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Interaction of Aminoglycoside Resistance Methyltransferase Sgm with Ribosomal RNA

OBJECTIVES: Aminoglycoside resistance methyltransferase (MTase) Sgm is a member of Arm family of MTases that confer high level resistance to aminoglycoside antibiotics. Some members of Arm family have been shown to act as m7G 1405 methyltransferases (16S rRNA, E. coli numbering). In our previous work, we modeled the Sgm structure and analyzed sequence– structure-function relationships by limited proteolysis and mutagenesis. Our further aim is to define the exact position, nature and extent of the Sgm methylation, as well as the substrate requirements of the enzyme. METHODS: Ribosomes or ribosomal subunits were purified from E. coli C41(DE3) cells carrying the wild-type or mutant sgm variants in the pET-25b(+) vector. rRNA was extracted from ribosomes or ribosomal subunits. To define the substrate requirements we tried several substrates: entire ribosomes, individual subunits and free 23S and 16S rRNA. The substrate of Sgm MTase was determined by both MALDI-TOF mass spectrometry (MS) and methylation assay. The identity of methylated nucleotide was analysed by primer extension analysis. rRNA methylated with the wild type or the mutant forms of Sgm enzyme was analysed by MALDI-TOF MS. RESULTS: 30S subunits were shown to be the substrate for Sgm MTase, while entire ribosome or naked RNA could not be methylated. Both primer extension analysis and MALDI-TOF MS confirmed that the methylation occur at G1405 in 16S rRNA. It was shown that ribosomes are completely modified in the cells expressing Sgm protein. Methylation pattern of all the Sgm mutants was determined using MALDI-TOF MS. It was shown that the extent of methylation at C1407 is decreased in the cells expressing Sgm, suggesting possible steric interference with YebU. CONCLUSIONS: We defined the Sgm substrate and enzyme methylation site and described the interaction of the wild type and mutant forms of the enzyme with ribosomal RNA. Our results will set the basis for more detailed studies of the reaction mechanism and recognition elements in 16S rRNA, which will all help to find the specific inhibitor of Arm MTases and thus fight the aminoglycoside resistance.

Arm methyltransferases; 16S rRNA methylation; G1405; antibiotic resistance

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