engleski

Sgm methyltransferase exhibits a unique ribosomal A site binding pattern among Arm family of enzymes.

Sgm methyltransferase was isolated from the natural producer of aminoglycoside antibiotic G-52 (6-N-methyl-sisomicin), Micromonospora zionensis. This enzyme is a member of Arm family of enzymes and modifies nucleotide G1405 in the A site of 16S rRNA, causing highlevel resistance to aminoglycoside antibiotics. Recently, members of Arm family of enzymes were found to be spreading by horizontal transfer in growing number of clinical strains, which significantly reduces the odds of a successful treatment of various bacterial infections. In this work, we explored the ribosomal A site binding pattern of the Sgm methyltransferase from the natural producer of aminoglycosides and the Arm enzymes isolated from clinical pathogens, ArmA, RmtA, RmtB, RmtC and RmtD. In our experiments we introduced various 16S rRNA mutations in E. coli strain carrying only one copy of 16S rRNA gene. Those cells had a uniform population of mutated ribosomes. We investigated the ability of these cells to grow in the presence of various concentrations of aminoglycoside kanamycin. We then introduced actively expressing Arm methyltransferases into these cells and monitored the impact of the mutations on the methyltransferase activity by determining minimal inhibitory concentration of kanamycin for these cells and analyzing the target nucleotide methylation with primer extension. Our results indicate that the mutations introduced in the A site of 16S rRNA affect the interaction between aminoglycoside resistance methyltransferase Sgm and bacterial ribosome. However, Arm methyltransferases isolated from clinical pathogens do not show the same ribosomal binding pattern, which suggests that Sgm methyltransferase exhibits a unique ribosomal A site binding pattern among Arm enzymes. So even though Arm enzymes from clinical strains and natural producer of aminoglycosides methylate the same target nucleotide, thereby causing high-level aminoglycoside resistance, their mode of action is slightly different. It is therefore of great importance to functionally characterize more members of the family in order to construct effective inhibitors of the members of the whole Arm family, thus fighting aminoglycoside resistance.

aminoglycoside resistance; methyltransferases; Arm; Kam; Ribosome; A site; mutagenesis

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