Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues
Christopher J Taylor1, Florence J Hardy1, Ashleigh J Burke1
1Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester, UK.
Protein Science : a Publication of the Protein Society
|April 13, 2023
Summary
Researchers engineered new translation components to genetically encode two histidine analogues, 3-methyl-L-histidine and 3-(3-pyridyl)-L-alanine, in proteins. This advances enzyme design by expanding the accessible chemistry within enzyme active sites.
Area of Science:
- Synthetic biology
- Protein engineering
- Biochemistry
Background:
- The genetic code can be expanded to incorporate non-canonical amino acids, offering new possibilities for enzyme design.
- Histidine analogues are valuable in enzyme engineering, acting as ligands or catalytic groups.
Purpose of the Study:
- To develop mutually orthogonal translation systems for the selective genetic encoding of two histidine analogues.
- To engineer enzymes with enhanced and novel functions through expanded amino acid incorporation.
Main Methods:
- Mutational analysis of pyrrolysyl-tRNA synthetases (PylRS) from Methanosarcina mazei (MmPylRS) and Methanomethylophilus alvus (MaPylRS).
- In vitro biochemical assays and X-ray crystallography for characterization.
- Engineering of orthogonal PylRS variants for specific amino acid incorporation.
Main Results:
- A Methanomethylophilus alvus PylRS (MaPylRSIFGFF) variant was developed with improved efficiency and specificity for 3-methyl-L-histidine (MeHis) incorporation.
- An orthogonal Methanosarcina mazei PylRS (MmPylRS) was engineered for 3-(3-pyridyl)-L-alanine (3-Pyr) incorporation.
- Proteins containing both 3-Pyr and MeHis were successfully produced using the engineered orthogonal systems.
Conclusions:
- The developed orthogonal translation components enable the simultaneous genetic encoding of two distinct histidine analogues.
- This work provides tools for creating novel enzymes with expanded chemical capabilities, advancing enzyme design and engineering.
Keywords:
3-methyl-histidine3-pyridylalaninedual incorporationgenetic code expansionhistidine analoguesMore Related Videos
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