A rationally engineered misacylating aminoacyl-tRNA synthetase

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Aminoacyl-tRNA synthetases (aaRS) are crucial for protein synthesis, linking specific amino acids to their cognate transfer RNAs (tRNAs).
• While the structures of 22 tRNA synthetase families are known, the specific enzyme signatures dictating amino acid selectivity remain largely elusive.
• Understanding these signatures is key to deciphering the fidelity of genetic information transfer.

Purpose of the Study:

• To investigate the enzyme signatures responsible for amino acid selectivity in aminoacyl-tRNA synthetases.
• To explore the structural basis for glutamyl-tRNA synthetase (GluRS) specificity and its potential transfer to glutaminyl-tRNA synthetase (GlnRS).
• To understand the role of tRNA in mediating amino acid specificity within the aaRS-tRNA complex.

Main Methods:

• Site-directed mutagenesis was used to introduce specific amino acid changes, such as transplanting arginine residues.
• X-ray crystallography was employed to determine the structures of wild-type and mutant GlnRS enzymes.
• Biochemical assays were performed to measure enzyme kinetics (K(M)) and the capacity for misacylation.

Main Results:

• Transplanting a single arginine residue from GluRS to GlnRS partially improved glutamate binding but disrupted structural integrity.
• Extensive modifications, including substitution of the primary binding site and surface loops, resulted in a 16,000-fold increase in GlnRS's ability to misacylate tRNA with glutamate.
• The engineered GlnRS mimicked the misacylating activity of GluRS found in organisms with alternative Gln-tRNA(Gln) synthesis pathways.

Conclusions:

• Single amino acid substitutions are insufficient to confer the substrate specificity of a different tRNA synthetase family.
• Extensive engineering of the GlnRS active site and surrounding regions is required to achieve significant misacylation with glutamate.
• The tRNA molecule itself plays a critical role in mediating amino acid specificity, potentially representing an ancient mechanism from the early evolution of the genetic code.