Abstract
Aminoacyl-tRNA synthetases maintain the fidelity during protein synthesis by selective activation of cognate amino acids at the aminoacylation site and hydrolysis of misformed aminoacyl-tRNAs at the editing site. Threonyl-tRNA synthetase (ThrRS) misactivates serine and utilizes an editing site cysteine (C182 in Escherichia coli) to hydrolyze Ser-tRNAThr. Hydrogen peroxide oxidizes C182, leading to Ser-tRNAThr production and mistranslation of threonine codons as serine. The mechanism of C182 oxidation remains unclear. Here we used a chemical probe to demonstrate that C182 was oxidized to sulfenic acid by air, hydrogen peroxide and hypochlorite. Aminoacylation experiments in vitro showed that air oxidation increased the Ser-tRNAThr level in the presence of elongation factor Tu. C182 forms a putative metal binding site with three conserved histidine residues (H73, H77 and H186). We showed that H73 and H186, but not H77, were critical for activating C182 for oxidation. Addition of zinc or nickel ions inhibited C182 oxidation by hydrogen peroxide. These results led us to propose a model for C182 oxidation, which could serve as a paradigm for the poorly understood activation mechanisms of protein cysteine residues. Our work also suggests that bacteria may use ThrRS editing to sense the oxidant levels in the environment.
Highlights
Aminoacyl-transfer ribonucleic acid synthetases are essential enzymes that ligate cognate amino acids to tRNAs, thereby providing the ribosome with correct aminoacyl-tRNAs as building blocks for protein synthesis [1]
Our previous studies show that hydrogen peroxide (H2O2) impairs the editing function of Escherichia coli threonyl-tRNA synthetase (ThrRS) and causes serine (Ser) misincorporation at threonine (Thr) codons [29]
DAz-2 was unable to detect RSOH formation in the C182A mutant of ThrRS treated with H2O2 or NaOCl (Figure 2), supporting the notion that C182 is the sensitive target for oxidation in ThrRS
Summary
Aminoacyl-transfer ribonucleic acid (tRNA) synthetases (aaRSs) are essential enzymes that ligate cognate amino acids to tRNAs, thereby providing the ribosome with correct aminoacyl-tRNAs (aa-tRNAs) as building blocks for protein synthesis [1]. AaRSs use pre- and post-transfer editing mechanisms to hydrolyze misactivated amino acids and misacylated aatRNAs, respectively [2,3]. Impairing post-transfer editing activity causes increased translational errors (mistranslation) in bacterial and eukaryotic cells [10,15,16]. Our previous studies show that hydrogen peroxide (H2O2) impairs the editing function of Escherichia coli threonyl-tRNA synthetase (ThrRS) and causes serine (Ser) misincorporation at threonine (Thr) codons [29]. The editing site of bacterial and eukaryotic ThrRSs contains a conserved cysteine residue (C182 in E. coli ThrRS) that is critical for the hydrolysis of misacylated Ser-tRNAThr [30,31]. We have shown that C182 is the target for H2O2-mediated oxidation, but how C182 is activated for oxidation remains unclear [29]
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