Crystal structure of the bifunctional tRNA modification enzyme MnmC from Escherichia coli

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 (xm 5 U). These xm 5 U34-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 (cmnm 5 U) to 5-methylaminomethyl uridine (mnm 5 U). The C-terminal domain of MnmC (MnmC1) is responsible for the flavin adenine dinucleotide (FAD)-dependent deacetylation of cmnm 5 U to 5-aminomethyl uridine (nm 5 U), whereas the N-terminal domain (MnmC2) catalyzes the subsequent S-adenosyl-L-methionine-dependent methylation of nm 5 U, leading to the final product, mnm 5 U34. 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 cmnm 5 U 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 Aya Kitamura and Toru Sengoku contributed equally to this work. MnmC1 and MnmC2 domains are physically linked, they could catalyze the two consecutive reactions in a rather independent manner.

Characterization and Structure of the Aquifex aeolicus Protein DUF752

“…In summary, the overall structure of A. aeolicus DUF752, in complex with its cofactor AdoMet, is very similar to that of the MnmC2 domain of E. coli MnmC, as we determined previously (27). All of the observations mentioned above support the idea that the monofunctional methyltransferase DUF752 of A. aeolicus functions in a similar manner to E. coli MnmC2, fused to its oxidase domain MnmC1, in the bifunctional MnmC.…”

“…3 A ) shares high similarity with the N-terminal (MnmC2) domain of the bifunctional E. coli MnmC we reported previously (supplemental Table S1) (27). However, a few other MTases also show significant structural similarities, including guanidinoacetate N -methyltransferase interacting with a small cellular metabolite and various MTases interacting with RNA, such as TrmI (tRNA-m 1 A58 N -methyltransferase), mRNA cap guanine-N7 methyltransferase, RsmC (16 S rRNA-m 2 G1207 N -methyltransferase), and Trm1 (tRNA-m 2 2 G N -methyltransferase (supplemental Table S1).…”

“…The features include the partially conserved (G X )G X G sequence within motif I, followed by a conserved Asn residue at the end of the β4 region. In many class I MTases, including the N-terminal domain of MnmC and the present A. aeolicus DUF752, this motif constitutes an essential element of the nucleotide binding pocket (27). …”

“…The expression vector containing MnmC, bearing a mutation in the essential Asp-178 (D178A), was prepared using a QuikChange TM site-directed mutagenesis kit (Stratagene) and expressed in the same E. coli Rosetta2(DE3) strain (Merck Novagen). The purification procedures for these recombinant proteins were described previously (27). …”

Modification of the wobble uridine in bacterial and mitochondrial tRNAs reading NNA/NNG triplets of 2-codon boxes

“…67,68 The catalytic centers of MnmC(o) and MnmC(m) face opposite sides of the protein, thus favoring a model in which the 2 domains can function in a relatively independent manner. However, given the nature of their interface, the possibility that conformational changes within the entire MnmC protein may occur in vivo and facilitate the functional cooperation of the domains could not be excluded.…”