Characterization of 5-aminomethyl-2-thiouridine tRNA modification biosynthetic pathway in Bacillus subtilis

Abstract

Post-transcriptional modifications, particularly at the uridine at position 34 of tRNA (known as the wobble uridine), play a crucial role in enhancing the accuracy and efficiency of the decoding system. 5-methyluridine derivatives (xm5U), including methylaminomethyl (mnm-) or carboxymethylaminomethyl (cmnm-) groups at the C5 atom of a uracil base, are widely conserved in prokaryotes at position 34 of tRNA and play a crucial role in reading NNA/G codons. In Escherichia coli (E. coli) tRNA, 5-methylaminomethyl(-2-thio)uridine (mnm5(s2)U) is modified through sequential enzymatic reactions. The MnmE-MnmG enzyme complex initially converts wobble uridine into 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm5(s2)U) through the glycine pathway. The bifunctional MnmC protein further modifies cmnm5(s2)U to mnm5(s2)U. The FAD-dependent oxidase domain (MnmC(o)) transforms cmnm5(s2)U into 5-aminomethyluridine (nm5(s2)U). Following this, the S-adenosylmethionine-dependent methyltransferase domain (MnmC(m)) methylates the free amino group, resulting in mnm5(s2)U34. Although mnm5(s2)U modification have been identified in Bacillus subtilis (B. subtilis), the presence of MnmC orthologs remains elusive. Here, I characterized both MnmC(o) and MnmC(m)-like enzymes in B. subtilis, namely YurR and MnmM, through a comparative genomics, and confirmed their biochemical activities through complementation and in vitro assays. YurR, an FAD-dependent oxidoreductase, converts cmnm5(s2)U to nm5(s2)U and MnmM, a methyltransferase, converts nm5(s2)U to mnm5(s2)U. Additionally, the orthologs from other species were capable of producing mnm5(s2)U synthesis. Moreover, I determined the X-ray crystal structures of YurR and MnmM. YurR structure suggest glycine moiety binding residue through structural comparison with other oxidoreductase enzymes and also propose tRNA binding site by electrostatic calculation. In complexed structure of MnmM with the anticodon stem-loop of tRNAGlu, conformational changes in both the tRNA and protein are observed. The U33-U34 nucleotides in tRNA are flipped out towards the active site, and in the protein, loops that are disordered in the SAH bound structure interact with the tRNA. Site-directed mutagenesis assays assist in understanding the role of individual amino acid residues in both enzymes. These collective results strongly support the conclusion that YurR and MnmM are accountable for the biosynthesis of mnm5(s2)U in B. subtilis.