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

Abstract

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.

Keywords: IMP-1 antibody; antibiotic resistance; enzyme evolution; metallo-β-lactamase; point mutation.

Figure 1

IMP-1 active site with Zn(II) ligands and residue 262. Serine 262 is highlighted with a red circle. The six remaining residues are Zn(II) ligands coordinating their respective Zn(II) ion (depicted as gray spheres). The enzyme backbone is represented as a cyan cartoon and residues as sticks with atoms colored in green (carbon), red (oxygen), blue (nitrogen), and yellow (sulfur). The top left Zn(II) ion is Zn1 in the 3H site and the bottom right one Zn2 in the DCH site. The image was created using PyMOL (www.pymol.org/) and PDB entry 1DD6 (Ref.21).

Figure 2

Structures of selected β-lactam substrates. Type I substrates (nitrocefin, cephalothin, and cefotaxime) possess R₂ groups with atoms of high electron density (indicated by black boxes); Type II substrates (ceftazidime, benzylpenicillin, ampicillin, imipenem, and meropenem) have axial methyl groups or positively charged and/or bulky R₂ groups.

Figure 3

Structural characteristics of amino acids at position 262. The amino acid structures of serine (left), threonine (middle), and valine (right) are depicted in the ionized form with shared functional groups highlighted in boxes. Hydroxyl groups are highlighted in dashed boxes and branched (bulky) groups in solid boxes.

Figure 4

Circular dichroism scans of IMP-1 (black line), IMP-1-S262T (green line), and IMP-1-S262V (red line).

Figure 5

Image of a representative western blot of whole cells (E. coli DH10B) expressing IMP-1, IMP-1-S262T, and IMP-1-S262V. DnaK at ∼70 kDa served as a constitutively expressed control. The band at ∼25 kDa corresponds to the MBLs. Fifty ng of purified IMP-1 were loaded as positive control in the left lane. Expression levels of IMP-1 and IMP-1-S262T are comparable and that of IMP-1-S262V is about 10% of that.