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TrmL is a <jats:styled-content style=\"fixed-case\">tRNA<\/jats:styled-content>\u2010modification enzyme that catalyzes the transfer of a methyl group to the 2\u2032\u2010hydroxyl group of the pyrimidines at the wobble position 34 in two <jats:styled-content style=\"fixed-case\">tRNA<jats:sup>L<\/jats:sup><\/jats:styled-content><jats:sup>eu<\/jats:sup> isoacceptors, but the mechanism remains elusive. In this study, we determined the crystal structure of TrmL from <jats:italic>Thermus thermophilus<\/jats:italic> (TtTrmL) to 1.7 \u00c5. The enzyme contains only the conserved minimal <jats:styled-content style=\"fixed-case\">SPOUT<\/jats:styled-content> fold, but displays distinct biochemical behavior from its <jats:italic>Escherichia coli<\/jats:italic> counterpart, EcTrmL. Interestingly, a fortuitous ligand of 5\u2032\u2010methylthioadenosine was consistently found at the <jats:styled-content style=\"fixed-case\">SAM<\/jats:styled-content>\u2010binding pocket in the crystal structures, which probably came from the expression host. Both TtTrmL and EcTrmL were capable of methylating each other's <jats:styled-content style=\"fixed-case\">tRNA<\/jats:styled-content> substrates, but the latter exhibited much higher activity than the former. Enzymatic activity assays showed that the reaction catalyzed by TtTrmL greatly depends on the reaction <jats:styled-content style=\"fixed-case\">pH<\/jats:styled-content> and is also affected by salt concentration. Via sequence alignment and structural superposition, we discovered that a universally conserved glutamate residue is likely to fulfill the role of the general base for the initial proton abstraction from the 2\u2032\u2010hydroxyl group of pyrimidines 34. Lastly, based on our structural and biochemical data, we proposed the dimer interface to be the <jats:styled-content style=\"fixed-case\">tRNA<\/jats:styled-content>\u2010binding site for TtTrmL.<\/jats:p><\/jats:sec><jats:sec><jats:title>Database<\/jats:title><jats:p>The atomic coordinates and structural factors have been deposited in the Protein Data Bank with accession number <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.rcsb.org\/pdb\/search\/structidSearch.do?structureId=5CO4\">5CO4<\/jats:ext-link>.<\/jats:p><\/jats:sec>","DOI":"10.1111\/febs.14120","type":"journal-article","created":{"date-parts":[[2017,5,25]],"date-time":"2017-05-25T17:15:44Z","timestamp":1495732544000},"page":"2251-2263","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Structural and biochemical insights into the 2\u2032\u2010<i>O<\/i>\u2010methylation of pyrimidines 34 in 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