Erschienen:
Springer Science and Business Media LLC, 2018
Erschienen in:Scientific Reports
Sprache:
Englisch
DOI:
10.1038/s41598-018-27118-5
ISSN:
2045-2322
Entstehung:
Anmerkungen:
Beschreibung:
<jats:title>Abstract</jats:title><jats:p>Dnmt2 methylates cytosine at position 38 of tRNA<jats:sup>Asp</jats:sup> in a variety of eukaryotic organisms. A correlation between the presence of the hypermodified nucleoside queuosine (Q) at position 34 of tRNA<jats:sup>Asp</jats:sup> and the Dnmt2 dependent C38 methylation was recently found <jats:italic>in vivo</jats:italic> for <jats:italic>S. pombe</jats:italic> and <jats:italic>D. discoideum</jats:italic>. We demonstrate a direct effect of the Q-modification on the methyltransferase catalytic efficiency <jats:italic>in vitro</jats:italic>, as V<jats:sub>max</jats:sub>/K<jats:sub>0.5</jats:sub> of purified <jats:italic>S. pombe</jats:italic> Dnmt2 shows an increase for <jats:italic>in vitro</jats:italic> transcribed tRNA<jats:sup>Asp</jats:sup> containing Q34 to 6.27 ∗ 10<jats:sup>–3</jats:sup> s<jats:sup>−1</jats:sup> µM<jats:sup>−1</jats:sup> compared to 1.51 ∗ 10<jats:sup>–3</jats:sup> s<jats:sup>−1</jats:sup> µM<jats:sup>−1</jats:sup> for the unmodified substrate. Q34tRNA<jats:sup>Asp</jats:sup> exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA. In order to get insight into the structural basis for the Q-dependency, the crystal structure of <jats:italic>S. pombe</jats:italic> Dnmt2 was determined at 1.7 Å resolution. It closely resembles the known structures of human and <jats:italic>E. histolytica</jats:italic> Dnmt2, and contains the entire active site loop. The interaction with tRNA was analyzed by means of mass-spectrometry using UV cross-linked Dnmt2-tRNA complex. These cross-link data and computational docking of Dnmt2 and tRNA<jats:sup>Asp</jats:sup> reveal Q34 positioned adjacent to the S-adenosylmethionine occupying the active site, suggesting that the observed increase of Dnmt2 catalytic efficiency by queuine originates from optimal positioning of the substrate molecules and residues relevant for methyl transfer.</jats:p>