AbstractThe availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl‐tRNA synthetase (MmPylRSIFGFF) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF) provided a variant with improved efficiency and specificity for 3‐methyl‐L‐histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and x‐ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3‐(3‐pyridyl)‐L‐alanine (3‐Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3‐Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.