In Gram-negative bacteria, resistance to β-lactam antibacterials is largely due to β-lactamases and is a growing public health threat. One of the most concerning β-lactamases to evolve in bacteria are the Class B enzymes, the metallo-β-lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel β-lactams and inhibitors. Two MBLs, the wild type IMP-1 with serine at position 262, and an engineered variant with valine at the same position (IMP-1-S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP-1-S262T) on the substrate spectrum. Here, we explore MBL sequence-structure-activity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP-1. Using site-directed mutagenesis, threonine was introduced at position 262, and the IMP-1-S262T enzyme, as well as the other two enzymes IMP-1 and IMP-1-S262V, were purified and kinetic constants were determined against a range of β-lactam antibacterials. Catalytic efficiencies (kcat /KM ) obtained with IMP-1-S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP-1 and IMP-1-S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in β-lactam turnover and support this approach to predict activities of certain novel MBL variants.
Submitter: Peter Oelschlaeger
Submission Date: Aug. 22, 2019, 12:36 p.m.
According to the standard numbering scheme (PMID 15215079) residue 196 is residue 262. ND indicates not detectable. S, I, and R indicate susceptible, intermediate, and resistant, respectively.
|Number of data points||234|
|Proteins||Metallo-beta-lactamase type 2|
|Assays/Quantities/Protocols||Experimental Assay: kcat ; Experimental Assay: Km ; Experimental Assay: kcat/Km ; Experimental Assay: MIC (absolute) ; Experimental Assay: MIC (relative) ; Experimental Assay: Resistance ; Derived Quantity: SD of kcat ; Derived Quantity: SD of Km ; Derived Quantity: SD of kcat/Km|
|Libraries||Activity data (Tables II & III)|