title: EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance.
abstract: Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.
authors.affiliation: Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Mann
authors.forename: L
authors.initials: L
authors.lastname: Xiong
authors.forename: A S
authors.initials: AS
authors.lastname: Mankin
authors.forename: A S
authors.initials: AS
authors.lastname: Chau
authors.forename: C A
authors.initials: CA
authors.lastname: Mendrick
authors.forename: D J
authors.initials: DJ
authors.lastname: Najarian
authors.forename: C A
authors.initials: CA
authors.lastname: Cramer
authors.forename: D
authors.initials: D
authors.lastname: Loebenberg
authors.forename: E
authors.initials: E
authors.lastname: Coates
authors.forename: N J
authors.initials: NJ
authors.lastname: Murgolo
authors.forename: F M
authors.initials: FM
authors.lastname: Aarestrup
authors.forename: R V
authors.initials: RV
authors.lastname: Goering
authors.forename: T A
authors.initials: TA
authors.lastname: Black
authors.forename: R S
authors.initials: RS
authors.lastname: Hare
authors.forename: P M
authors.initials: PM
authors.lastname: McNicholas
mesh_terms: D000617:Aminoglycosides; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics / Q000502:physiology; D016984:Enterococcus faecium / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; C001202:evernimicin; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase