Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

Abstract

The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfrÀ strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m 2 A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules.

Evolution of tRNAPhe:imG2 methyltransferases involved in the biosynthesis of wyosine derivatives in Archaea

“…The PAB2272 protein differs from NEQ228 by the absence of the D1 domain (de Crécy-Lagard et al 2010), yet both enzymes clearly demonstrate the dual specificity, suggesting that this domain is not responsible for the dual activity of the aTrm5a proteins. Similar observations have been made for the aTrm5b-type enzyme MJ0883 (Goto-Ito et al 2008), where D1 was shown to be dispensable for the methylation of the G37 tRNA substrate. Nevertheless, one cannot exclude that the D1 domain is important at physiological temperatures above 50°C, which was used for the methylation assays during our experiments.…”

“…No spots corresponding to m 1 G or imG2 were detected in the absence of SSO2439 (data not shown). Since the migration profile for likely pimG2pA on TLC plates was the same as that obtained for PAB2272 and NEQ228, we concluded that the SSO2439 protein exhibits the tRNA Amino acid residues of PAB2272 that are important for the formation of m1G and imG2 derivatives A previously conducted bioinformatic analysis (de Crécy-Lagard et al 2010) in conjunction with the structural work on Methanocaldococcus jannashii Trm5b (MJ0883) and Taw2 (MJ1157) as well as Pyrococcus horikoshii PH0793 enzyme Taw2 (Goto-Ito et al 2008Umitsu et al 2009) allowed identification of several amino acid residues, critical for the recognition of the S-AdoMet cofactor and the G/imG-14 substrate at position 37 in tRNA Phe . Here, we performed the bioinformatic analysis of the archaeal Trm5a/b/c family of enzymes (Fig.…”

“…Interestingly, while the crystal structure of aTrm5b (MJ0883) has been solved using either sinefungin (analog of S-AdoMet) or S-AdoMet as a methyl donor, and tRNA Cys or tRNA Leu as a substrate (Goto-Ito et al 2008, no crystal structure of any enzyme in complex with tRNA…”

Biosynthesis of wyosine derivatives in tRNAPhe of Archaea: role of a remarkable bifunctional tRNAPhe:m1G/imG2 methyltransferase

“…The first is Cfr from E. coli that normally mediates the single methylation of the 8-amidine carbon of A2503 of 23S rRNA to form 8-methyadenosine (m 8 A). However, when E. coli lacks the methyltransferase RlmN that catalyzes the methylation of the 2-amidine carbon of the same A2503 in 23S rRNA, Cfr can compensate such deficiency and catalyzes the formation of doubly methylated 2,8-dimethyladenosine (m 2,8 A) (Giessing et al 2009). Similarly, a single bifunctional methyltransferase NS5 of flaviviruses catalyzes the sequential methylation of the N 7 -atom of the 5 ′ -terminal guanosine and 2 ′ -hydroxyl group of the neighboring adenosine, resulting in the formation of the type 1 cap m 7…”

“…However, at variance with the yeast Tyw1, archaeal Taw1 does not have the flavin-binding domain but contains a second Fe-S cluster instead (Perche-Letuvée et al 2012). The corresponding recombinant Taw1 enzymes of Pyrococcus horikoschii (PH1705), Methanocaldococcus jannaschii (MJ0257), and Pyrococcus abyssi (PAB2039) have been purified; and in the two last cases, their enzymatic activities have been successfully demonstrated in vitro using pyruvate as the key substrate, AdoMet as the cofactor, and dithionite as the source of reducing power (Goto-Ito et al 2007;Young and Bandarian 2011;Perche-Letuvée et al 2012).…”