Redesigning the stereospecificity of tyrosyl-tRNA synthetase - École polytechnique Access content directly
Journal Articles Proteins: Structure, Function, and Genetics Year : 2016

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.

Dates and versions

hal-01315193 , version 1 (12-05-2016)



Thomas Simonson, Shixin Ye-Lehmann, Zoltan Palmai, Najette Amara, Sandra Wydau-Dematteis, et al.. Redesigning the stereospecificity of tyrosyl-tRNA synthetase. Proteins: Structure, Function, and Genetics, 2016, 84 (2), pp.240-253. ⟨10.1002/prot.24972⟩. ⟨hal-01315193⟩
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