Published on Oct. 1, 2024 in Biochemistry volume 63 (19).
PubMed ID: 39350642
DOI: 10.1021/acs.biochem.4c00401
Abstract:
5-Methyluridine (m<sup>5</sup>U) rRNA modifications frequently occur at U747 and U1939 (<i>Escherichia coli</i> numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of <i>S</i>-adenosyl-l-methionine (SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively. In contrast, Gram-positive bacteria utilize a single SAM-dependent rRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain archaea, specifically within the <i>Thermococcales</i> group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m<sup>5</sup>U (Arm<sup>5</sup>U) MTases. Among these, a tRNA-specific Arm<sup>5</sup>U MTase (<sub>Pab</sub>TrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm<sup>5</sup>U MTase from the hyperthermophilic archaeon <i>Pyrococcus horikoshii</i> (<i>Ph</i>RlmCD). An in-depth structural examination revealed a dynamic hinge movement induced by the replacement of the iron-sulfur cluster with disulfide bonds, obstructing the substrate-binding site. It revealed distinctive characteristics of <i>Ph</i>RlmCD, including elongated positively charged loops in the central domain and rotational variations in the TRAM domain, which influence substrate selectivity. Additionally, the results suggested that two potential mini-rRNA fragments interact in a similar manner with <i>Ph</i>RlmCD at a positively charged cleft at the interface of domains and facilitate dual MTase activities akin to the protein RlmCD. Altogether, these observations showed that Arm<sup>5</sup>U MTases originated from horizontal gene transfer events, most likely from Gram-positive bacteria.