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Anticodon‐binding domain swapping in a nondiscriminating aspartyl‐tRNA synthetase reveals contributions to tRNA specificity and catalytic activity
Author(s) -
Chuawong Pitak,
Likittrakulwong Wirot,
Suebka Suwimon,
Wiriyatanakorn Nuttapon,
Saparpakorn Patchreenart,
Taweesablamlert Amata,
Sudprasert Wanwisa,
Hendrickson Tamara,
Svasti Jisnuson
Publication year - 2020
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.25881
Subject(s) - chimera (genetics) , transfer rna , enzyme , biochemistry , escherichia coli , biology , amino acid , aminoacyl trna synthetase , heterologous , chemistry , rna , microbiology and biotechnology , gene
The nondiscriminating aspartyl‐tRNA synthetase (ND‐AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNA Asp and tRNA Asn generating a correctly charged Asp‐tRNA Asp and an erroneous Asp‐tRNA Asn . 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 tRNA Asp over tRNA Asn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera‐N exhibited low catalytic activity toward tRNA Asp and was unable to aspartylate tRNA Asn . 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.