Nucleotide 34 in tRNA is extensively modified to ensure translational fidelity and efficacy in cells. The deamination of adenosine at this site catalyzed by the enzyme TadA gives rise to inosine (I), which serves as a typical example of the wobble hypothesis due to its diverse basepairing capability. However, recent studies have shown that tRNAArgACG in Mycoplasma capricolum contains unmodified adenosine, in order to decode the CGG codon. The structural basis behind the poorly performing enzyme M. capricolum TadA (McTadA) is largely unclear. Here we present the structures of the WT and a mutant form of McTadA determined at high resolutions. Through structural comparison between McTadA and other active TadA enzymes as well as modeling efforts, we found that McTadA presents multiple structural conflicts with RNA substrates and thus offered support to previous studies from a structural perspective. These clashes would potentially lead to reduced substrate binding affinity of McTadA, consistent with our in vitro deamination activity and binding assays. To rescue the deamination activity of McTadA, we carried out two rounds of protein engineering through structure-guided design. The unsuccessful attempts of the activity restoration could be attributed to the altered dimer interface and stereo hindrance from the non-catalytic subunit of McTadA, which could be the inevitable outcome of the natural evolution. Our study provides structural insight into an alternative decoding and evolutionary strategy by a compromised TadA enzyme at a molecular level.
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Cover Image
Cover Image
Glutamine transport across cell membranes is performed by a variety of transporters, including the alanine serine cysteine transporter 2 (ASCT2). The substrate-binding site of ASCT2 was proposed to be specific for small amino acids with neutral side chains, excluding basic substrates such as lysine. In this issue Ndaru and co-workers (pp. 1443–1457) expand the substrate specificity of ASCT2 to include amino acid substrates with positively charged side chains. The image shows the docking pose of L-DAB and L-DAP, from the side view of the ASCT2 homology model in the outward-open conformation.The image was provided by Christof Grewer.
Structure of a tRNA-specific deaminase with compromised deamination activity
Huijuan Liu, Saibin Wu, Dewei Ran, Wei Xie; Structure of a tRNA-specific deaminase with compromised deamination activity. Biochem J 30 April 2020; 477 (8): 1483–1497. doi: https://doi.org/10.1042/BCJ20190858
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