Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit

Abstract

NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin–apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics.

Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase

“…5A). As this ␣-helical ␤5/6 linker structure appears to be unique to NpmA among the known m 1 A1408 methyltransferase structures (19)(20)(21)(22), we individually replaced two central residues, A148 and E149, with proline to test whether disrupting the ␣-helix would impact NpmA activity. However, both variants conferred wild-type MICs ( Table 1), suggesting that the precise structure formed by the ␤5/6 linker is not critical for NpmA activity.…”

Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host

“…To study the putative biological cost associated with expression of resistance, the armA and npmA genes and their inactive point mutants armA * and npmA * were cloned with their own promoters in the stable low-copy-number plasmid pGB2 (Churchward et al 1984; Supplemental Table S1) into E. coli. Methyltransferase mutants unable to bind the S-adenosyl-Lmethionine cofactor were constructed by site-directed mutagenesis; for ArmA * , Lys 180 was changed to Ala (Schmitt et al 2009), and for NpmA, Asp 30 and Asp 55 were both changed to Ala (Husain et al 2011). …”

“…As opposed to ArmA, the interaction of NpmA with the 30S subunit does not induce any global structural rearrangement but rather only local changes (Husain et al 2011). It has been proposed that nucleotide A1408 may be flipped out and more solvent-accessible in complex with NpmA than in the 30S subunit before modification (Husain et al 2011).…”

“…It has been proposed that nucleotide A1408 may be flipped out and more solvent-accessible in complex with NpmA than in the 30S subunit before modification (Husain et al 2011). Hence, it is likely that interference between NpmA and RsmF, which methylate adjacent nucleotides, could be due to a clash between exogenous and endogenous methyltransferases that affects access to their target bases A1408 and C1407.…”

“…It was shown that mutations in the decoding center promote stop codon readthrough and frameshifting during elongation (O'Connor et al 1997). Lack of post-transcriptional modification also affects translation initiation, frameshifting, and codon readthrough (Kimura and Suzuki 2010;Husain et al 2011). However, to the best of our knowledge, the effect of resistance methylation at G1405 and A1408 on ribosome function has not yet been studied.…”