A Designed, Highly Efficient Pyrrolysyl-tRNA Synthetase Mutant Binds o-Chlorophenylalanine Using Two Halogen Bonds

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Pyrrolysyl-tRNA synthetase (PylRS) is a key tool for genetic code expansion.
• PylRS is structurally similar to phenylalanyl-tRNA synthetase (PheRS).

Purpose of the Study:

• To design a PylRS mutant capable of incorporating o-substituted phenylalanines.
• To investigate the mechanism of recognition and incorporation of o-chlorophenylalanine (o-ClF).

Main Methods:

• Mutagenesis of PylRS to mimic PheRS ligand interactions, creating the oClFRS mutant.
• Crystallographic analysis of oClFRS bound to o-ClF.
• Assessing the efficiency of genetic incorporation of o-ClF.

Main Results:

• The engineered oClFRS mutant efficiently incorporates various o-substituted phenylalanines at the amber codon.
• The crystal structure reveals o-ClF binding in a deep, hydrophobic pocket via two halogen bonds.
• o-ClF binding interactions are strong but in a catalytically inactive site, requiring repositioning for activation.

Conclusions:

• This study presents the first aminoacyl-tRNA synthetase utilizing two halogen bonds for ligation recognition.
• The oClFRS mutant demonstrates improved efficiency for o-ClF incorporation compared to native PylRS substrates.
• This work provides a novel strategy for developing aminoacyl-tRNA synthetase mutants with enhanced selectivity for noncanonical amino acids.