RESUMEN
In this study, we present an extensive 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 1722 (DUF1722). RudS enzymes exhibit specific de-modification activity towards the 4-thiouridine modification (s4U) in tRNA molecules, as indicated by our experimental findings. The heterologous overexpression of RudS genes in Escherichia coli significantly reduces the tRNA 4-thiouridine content and diminishes UVA-induced growth delay, indicating the enzyme's role in regulating photosensitive tRNA s4U modification. 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 that RudS tRNA modification eraser proteins may play a role in regulating tRNA during prokaryotic stress responses.
RESUMEN
In all domains of life, transfer RNAs (tRNAs) contain post-transcriptionally sulfur-modified nucleosides such as 2- and 4-thiouridine. We have previously reported that a recombinant [4Fe-4S] cluster-containing bacterial desulfidase (TudS) from an uncultured bacterium catalyzes the desulfuration of 2- and 4-thiouracil via a [4Fe-5S] cluster intermediate. However, the in vivo function of TudS enzymes has remained unclear and direct evidence for substrate binding to the [4Fe-4S] cluster during catalysis was lacking. Here, we provide kinetic evidence that 4-thiouridine-5'-monophosphate rather than sulfurated tRNA, thiouracil, thiouridine or 4-thiouridine-5'-triphosphate is the preferred substrate of TudS. The occurrence of sulfur- and substrate-bound catalytic intermediates was uncovered from the observed switch of the S = 3/2 spin state of the catalytic [4Fe-4S] cluster to a S = 1/2 spin state upon substrate addition. We show that a putative gene product from Pseudomonas putida KT2440 acts as a TudS desulfidase in vivo and conclude that TudS-like enzymes are widespread desulfidases involved in recycling and detoxifying tRNA-derived 4-thiouridine monophosphate nucleosides for RNA synthesis.