engleski

Aminoglycoside resistance methyltrasnferase Sgm: functional characterization and interaction with ribosomal RNA

Sgm methyltransferase (MTase) is a member of Arm family of enzymes that confer high level resistance to aminoglycoside antibiotics. This enzyme is originally found in Micromonospora zionensis, a natural producer of aminoglycoside antibiotic sisomicin. In recent time, members of this family have emerged in clinical practice, posing a serious threat to a successful treatment of severe bacterial infections. The available data on Sgm structure, function and mechanism of action are still very limited. In order to fight this fast spreading and highly dangerous mode of bacterial action against aminoglycosides, more information about these enzymes is needed. Recently, we modeled the Sgm structure and analyzed sequence– structure-function relationships by limited proteolysis and mutagenesis. In this work we defined the position of the Sgm methylation, as well as the substrate requirements of the enzyme. We also investigated how the presence of the Sgm enzyme affects the exponential growth of E. coli by growing cells in rich and minimal media at different temperatures. In competition growth assays we examined the ability of the cells carrying the Sgm methyltransferase to compete with the cells lacking the enzyme. 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. Also, E. coli cells expressing the Sgm grow slightly faster compared with the non expressing strain and the effect is shown both in competition experiments and in individual strain growth. Our results have set the basis for current kinetic 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.

aminoglycoside resistance; 16S rRNA methylation; Sgm methyltransferase; MALDI-TOF MS

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