Full Implementation of the Genetic Code by Tryptophanyl-tRNA Synthetase Requires Intermodular Coupling

Li, Li; Carter, Charles W.

doi:10.1074/jbc.m113.510958

Cited by 38 publications

(107 citation statements)

References 34 publications

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“…The designed 46-mers behave much more like generalized ATP activators. The smaller turnover numbers of the WT 46-mers, in contrast, likely reflect the fact that much of the contemporary aaRS catalytic machinery involves interactions between the ATP-binding sites and other modular components (42,43), which have been deleted.…”

Section: Design Altered the Enzymatic Properties Of The Sense-antisenmentioning

confidence: 99%

“…Furthermore, complementation can be characterized in greater detail by measuring the energetic coupling between them using modular thermodynamic cycles (7,43). Characterization of additional pairs of WT 46-mers and designed sense/antisense peptides also may help resolve the question of whether the constraint of genetic complementarity is inconsistent with high amino acid affinity.…”

Section: Experimental Recapitulation Of Possible Assembly Of Other Momentioning

confidence: 99%

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Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

Martínez‐Rodríguez

Erdoğan

Jimenez-Rodriguez

et al. 2015

Journal of Biological Chemistry

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121

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Section: Design Altered the Enzymatic Properties Of The Sense-antisenmentioning

confidence: 99%

Section: Experimental Recapitulation Of Possible Assembly Of Other Momentioning

confidence: 99%

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

Martínez‐Rodríguez

Erdoğan

Jimenez-Rodriguez

et al. 2015

Journal of Biological Chemistry

Self Cite

121

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“…2A). Energetic coupling (about Ϫ6 kcal/mol) between these two modules enhances all three TrpRS functions, Mg 2ϩ -dependent rate acceleration, specificity, and tRNA Trp aminoacylation to approximately the same degree (8).…”

Section: Modular Trprs Construction and The Conformational Cycle-mentioning

confidence: 99%

“…The TrpRS Urzyme (18,19) enabled us to examine this intrinsic modularity directly (8). The Urzyme contains a functional active site without either CP1 or the ABD.…”

Section: Modular Trprs Construction and The Conformational Cycle-mentioning

confidence: 99%

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Enhanced Amino Acid Selection in Fully Evolved Tryptophanyl-tRNA Synthetase, Relative to Its Urzyme, Requires Domain Motion Sensed by the D1 Switch, a Remote Dynamic Packing Motif

Weinreb

Chandrasekaran

et al. 2014

Journal of Biological Chemistry

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Background: Amino acid selection by tryptophanyl-tRNA synthetase (TrpRS) requires intermodular coupling. Results: Dynamic repacking of four side chains increases amino acid specificity 500-fold in contemporary TrpRS by reducing pocket size near the transition state. Conclusion: An ancient tertiary packing motif not only activates the catalytic Mg 2ϩ ion during catalysis, but also determines cognate amino acid specificity. Significance: Allosteric enforcement of specificity increases robustness to mutation.

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Redesigning the stereospecificity of tyrosyl-tRNA synthetase

Simonson

Ye-Lehmann²,

Palmai

et al. 2016

Proteins

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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.

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Full Implementation of the Genetic Code by Tryptophanyl-tRNA Synthetase Requires Intermodular Coupling

Cited by 38 publications

References 34 publications

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

Enhanced Amino Acid Selection in Fully Evolved Tryptophanyl-tRNA Synthetase, Relative to Its Urzyme, Requires Domain Motion Sensed by the D1 Switch, a Remote Dynamic Packing Motif

Redesigning the stereospecificity of tyrosyl-tRNA synthetase

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