Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion
Thomas L Williams1, Irshad M Taily1, Lewis Hatton1
1School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, United Kingdom.
Angewandte Chemie (International Ed. in English)
|March 28, 2024
Summary
Genetic code expansion enables secondary amines to be incorporated into diverse proteins, creating novel artificial enzymes. This breakthrough expands enzyme design possibilities for biocatalysis and evolution studies.
Area of Science:
- Biochemistry
- Synthetic Biology
- Enzyme Engineering
Background:
- Secondary amines are reactive and can create artificial enzymes.
- Current methods for incorporating secondary amines into proteins are limited in scope.
- Genetic code expansion offers a way to overcome these limitations.
Purpose of the Study:
- To develop a method for incorporating secondary amines into proteins at desired positions using genetic code expansion.
- To create novel artificial enzymes with transfer hydrogenation activity.
- To explore the potential of these engineered enzymes in biocatalysis.
Main Methods:
- Utilized genetic code expansion to incorporate pyrrolysine analogues with secondary amines into proteins.
- Engineered superfolder green fluorescent protein (sfGFP), LmrR, and dihydrofolate reductase (DHFR) variants.
- Assessed proteolytic stability and catalytic activity of the engineered proteins.
Main Results:
- Successfully incorporated secondary amines into sfGFP, LmrR, and DHFR.
- Engineered LmrR and DHFR variants exhibited transfer hydrogenation activity.
- A D-proline containing analogue showed proteolytic stability and catalytic function.
- DHFR variants demonstrated stereoselective reactions using NADPH as a hydride source.
- DHFR-based catalysis was integrated into an enzymatic recycling scheme.
Conclusions:
- Genetic code expansion provides a versatile platform for engineering artificial enzymes with secondary amines.
- Protein template selection influences nucleophile options and catalytic efficiency.
- The developed method has broad applications in enzyme design, evolution, and biocatalyst development.
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