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Redesigning the stereospecificity of tyrosyl-tRNA synthetase.

TitleRedesigning the stereospecificity of tyrosyl-tRNA synthetase.
Publication TypeJournal Article
Year of Publication2016
AuthorsSimonson, T, Ye-Lehmann, S, Palmai, Z, Amara, N, Wydau-Dematteis, S, Bigan, E, Druart, K, Moch, C, Plateau, P
JournalProteins
Volume84
Issue2
Pagination240-53
Date Published2016 Feb
ISSN1097-0134
KeywordsEscherichia coli Proteins, Molecular Dynamics Simulation, Protein Engineering, Stereoisomerism, Thermodynamics, Tyrosine-tRNA Ligase
Abstract

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.

DOI10.1002/prot.24972
Alternate JournalProteins
PubMed ID26676967