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- MakaleAçık Erişim2018Scopus SJRQ3Effect of amino acid substitution in New Delhi metallo-β-lactamase on carbapenem susceptibility
Düzgün, Azer Özad
The aim of this work was investigation of clinically important amino acid substitutions of NDM-1 variants. A blaNDM-1 gene was cloned into expression vector pET100/D-TOPO. The sequence of NDM-1 variants with substituted amino acids was determined by ClustalW program. A pET100/D-TOPO + blaNDM-1 was used to generate the alanine mutations at different positions, such as NDM-2 (P28A), NDM-3 (D95A), NDM-4 (M154A), NDM-5 (V88A), NDM-7 (D130A), and NDM-9 (E152A). The mutant variants were transformed into Escherichia coli DH5α. Changes in the activities of alanine mutation variants were determined by E-test. All samples had 32 μg/ml MIC values against ampicillin. The 28th amino acid mutation sample had the highest MIC value against ceftazidime, whereas decreased MIC value for piperacillin. It was observed that the resistance to imipenem was increased in mutant variants D95A, M154A, D130A, and E152A, comparing with P28A and V88A. It was found that NDM-1 has 0.64 μg/ml and the 130th amino acid mutation sample has 0.75 μg/ml meropenem MIC value.
AtıfScopus5WoS5OpenAlex6İncele - MakaleYalnız künye2017Molecular docking analysis and determination of minimum inhibition concentration of VIM-38/R228S
Duzgun, Azer Ozad; Saral, Aysegul
Objective: The aim of this study was to determine the effects of residues at 228th positions of VIM-38 on minimum inhibition concentration (MIC) and molecular docking analysis. Materials and methods: blaVIM-38 gene was cloned into expression vector pET100/D-TOPO. Then pET100/D-TOPO-VIM-38 was used to generate the R228S mutation by site-directed mutagenesis. The mutant was transformed into E. coli Dh5α. Changes in the activity of mutation R228S was determined by E-test. Molecular docking analysis of the binding affinity of meropenem and imipenem with VIM-38 and VIM-38 R228S was performed using AutoDock Vina_1_1_2. Results: According to E-test results, pET100/D-TOPO-VIM-38 and pET100/D-TOPO-VIM-38/ R228S have same MIC value for amoxicillin, ticarcillin/clavulanic acid, ampicillin and ticarcillin. It was determined decrease in pET100/D-TOPO-VIM-38/ R228S of MIC for aztreonam, amikacin and cefixine. There are 3-fold deference in MIC between pET100/D-TOPO-VIM-38 and pET100/D-TOPO-VIM-38/ R228S for cefoxitin. pET100/D-TOPO-VIM-38 has 0.5 µg/ml and pET100/D-TOPO-VIM-38/ R228S has 0.125 µg/m for imipenem, we observed 4-fold deference. Also, there is about 170-fold deference between pET100/D-TOPO-VIM-38 (16 µg/ml) and pET100/D-TOPO-VIM-38/ R228S (0.094 µg/ml) for meropenem. VIM-38 and VIM-38 R228S have very close to the negative low free energies of binding of the meropenem and imipenem. Conclusion: These results showed that substitution (R228S) slightly have effect on enzyme activity.