Accuracy during translation ensures faithful conversion of the genetic code. Aminoacyl tRNA-synthetases (aaRS) are essential enzymes that attach an amino acid to a cognate tRNA. The aaRS first activates an amino acid in an ATP-dependent manner to form an aminoacyl adenylate. Next, the aaRS attaches the aminoacyl adenylate to the 3’-OH of the terminal adenosine residue of the cognate tRNA. The aminoacylated tRNA is transported to the ribosome by the elongation factor Tu and GTP to participate in translation. AaRSs play a major role in maintaining translational accuracy by attaching the correct amino acids to their cognate tRNAs. Although the active site pockets of many aaRSs are able to discriminate against the wrong amino acid, the shared chemicophysical structure of some amino acids can lead to mis-activated amino acids. To maintain translation accuracy, aaRS have evolved proofreading strategies that can hydrolyze a mis-activated aminoacyl adenylate in the active site or translocate a misacylated tRNA to a separate editing domain capable of hydrolysis. The proofreading strategies ensure fidelity during aminoacylation and prevent mistranslation. It has been previously shown that disruption of the editing domain of alanyl-tRNA synthetase (AlaRS) in mice causes neurodegeneration. In E. coli, perturbation to the editing domain through mutagenesis of a critical cysteine residue, Cys666, leads to the global dysregulation of the E. coli proteome resulting in a wide range of phenotypes including growth defects, impaired motility, and antibiotic sensitivity.Here we show that the stress caused by the replacement of the critical cysteine in the editing domain of E. coli AlaRS selects for spontaneous second-site suppressor mutations that alter the editing domain to alleviate the phenotypic defects. This work highlights the importance of maintaining AlaRS fidelity during aminoacylation and characterizes novel features of the AlaRS editing domain.Recent studies showed that environmental challenges such as exposure to reactive oxygen species (ROS) can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. Here we show that oxidative stress does not affect the proofreading function of E. coli AlaRS but leads to a decrease in the rate of tRNAAla aminoacylation. Additionally, residues that undergo oxidative modification were identified using mass spectrometry and their contribution to the regulation of E. coli AlaRS under oxidative stress were analyzed. This study indicates that E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.