteins with improved functional properties.
- The developed ancestral HLDs serve as robust templates for directed evolution and potential biocatalysts.
- This study underscores the power of ASR in achieving significant protein engineering outcomes, including enhanced stability and altered substrate specificity, which are challenging via rational design.