The physiological target for LeuRS translational quality control is norvaline

The fidelity of protein synthesis depends on the capacity of aminoacyl‐tRNA synthetases (AARSs) to couple only cognate amino acid‐tRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here, we show that the editing activity of Escherichia coli leucyl‐tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis‐incorporation of the non‐standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing‐deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS‐based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non‐essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site. Synopsis Aminoacyl‐tRNA‐synthetase (AARS) editing hydrolyses mischarged tRNAs. LeuRS editing is not a response to poor selectivity between Leu and Ile, as previously suggested, but targets the non‐standard amino acid norvaline during hypoxic growth. LeuRS discriminates against isoleucine with more than 10 4 ‐fold specificity arising from weak ground state binding and decreased rate of the chemical step. The major threat for the accuracy of Leu‐tRNALeu synthesis is posed by a non‐standard amino acid norvaline. The prime role of the LeuRS post‐transfer editing domain is to prevent participation of norvaline in protein synthesis. LeuRS post‐transfer editing is not required under normal or isoleucine‐rich growth conditions, but rescues Escherichia coli growth under norvaline‐rich conditions. Low oxygen levels trigger accumulation of norvaline in Escherichia coli , providing a biological rationale for evolution of efficient LeuRS editing. Graphical Abstract Aminoacyl‐tRNA‐synthetase (AARS) editing hydrolyses mischarged tRNAs. LeuRS editing is not a response to poor selectivity between Leu and Ile, as previously suggested, but targets the non‐standard amino acid norvaline during hypoxic growth.