Decoding Substrate\nSelectivity of an Archaeal RlmCD-like\nMethyltransferase Through Its Salient Traits

5-Methyluridine (m<sup>5</sup>U) rRNA modifications frequently\noccur at U747 and U1939 (Escherichia coli numbering) in domains II and IV of the 23S rRNA in Gram-negative\nbacteria, with the help of <i>S</i>-adenosyl-l-methionine\n(SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively.\nIn contrast, Gram-positive bacteria utilize a single SAM-dependent\nrRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain\narchaea, specifically within the <i>Thermococcales</i> group,\nhave been found to possess two genes encoding SAM-dependent archaeal\n(tRNA and rRNA) m<sup>5</sup>U (Arm<sup>5</sup>U) MTases. Among these,\na tRNA-specific Arm<sup>5</sup>U MTase (<sub>Pab</sub>TrmU54) has\nalready been characterized. This study focused on the structural and\nfunctional characterization of the rRNA-specific Arm<sup>5</sup>U\nMTase from the hyperthermophilic archaeon Pyrococcus\nhorikoshii (<i>Ph</i>RlmCD). An in-depth\nstructural examination revealed a dynamic hinge movement induced by\nthe replacement of the iron–sulfur cluster with disulfide bonds,\nobstructing the substrate-binding site. It revealed distinctive characteristics\nof <i>Ph</i>RlmCD, including elongated positively charged\nloops in the central domain and rotational variations in the TRAM\ndomain, which influence substrate selectivity. Additionally, the results\nsuggested that two potential mini-rRNA fragments interact in a similar\nmanner with <i>Ph</i>RlmCD at a positively charged cleft\nat the interface of domains and facilitate dual MTase activities akin\nto the protein RlmCD. Altogether, these observations showed that Arm<sup>5</sup>U MTases originated from horizontal gene transfer events,\nmost likely from Gram-positive bacteria.