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: Resistance ; Experimental Assay: MIC (relative) ; Experimental Assay: MIC (absolute) ; Experimental Assay: kcat/Km ; Experimental Assay: Km ; Experimental Assay: kcat ; Derived Quantity: SD of kcat/Km ; Derived Quantity: SD of Km ; Derived Quantity: SD of kcat|
|Libraries||Activity data (Tables II & III)|
|Structure ID||Release Date||Resolution||Structure Title|
|1DD6||1999-11-08T00:00:00+0000||2.0||IMP-1 METALLO BETA-LACTAMASE FROM PSEUDOMONAS AERUGINOSA IN COMPLEX WITH A MERCAPTOCARBOXYLATE INHIBITOR|
|1VGN||2004-04-27T00:00:00+0000||2.63||Structure-based design of the irreversible inhibitors to metallo--lactamase (IMP-1)|
|1WUO||2004-12-08T00:00:00+0000||2.01||Crystal structure of metallo-beta-lactamase IMP-1 mutant (D81A)|
|1WUP||2004-12-08T00:00:00+0000||3.0||Crystal structure of metallo-beta-lactamase IMP-1 mutant (D81E)|
|2DOO||2006-05-01T00:00:00+0000||2.43||The structure of IMP-1 complexed with the detecting reagent (DansylC4SH) by a fluorescent probe|
|4C1F||2013-08-12T00:00:00+0000||2.01||Crystal structure of the metallo-beta-lactamase IMP-1 with L-captopril|
|4C1G||2013-08-12T00:00:00+0000||1.71||Crystal structure of the metallo-beta-lactamase IMP-1 with D-captopril|
|5EV6||2015-11-19T00:00:00+0000||1.98||Crystal structure of the native, di-zinc metallo-beta-lactamase IMP-1|
|5EV8||2015-11-19T00:00:00+0000||2.3||Crystal structure of the metallo-beta-lactamase IMP-1 in complex with the bisthiazolidine inhibitor D-CS319|
|5EWA||2015-11-20T00:00:00+0000||2.3||Crystal structure of the metallo-beta-lactamase IMP-1 in complex with the bisthiazolidine inhibitor L-VC26|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|100.0||A||Metallo-beta-lactamase type 2||P52699||BLAB_SERMA|