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Directed Evolution of an Artificial Imine Reductase
Martina Hestericová1, Tillman Heinisch1, Lur Alonso-Cotchico2
1Department Chemistry, University of Basel, Mattenstrasse 24a, BPR 1096, Basel, 4002, Switzerland.
Angewandte Chemie (International Ed. in English)
|December 22, 2017
Summary
Directed evolution of artificial transfer hydrogenases (ATHases) using biotin-streptavidin technology yielded improved catalysts. These engineered ATHases exhibit enhanced activity, selectivity, and stability for cyclic imine reduction.
Area of Science:
- Biochemistry
- Protein Engineering
- Catalysis
Background:
- Artificial metalloenzymes integrate metal cofactors into host proteins, gaining research interest.
- Biotin-streptavidin technology offers a platform for developing artificial enzymes.
- Directed evolution enables protein optimization for specific catalytic functions.
Purpose of the Study:
- To engineer an artificial transfer hydrogenase (ATHase) with improved performance using directed evolution.
- To investigate the structural and mechanistic basis for enhanced catalytic activity and selectivity.
- To validate findings through structural analysis and computational methods.
Main Methods:
- Directed evolution of an artificial transfer hydrogenase (ATHase) based on biotin-streptavidin technology.
- Screening of engineered variants in cell-free extracts for improved catalytic activity and selectivity.
- X-ray crystallography for structural analysis of evolved ATHases.
- Multiscale computational approaches including molecular dynamics and protein-ligand docking.
Main Results:
- Two streptavidin isoforms with enhanced catalytic activity and selectivity for cyclic imine reduction were obtained.
- Evolved ATHases demonstrated stability under biphasic catalytic conditions.
- Structural analysis revealed that bulky active site residues alter cofactor flexibility, increasing metal exposure and reversing enantioselectivity.
- Computational studies confirmed the proposed mechanism of altered enantioselectivity.
Conclusions:
- Directed evolution is effective for optimizing artificial metalloenzymes like ATHases.
- Structural modifications within the active site significantly influence cofactor behavior and catalytic outcomes.
- The study provides insights into the rational design of artificial enzymes with tailored properties.
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