RudS: Bacterial Desulfidase Responsible for tRNA 4-Thiouridine De-modification

In this study, we present a comprehensive analysis of a widespread group of bacterial tRNA de-modifying enzymes, dubbed RudS, which consist of a TudS desulfidase fused to a domain of unknown function (DUF1722). RudS enzymes exhibit specific de-modification activity towards the 4-thiouridine modification (s4U) in tRNA molecules, as indicated by our experimental findings. Notably, heterologous overexpression of RudS genes in Escherichia coli leads to a significant reduction in tRNA 4-thiouridine content, highlighting the enzyme's role in tRNA s4U modification regulation. Through a combination of protein modeling, docking studies, and molecular dynamics simulations, we have identified amino acid residues involved in catalysis and tRNA binding. Experimental validation through targeted mutagenesis confirms the TudS domain as the catalytic core of RudS, with the DUF1722 domain facilitating tRNA binding in the anticodon region. Our results suggest a potential role for RudS tRNA modification eraser proteins in prokaryotic tRNA regulation pathways.