engleski

Functional properties of aminoglycoside-resistant methyltransferase Sgm

Objective: Aminoglycoside resistance methyltransferase Sgm is a member of the Arm family of enzymes that confer high level resistance to 4, 6-disubstituted deoxystreptamines. Recently we have modelled the Sgm structure and analysed the sequence– function relationship by limited proteolysis and mutagenesis. Here we aimed at defining the conditions of methyl transfer, substrate requirements and kinetic parameters of the Sgm enzyme, as well as at its influence on the bacterial cell growth and survival. Methods: The sgm gene with N-terminal His-tag was cloned into expression vector pET-25b(+), expressed in Escherichia coli C41(DE3)pLysS cells and purified to homogeneity. The amount of co-purified cofactor S-adenosyl-methionine (AdoMet) was determined by measuring the A280/A260 ratio. Enzymatic properties of the Sgm enzyme were analysed by methylation test using variable concentrations of Mg(2+) and NH(4)Cl at different pH values. To define the substrate requirements we tried several substrates: entire ribosome, individual subunits and free 23S and 16S rRNA. Kinetic parameters were determined by varying concentrations of cofactor AdoMet and the substrate, while keeping the amount of the enzyme constant. We 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. Results: Nearly 10% of the purified enzyme contains bound AdoMet. The enzyme is most active in the presence of 10– 15 mM Mg(2+) and 90 mM NH(4)Cl at pH 7– 8. Small ribosomal subunits are readily methylated by the Sgm enzyme, but neither intact ribosome and 50S subunit or free 23S and 16S rRNA can serve as a substrate. The Sgm follows Michaelis-Menten kinetics with KM for AdoMet of 38 mM and kcat of 0.2 min-1. E. coli cells expressing the Sgm grow slightly faster compared with the non expressing strain. The effect is shown both in competition experiments and in individual strain growth. Conclusions: We defined the enzymatic properties and the substrate of the Sgm enzyme and observed its positive influence on cell growth. Our results 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 methyltransferases and thus fight the aminoglycoside resistance.

aminoglycoside antibiotics; 16S rRNA methylation; methyltransferase Sgm

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