Abstract
Post-transcriptional modifications of bases within the transfer RNAs (tRNA) anticodon significantly affect the decoding system. In bacteria and eukaryotes, uridines at the wobble position (U34) of some tRNAs are modified to 5-methyluridine derivatives (xm⁵U). These xm⁵U34-containing tRNAs read codons ending with A or G, whereas tRNAs with the unmodified U34 are able to read all four synonymous codons of a family box. In Escherichia coli (E.coli), the bifunctional enzyme MnmC catalyzes the two consecutive reactions that convert 5-carboxymethylaminomethyl uridine (cmnm⁵U) to 5-methylaminomethyl uridine (mnm⁵U). The C-terminal domain of MnmC (MnmC1) is responsible for the flavin adenine dinucleotide (FAD)-dependent deacetylation of cmnm⁵U to 5-aminomethyl uridine (nm⁵U), whereas the N-terminal domain (MnmC2) catalyzes the subsequent S-adenosyl-L-methionine-dependent methylation of nm⁵U, leading to the final product, mnm⁵U34. Here, we determined the crystal structure of E.coli MnmC containing FAD, at 3.0 Å resolution. The structure of the MnmC1 domain can be classified in the FAD-dependent glutathione reductase 2 structural family, including the glycine oxidase ThiO, whereas the MnmC2 domain adopts the canonical class I methyltransferase fold. A structural comparison with ThiO revealed the residues that may be involved in cmnm⁵U recognition, supporting previous mutational analyses. The catalytic sites of the two reactions are both surrounded by conserved basic residues for possible anticodon binding, and are located far away from each other, on opposite sides of the protein. These results suggest that, although the MnmC1 and MnmC2 domains are physically linked, they could catalyze the two consecutive reactions in a rather independent manner.