Escherichia coli Alanyl-tRNA Synthetase Maintains Proofreading Activity and Translational Accuracy Under Oxidative Stress

Authors

Arundhati Kavoor, The Ohio State University
Paul Kelly, The Ohio State University
Michael Ibba, Chapman UniversityFollow

Document Type

Article

Publication Date

2-15-2022

Abstract

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in Escherichia coli AlaRS, unlike in other aaRSs, this does not affect proofreading activity against the noncognate substrates serine and glycine and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNA^Ala formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNA^Ala aminoacylation observed under oxidative stress, suggesting that these residues may act as reactive oxygen species “sinks” to protect catalytically critical sites from oxidative damage. Combined, our results indicate 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.

Comments

This article was originally published in Journal of Biological Chemistry, volume 298, issue 3, in 2022. https://doi.org/10.1016/j.jbc.2022.101601

Recommended Citation

Kavoor A, Kelly P, and Ibba M. (2022) Escherichia coli alanyl-tRNA synthetase maintains proofreading activity and translational accuracy under oxidative stress. J Biol Chem. 298:101601. https://doi.org/10.1016/j.jbc.2022.101601

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