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.
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.
Department of Biological Sciences, 14 Science drive 4, National University of Singapore, Singapore.
Nilofer
N
Husain
Sonja
S
Obranic
Lukasz
L
Koscinski
J
J
Seetharaman
Fedora
F
Babic
Janusz M
JM
Bujnicki
Gordana
G
Maravic-Vlahovicek
J
J
Sivaraman
D000225:Adenine / Q000737:chemistry; D001483:Base Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007613:Kanamycin Resistance; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D023302:Protein Footprinting; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry* / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000737:chemistry; D012436:S-Adenosylmethionine / Q000737:chemistry
D029968:Escherichia coli Proteins; D012336:RNA, Ribosomal, 16S; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C554940:NpmA protein, E coli; D000225:Adenine