title: The avilamycin resistance determinants AviRa and AviRb methylate 23S rRNA at the guanosine 2535 base and the uridine 2479 ribose.
abstract: Avilamycin is an orthosomycin antibiotic that has shown considerable potential for clinical use, although it is presently used as a growth promoter in animal feed. Avilamycin inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit. The ribosomes of the producer strain, Streptomyces viridochromogenes Tü57, are protected from the drug by the action of three resistance factors located in the avilamycin biosynthetic gene cluster. Two of the resistance factors, aviRa and aviRb, encode rRNA methyltransferases that specifically target 23S rRNA. Recombinant AviRa and AviRb proteins retain their activity after purification, and both specifically methylate in vitro transcripts of 23S rRNA domain V. Reverse transcriptase primer extension indicated that AviRa is an N-methyltransferase that targets G2535 within helix 91 of the rRNA, whereas AviRb modified the 2'-O-ribose position of nucleotide U2479 within helix 89. MALDI mass spectrometry confirmed the exact positions of each of these modifications, and additionally established that a single methyl group is added at each nucleotide. Neither of these two nucleotides have previously been described as a target for enzymatic methylation. Molecular models of the 50S subunit crystal structure show that the N-1 of the G2535 base and the 2'-hydroxyl of U2479 are separated by approximately 10 A, a distance that can be spanned by avilamycin. In addition to defining new resistance mechanisms, these data refine our understanding of the probable ribosome contacts made by orthosomycins and of how these antibiotics inhibit protein synthesis.
authors.affiliation: Department of Pharmaceutical Biology, Albert Ludwigs University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Irina
authors.initials: I
authors.lastname: Treede
authors.forename: Lene
authors.initials: L
authors.lastname: Jakobsen
authors.forename: Finn
authors.initials: F
authors.lastname: Kirpekar
authors.forename: Birte
authors.initials: B
authors.lastname: Vester
authors.forename: Gabriele
authors.initials: G
authors.lastname: Weitnauer
authors.forename: Andreas
authors.initials: A
authors.lastname: Bechthold
authors.forename: Stephen
authors.initials: S
authors.lastname: Douthwaite
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002240:Carbohydrate Sequence; D024881:Drug Resistance, Bacterial; D006151:Guanosine / Q000737:chemistry / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000378:metabolism; D014529:Uridine / Q000737:chemistry / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D009844:Oligosaccharides; D012338:RNA, Ribosomal, 23S; D006151:Guanosine; C006653:avilamycin; D008780:Methyltransferases; D014529:Uridine