Directed Evolution of an Artificial Imine Reductase

Artificial metalloenzymes, resulting from incorporation of a metal cofactor within a host protein, have received increasing attention in the last decade. The directed evolution is presented of an artificial transfer hydrogenase (ATHase) based on the biotin‐streptavidin technology using a straightfor...

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Bibliographic Details
Published inAngewandte Chemie International Edition Vol. 57; no. 7; pp. 1863 - 1868
Main Authors Hestericová, Martina, Heinisch, Tillman, Alonso‐Cotchico, Lur, Maréchal, Jean‐Didier, Vidossich, Pietro, Ward, Thomas R.
Format Journal Article
LanguageEnglish
Published Germany Wiley Subscription Services, Inc 12.02.2018
EditionInternational ed. in English
Subjects
artificial metalloenzymes
Biotin
Catalysis
Catalytic activity
cyclic imines
Directed evolution
Enantiomers
enzyme catalysis
Evolution
Hydrogenase
Imines
Isoforms
Metals
Molecular dynamics
Proteins
Reductase
Streptavidin
Structural analysis
transfer hydrogenation
enzyme catalysis
transfer hydrogenation
cyclic imines
artificial metalloenzymes
directed evolution
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Summary:Artificial metalloenzymes, resulting from incorporation of a metal cofactor within a host protein, have received increasing attention in the last decade. The directed evolution is presented of an artificial transfer hydrogenase (ATHase) based on the biotin‐streptavidin technology using a straightforward procedure allowing screening in cell‐free extracts. Two streptavidin isoforms were yielded with improved catalytic activity and selectivity for the reduction of cyclic imines. The evolved ATHases were stable under biphasic catalytic conditions. The X‐ray structure analysis reveals that introducing bulky residues within the active site results in flexibility changes of the cofactor, thus increasing exposure of the metal to the protein surface and leading to a reversal of enantioselectivity. This hypothesis was confirmed by a multiscale approach based mostly on molecular dynamics and protein–ligand dockings. Skipping steps: An artificial imine reductase is generated by incorporation of a biotinylated iridium piano‐stool complex within streptavidin. A streamlined process allows a faster screening in cell‐free extracts. With this system, both enantiomers of the product can be obtained in high ee after only a few rounds of saturation mutagenesis.
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ISSN:1433-7851
1521-3773
DOI:10.1002/anie.201711016