Significance A key step in protein synthesis is catalyzed by aminoacyl-tRNA synthetases (ARSs), which attach specific amino acids to tRNAs. Errors caused by ARS amino acid misactivation require proofreading activities encoded in editing domains. Approximately half of the ARSs possess intrinsic editing functions, and single-domain editing proteins also are encoded in some organisms. The present study used an in vivo screen coupled with in vitro assays to elucidate the substrate specificity of two free-standing editing domains, revealing that they are multifunctional Ser/Thr-tRNA deacylases with the ability to prevent errors caused by a number of ARSs. Trans -editing proteins with broad substrate specificities provide a growth advantage to microbes under conditions in which amino acid pools are altered and may represent novel targets.