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.

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria

“…Bioinformatic analysis shows the occurrence of diverse biochemical strategies for hypermodification of U34 in tRNA Glu, Gln, Lys in bacteria. Our results from the S. mutans and S. pneumoniae ΔmnmM strains confirm the functional assignment of Cho et al (22) that MnmM is required in the SAM-dependent methylation reaction nm 5 s 2 U to form mnm 5 s 2 U. Our analysis from B. subtilis ΔmnmM, however, did not show the same requirement, suggesting that, in this organism, other methylases may provide functional replacement under certain growth conditions.…”

“…The genomic co-localization of ytqA and mnmM observed in S. mutans is also observed in B. subtilis . Recently, MnmM has been identified as the SAM-dependent methyltransferase catalyzing the final step in mnm 5 s 2 U synthesis (22) In this report, Cho et showed that nmnM inactivation resulted in the loss of mnm 5 s 2 U with concomitant accumulation of nm 5 sU, a phenotype similar to that of S. mutans . However, earlier studies from the Armengod laboratory had shown that inactivation of mnmM ( ytqB ) does not eliminate the synthesis of mnm 5 s 2 U tRNA (20).…”

“…Analysis of the distribution of the known mnm 5 s 2 U34 synthesis protein family members in the bacterial genomes covered in the COG database revealed a patchy distribution for the latter part of the pathway (Fig. 2 First, in the subset of organisms that only use the ammonia pathway to directly synthesize nm 5 s 2 U, no MnmC1 is required, and the subsequent methylation to mnm 5 s 2 U can be performed by MnmC2 family proteins, like in A. aeolicus (9) or possibly by YtqB/MnmM proteins (22).…”

“…More recently, YtqB (MnmM) was identified as the enzyme responsible for the conversion of nm 5 s 2 U into mnm 5 s 2 U, performing an MnmC2-like reaction (22). This assignment was attributed to the depletion of mnm 5 s 2 U accompanied by the accumulation of nm 5 s 2 U tRNA in B. subtilis mnmM strain.…”

“…This assignment was attributed to the depletion of mnm 5 s 2 U accompanied by the accumulation of nm 5 s 2 U tRNA in B. subtilis ΔmnmM strain. Trans-complementation of not only B. subtilis mnmM , but also Staphylococcus aureus and Arabidopsis thaliana mnmM orthologs restored mnm 5 s 2 U levels (22). Furthermore, according to Cho and collaborators, B. subtilis mnmM expression in E. coli ΔmnmC shows detectable levels of mnm 5 s 2 U but at levels significantly lower than those of the wild type.…”