An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity
Douglas D Young1, Travis S Young, Michael Jahnz
1Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Biochemistry
|February 2, 2011
Summary
A novel aminoacyl-tRNA synthetase (aaRS) exhibits broad specificity for unnatural amino acids, enabling the incorporation of 18 diverse types into proteins. Structural analysis reveals binding site features controlling this substrate permissivity.
Area of Science:
- Biochemistry
- Molecular Biology
- Synthetic Biology
Background:
- Aminoacyl-tRNA synthetases (aaRSs) are crucial enzymes for protein synthesis, typically exhibiting high specificity for their cognate amino acids.
- Expanding the genetic code to include unnatural amino acids (uaas) in proteins requires orthogonal aaRSs with altered substrate specificities.
Purpose of the Study:
- To identify and characterize aminoacyl-tRNA synthetases (aaRSs) with broad substrate specificity for unnatural amino acids (uaas).
- To investigate the structural basis of polyspecificity in aaRSs for enhanced uaa incorporation into proteins.
Main Methods:
- Rapid fluorescence-based screening of aaRSs against a library of uaas.
- X-ray crystallography to determine the structure of a polyspecific aaRS-uaa complex.
- Site-directed mutagenesis and functional assays to probe substrate specificity determinants.
Main Results:
- A p-cyanophenylalanine-specific aaRS (pCNF-RS) demonstrated high polyspecificity, accepting 18 different uaas while discriminating against canonical amino acids.
- The crystal structure of pCNF-RS complexed with p-cyanophenylalanine revealed key binding site features contributing to uaa recognition.
- Comparison with other aaRS structures highlighted the roles of binding site size and subtle interactions in controlling substrate permissivity.
Conclusions:
- Engineered aaRSs can achieve broad substrate specificity for uaas, significantly expanding the toolkit for protein engineering.
- Understanding the structural determinants of aaRS polyspecificity is essential for designing novel enzymes for expanded genetic codes.
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