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

•With two mutations at the active site, M. mazei pyrrolysyl-tRNA synthetase is engineered to efficiently mediate o-chlorophenylalanine at amber codon.•The incorporation efficiency is much higher than that of Nε-Boc-lysine mediated by the wild-type enzyme.•The X-ray crystallography analysis of the engineered enzyme bound with o-chlorophenylalanine shows that o-chlorophenylalanine involves two halogen bonds to bind at an inactive pocket of the enzyme.•This is one of very few examples that a protein involves halogen bonds to bind its ligand.