Abstract
Using genetic code expansion, canonical amino acid residues can be site-specifically substituted by noncanonical amino acids (ncAAs) with modified chemical properties. This technique has enabled detailed enzymatic studies, the design of enzymes that catalyze novel reactions, and the engineering of enzymes with improved function. In proteins, histidine can play versatile roles in catalysis, including as an acid, a base, a nucleophile, and a coordinating ligand to a catalytic metal. However, the current scope of histidine-like ncAAs that can be incorporated is limited. Herein, we develop a toolkit consisting of nine new aminoacyl-tRNA synthetase/tRNA pairs for the site-specific genetic encoding of an expanded set of 12 new histidine-like ncAAs. The 12 ncAAs feature broadly tuned nitrogen pK(a)H, alternative heterocycles, and varying substitution patterns. We profile the substrate specificity of the developed aaRS/tRNA pairs and uncover many mutually orthogonal substrate specificities, which we validate for six combinations of dual encoded histidine-like ncAAs. We expect that the tools presented herein will be broadly applicable to study histidine residues in catalysis and to tune the properties of histidine residues for enzyme engineering and design.