Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity.

Pitak Chuawong, Wirot Likittrakulwong, Suwimon Suebka, Nuttapon Wiriyatanakorn, Patchreenart Saparpakorn, Amata Taweesablamlert, Wanwisa Sudprasert, Tamara Hendrickson, Jisnuson Svasti

Proteins 2020 Sep

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The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNAAsp and tRNAAsn generating a correctly charged Asp-tRNAAsp and an erroneous Asp-tRNAAsn . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNAAsp over tRNAAsn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNAAsp and was unable to aspartylate tRNAAsn . The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity. © 2020 Wiley Periodicals, Inc.

Citation

Pitak Chuawong, Wirot Likittrakulwong, Suwimon Suebka, Nuttapon Wiriyatanakorn, Patchreenart Saparpakorn, Amata Taweesablamlert, Wanwisa Sudprasert, Tamara Hendrickson, Jisnuson Svasti. Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity. Proteins. 2020 Sep;88(9):1133-1142