Identification of a novel 5-aminomethyl-2-thiouridine methyltransferase in tRNA modification

Abstract

The uridine at the 34th position of tRNA, which is able to base pair with the 3'-end codon on mRNA, is usually modified to influence many aspects of decoding properties during translation. Derivatives of 5-methyluridine (xm5U), which include methylaminomethyl (mnm-) or carboxymethylaminomethyl (cmnm-) groups at C5 of uracil base, are widely conserved at the 34th position of many prokaryotic tRNAs. In Gram-negative bacteria such as Escherichia coli, a bifunctional MnmC is involved in the last two reactions of the biosynthesis of mnm5(s2)U, in which the enzyme first converts cmnm5(s2)U to 5-aminomethyl-(2-thio)uridine (nm5(s2)U) and subsequently installs the methyl group to complete the formation of mnm5(s2)U. Although mnm5s2U has been identified in tRNAs of Gram-positive bacteria and plants as well, their genomes do not contain an mnmC ortholog and the gene(s) responsible for this modification is unknown. We discovered that MnmM, previously known as YtqB, is the methyltransferase that converts nm5s2U to mnm5s2U in Bacillus subtilis through comparative genomics, gene complementation experiments, and in vitro assays. Furthermore, we determined X-ray crystal structures of MnmM complexed with anticodon stem loop of tRNAGln. The structures provide the molecular basis underlying the importance of U33-nm5s2U34-U35 as the key determinant for the specificity of MnmM.

Graphical Abstract

A novel methyltransferase MnmM, formerly known as YtqB, has been identified to be involved in the biosynthesis of mnm5s2U in tRNAs of Gram-positive bacteria and plants.

Figure 1.

Biosynthetic pathway of mnm⁵s²U modification in E.coli. The s²U is modified to cmnm⁵s²U or nm⁵s²U by MnmEG complex with glycine or ammonium as a substrate, respectively. Then, MnmC(o) domain of bifunctional enzyme MnmC removes carboxymethyl group of cmnm⁵s²U to form nm⁵s²U. Finally, MnmC(m) domain methylates nm⁵s²U using SAM as methyl donor to generate mnm⁵s²U.

Figure 2.

Comparative genomics searching for MnmC-like enzyme(s). (Left) A Venn diagram representing the distribution of 198 genes commonly present in Bacillus subtilis subtilis 168, Staphylococcus aureus NCTC 8325, and Acetoanaerobium sticklandii DSM 519, but absent in Escherichia coli K-12 MG1655. (Right) A scheme describing the process for identifying the candidate gene(s) responsible for 5-mnm modification in this study. ORF, open reading frame; IMG/M, Integrated Microbial Genomes & Microbiomes system; COG, Clusters of Orthologous Genes.

Figure 3.

Gene complementation and in vitro assays of B. subtilis YtqB and its orthologs. (A) HPLC analyses of gene complementation experiments showing the xm⁵s²U contents of bulk tRNA extracted from B. subtilis wild-type, ΔytqB, ΔytqB transformed with a plasmid pHT-bs-ytqB, and ΔmnmG strains. (B) Extracted ion chromatograph (EIC) corresponding to an m/z value of mnm⁵s²U (m/z 304.09617 ± 0.01) from B. subtilis wild-type, ΔytqB, and ΔytqB transformed with a plasmid pHT-bs-ytqB strains. N.D., not detected. (C) Representative MS1 and MS2 spectra of mnm⁵s²U and fragmentation patterns of mnm⁵s²U. (D) HPLC analyses of in vitro methylation assay of heterologously expressed YtqBs of various species with nm⁵s²U-containing bulk tRNA extracted from B. subtilis ΔytqB strain. Reaction samples without enzyme was used as a negative control (control). bs, B. subtilis; sa, S. aureus; at, A. thaliana; os, O. sativa.

Figure 4.

Overall view of tRNA-MnmM interactions. The structure of MnmM-SAM-ASL complex. MnmM dimer is shown in cartoons, ASL in sticks, and SAM in balls. Fourier difference maps (2Fo – Fc) contoured at 1.0-σ show electron densities around ASL bound to (A) saMnmM (cyan) and (B)bsMnmM (navy). Electrostatic potential (blue is positive, red is negative, and white is neutral) is mapped on the surfaces of saMnmM-SAM-ASL (C, E) and bsMnmM-SAM-ASL (D, F).

Figure 5.

Base flipping of U33 and U34 in anticodon loop upon binding to saMnmM. (A) Active site of saMnmM-SAM-ASL highlighting the interactions with U33, U34, and U35 of bound ASL (carbon atoms in orange) and SAM (carbon atoms in grey), compared with a ‘canonical’ tRNA (4V7M, purple). Bases of both U33 and U34 are flipped-out in the ASL-saMnmM complex structure. (B) Close-up of the active site showing the molecular interactions among the anticodon loop of tRNA, saMnmM and SAM. (C) Schematic diagram of interactions between the ASL with amino acid residues of saMnmM. Oxygen is shown in red, nitrogen in blue, and sulfur in yellow.

Figure 6.

Mutagenesis assays of bsMnmM. (A) Conserved residues in bsMnmM and saMnmM. The residues targeted for site-directed mutagenesis are colored in red. (B) The conversion rate between nm⁵s²U to mnm⁵s²U was plotted, using the wild-type and mutant MnmMs, nm⁵s²U-rich tRNA extracted from B. subtilis ΔytqB, and SAM. Activities of K11E, D34A, N101D, Y104A, K110E and H144A were very low as those of N101A and Y141F, and were excluded for clarity. Relative activities of all mutants are summarized in the bottom table, where +++ denotes the highest, ++ for modest, + for marginal and – for the lowest activity.

Figure 7.

A proposed reaction mechanism for the methylation of bsMnmM. (A) A close-up of the active site of bsMnmM using a model with nm⁵s²U34 bearing tRNA. Hydrogen bonds are shown as dashed lines and the distances are labeled in Å. (B) In the first step, the oxyanionic form of the amide side chain of N98 (N101) deprotonates from the methyl ammonium group of nm⁵s²U34. Next, the activated amine in nm⁵s²U34 attacks the S-methyl group of SAM to complete the formation of mnm⁵s²U34 and SAH. The dotted lines represent hydrogen bonds. Ade, adenosine; hcy, homocysteine.