(1) Background: Metallo-β-lactamases (MBLs) have raised concerns due to their ability to inactivate carbapenems and newer generation cephalosporins and the absence of clinically available MBL inhibitors. Their genes are often transferred horizontally, and the number of MBL variants has grown exponentially, with many newer variants showing enhanced enzyme activity or stability. In this study, we investigated a closely related group of variants from the IMP family that all contain the combination of mutations S115T and S119G relative to IMP-1. (2) Methods: The effects of each individual mutation and their combination in the IMP-1 sequence background in comparison to IMP-1 were investigated. Their ability to confer resistance and their in-cell expression levels were determined. All enzymes were purified, and their secondary structure and thermal stability were determined with circular dichroism. Their Zn(II) content and kinetic constants with a panel of β-lactam antibiotics were determined. (3) Results: All four enzymes were viable and conferred resistance to all antibiotics tested except aztreonam. However, the single-mutant enzymes were slightly deficient, IMP-1S115T due to decreased enzyme activity and IMP-1-S119G due to decreased thermal stability and expression, while the double mutant did not show these defects. (4) Conclusions: These observations suggest that S119G was acquired due to its increased enzyme activity and S115T to suppress the thermal stability and expression defect introduced by S119G.
Submitter: Paulie Dang
Submission Date: Jan. 14, 2020, 11:51 a.m.
Explanation of amino acid numbering: Residues 76 and 80 that were mutated in this study are called residues 115 and 119, respectively, according to the standard numbering scheme (PMID 15215079). ND indicates not detectable.
|Number of data points||404|
|Proteins||Metallo-beta-lactamase type 2|
|Assays/Quantities/Protocols||Experimental Assay: Expression Level ; Experimental Assay: Resistance (SIR) ; Experimental Assay: Tm ; Experimental Assay: Inhibition Zone Diameters ; Experimental Assay: kcat/Km ; Experimental Assay: Kcat ; Experimental Assay: Km ; Experimental Assay: MIC (Relative) ; Experimental Assay: MIC (Absolute) ; Derived Quantity: SD of kcat/Km ; Derived Quantity: SD of Km ; Derived Quantity: SD of Kcat ; Derived Quantity: SD of Tm ; Derived Quantity: SD of Inhibition Zone Diameters|
|Libraries||Cell Protein Expression Level ; Agar Diffusion Assay ; Melting Temperature Data ; Activity Data|