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/Km ; Derived Quantity: SD of Km ; Derived Quantity: SD of kcat|
|Libraries||Activity data (Tables II & III)|
|Structure ID||Release Date||Resolution||Structure Title|
|6JED||2019-02-05T00:00:00+0000||1.57||Crystal structure of IMP-1 metallo-beta-lactamase in a complex with MCR|
|5Y5B||2017-08-08T00:00:00+0000||1.7||Crystal Structure Of IMP-1 Metallo-beta-lactamase|
|5HH4||2016-01-09T00:00:00+0000||2.0||Crystal structure of metallo-beta-lactamase IMP-1 in complex with a phosphonate-based inhibitor|
|1VGN||2004-04-27T00:00:00+0000||2.63||Structure-based design of the irreversible inhibitors to metallo--lactamase (IMP-1)|
|1DD6||1999-11-08T00:00:00+0000||2.0||IMP-1 METALLO BETA-LACTAMASE FROM PSEUDOMONAS AERUGINOSA IN COMPLEX WITH A MERCAPTOCARBOXYLATE INHIBITOR|
|4C1F||2013-08-12T00:00:00+0000||2.01||Crystal structure of the metallo-beta-lactamase IMP-1 with L-captopril|
|6JKA||2019-02-28T00:00:00+0000||2.01||Crystal structure of metallo-beta-lactamse, IMP-1, in complex with a thiazole-bearing inhibitor|
|4C1G||2013-08-12T00:00:00+0000||1.71||Crystal structure of the metallo-beta-lactamase IMP-1 with D-captopril|
|6ZYS||2020-08-02T00:00:00+0000||1.87||Structure of IMP-1 with 2-Mercaptomethyl-thiazolidine D-syn-1b|
|6LBL||2019-11-14T00:00:00+0000||1.68||Crystal structure of IMP-1 metallo-beta-lactamase in complex with NO9 inhibitor|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|100.0||A||Metallo-beta-lactamase type 2||P52699||BLAB_SERMA|