Redesigning the stereospecificity of tyrosyl‐tRNA synthetase

d ‐Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl‐tRNA synthetases (aaRSs), and this strategy might be applicable to d ‐amino acids. Several aaRSs can aminoacylate their tRNA with a d ‐amino acid; of these, tyrosyl‐tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its d ‐Tyr binding further, relative to l ‐Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl‐adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain l ‐Tyr/ d ‐Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge‐altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards d ‐Tyr; one of these has an inverted stereospecificity, with a large preference for d ‐Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled. Proteins 2016; 84:240–253. © 2015 Wiley Periodicals, Inc.