An ancient type of MnmA protein is an iron-sulfur cluster-dependent sulfurtransferase for tRNA anticodons.

Abstract

Transfer RNA (tRNA) is an adaptor molecule indispensable for assigning amino acids to codons on mRNA during protein synthesis. 2-thiouridine (s2U) derivatives in the anticodons (position 34) of tRNAs for glutamate, glutamine, and lysine are post-transcriptional modifications essential for precise and efficient codon recognition in all organisms. s2U34 is introduced either by (i) bacterial MnmA/eukaryote mitochondrial Mtu1 or (ii) eukaryote cytosolic Ncs6/archaeal NcsA, and the latter enzymes possess iron-sulfur (Fe–S) cluster. Here, we report the identification of novel-type MnmA homologs containing three conserved Cys residues, which could support Fe–S cluster binding and catalysis, in a broad range of bacteria, including thermophiles, Cyanobacteria, Mycobacteria, Actinomyces, Clostridium, and Helicobacter. Using EPR spectroscopy, we revealed that Thermus thermophilus MnmA (TtMnmA) contains an oxygen-sensitive [4Fe–4S]-type cluster. Efficient in vitro formation of s2U34 in tRNALys and tRNAGln by holo-TtMnmA occurred only under anaerobic conditions. Mutational analysis of TtMnmA suggested that the Fe–S cluster is coordinated by the three conserved Cys residues (Cys105, Cys108, and Cys200), and is essential for its activity. Evolutionary scenarios for the sulfurtransferases, including the Fe–S cluster containing Ncs6/NcsA s2U thiouridylases and several distantly related sulfurtransferases, are proposed.