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Understanding the determinants of substrate specificity in IMP family metallo‐β‐lactamases: The importance of residue 262

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.