Aminoacyl-tRNA synthetases (aaRSs) set up the genetic code by covalently attaching the amino acids to their cognate tRNAs with a high specificity. For several aaRSs, mismatched products are cleared by hydrolytic editing mechanisms, which are essential to maintain the fidelity of translation. These editing mechanisms include pre-transfer editing that targets misactivated adenylate, and post-transfer editing that hydrolyzes misacylated tRNA. We identified aaRSs in Mycoplasma parasites have point mutations and deletions in their respective editing domains. The deleterious effect on editing was confirmed with Mycoplasma mobile leucyl-tRNA synthetase (LeuRS) studied in vitro. In vivo mistranslation was determined by mass spectrometric analysis of proteins produced in the parasite. These mistranslations are uniformly cases where the predicted closely similar amino acid replaced the correct one. Thus, natural aaRS editing-domain mutations in Mycoplasma parasites cause mistranslation. We further studied the pre-transfer editing pathway in M. mobile LeuRS and Mycoplasma synoviae LeuRSs. In both cases, we identified a tRNA-dependent pre-transfer editing activity. However, the pre- transfer editing is not strong enough to fully suppress misaminoacylation and mistranslation in these organisms. Pre-transfer editing is considered as a more ancient proofreading pathway, and we investigated its detailed mechanism by unbiased molecular dynamics simulations and umbrella sampling in ∆CP1 LeuRS, an ideal system for pre-transfer editing investigation. Our results suggested a surprisingly simple model for pre-transfer editing, which only requires a small rotation of A76 that makes space for a water molecule to hydrolyze the non-cognate adenylate. As such, the aminoacylation site of the ∆CP1 LeuRS should be considered as the active site for both aminoacylation and pre-transfer editing reactions; it can be switched from one to the other by the adenylate bound. Finally, metadynamics and fluorescence spectroscopy was used to study the binding of ATP to aaRS, a millisecond process essential for the aminoacylation. The calculated minimum free energy pathway suggested a two-step binding mechanism, which consists of a fast initial binding of triphosphate, followed by a slow binding of the nucleoside. In the slow nucleoside binding step, a conserved histidine stabilizes the incoming ATP through base-stacking interaction with adenine. Mutation of this histidine leads to a 26-fold decrease of ATP binding affinity. Additional metadynamics simulations of the slow binding of nucleoside identify an intermediate quality control state that aaRS uses to select ATP over other nucleoside triphosphates.
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