Evolutionarily conserved proteins MnmE and GidA catalyze the formation of two methyluridine derivatives at tRNA wobble positions

Abstract

The wobble uridine of certain bacterial and mitochondrial tRNAs is modified, at position 5, through an unknown reaction pathway that utilizes the evolutionarily conserved MnmE and GidA proteins. The resulting modification (a methyluridine derivative) plays a critical role in decoding NNG/A codons and reading frame maintenance during mRNA translation. The lack of this tRNA modification produces a pleiotropic phenotype in bacteria and has been associated with mitochondrial encephalomyopathies in humans. In this work, we use in vitro and in vivo approaches to characterize the enzymatic pathway controlled by the Escherichia coli MnmE•GidA complex. Surprisingly, this complex catalyzes two different GTP- and FAD-dependent reactions, which produce 5-aminomethyluridine and 5-carboxymethylamino-methyluridine using ammonium and glycine, respectively, as substrates. In both reactions, methylene-tetrahydrofolate is the most probable source to form the C5-methylene moiety, whereas NADH is dispensable in vitro unless FAD levels are limiting. Our results allow us to reformulate the bacterial MnmE•GidA dependent pathway and propose a novel mechanism for the modification reactions performed by the MnmE and GidA family proteins.

Bacillus subtilis exhibits MnmC-like tRNA modification activities

“…Analysis of nucleosides by reverse-phase HPLC was performed as described [11]. The nucleosides were identified according to their UV spectra, relative retention times, and by comparison with appropriate controls, including synthetic markers [7,9,26]. …”

“…In particular, modified nucleosides of the anticodon loop transform the loop architecture and dynamics to meet the requirements that the ribosome places on all tRNAs [2,4,5]. In Escherichia coli , the MnmEG complex, formed by the dimeric proteins MnmE and MnmG [6], modifies the wobble uridine (U34) of tRNA Lys UUU , tRNA Glu UUC , tRNA Gln UUG , tRNA Leu UAA , tRNA Arg UCU , and tRNA Gly UCC [7,8]. MnmEG catalyzes the incorporation of either an aminomethyl (nm) or a carboxymethylaminomethyl (cmnm) group at position 5 of U34 using ammonium or glycine as substrate (Figure 1) [7].…”

“…In Escherichia coli , the MnmEG complex, formed by the dimeric proteins MnmE and MnmG [6], modifies the wobble uridine (U34) of tRNA Lys UUU , tRNA Glu UUC , tRNA Gln UUG , tRNA Leu UAA , tRNA Arg UCU , and tRNA Gly UCC [7,8]. MnmEG catalyzes the incorporation of either an aminomethyl (nm) or a carboxymethylaminomethyl (cmnm) group at position 5 of U34 using ammonium or glycine as substrate (Figure 1) [7]. The MmEG products can be converted into a 5-methylaminomethyl (mnm 5 ) group through the action of the two-domain, bifunctional enzyme MnmC [9].…”

YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNALeu isoacceptors

“…Loss of anticodon modifications, particularly at the 34 wobble position, disrupts gene expression and affects a range of phenotypic traits including virulence, pathogenicity, and cellular response to stress (Karita et al 1997;Forsyth et al 2002;Gong et al 2004;Sha et al 2004;Shin et al 2009). Formation of the more complex nucleotide modifications involves a series of steps by different enzymes, and the pathways for the majority of these have been characterized (e.g., Hagervall et al 1987;Björk and Hagervall 2005;Ikeuchi et al 2006;Lundgren and Björk 2006;El Yacoubi et al 2009;Moukadiri et al 2009). …”

“…Formation of the 5-carboxymethylaminomethyl modification (cmnm) on the base of uridine-34 in tRNA Leu cmnm5UmAA by the enzymes MnmE and MnmG (formerly GidA) has recently been described in detail (Moukadiri et al 2009); however, identification of the 29-O-methyltransferase(s) that modifies nucleotide 34 in this and the tRNA Leu CmAA isoacceptor has remained elusive (Purta et al 2006). In this study, we have applied a comparative genomics approach to prioritize E. coli gene candidates that could encode the undiscovered 29-O-methyltransferase(s).…”