Kinetic and Structural Characterization of the First B3 Metallo-β-Lactamase with an Active-Site Glutamic Acid

Abstract

The structural diversity in metallo-β-lactamases (MBLs), especially in the vicinity of the active site, has been a major hurdle in the development of clinically effective inhibitors. Representatives from three variants of the B3 MBL subclass, containing either the canonical HHH/DHH active site motif (present in the majority of MBLs in this subclass) or the QHH/DHH (B3-Q) or HRH/DQK (B3-RQK) variations were reported previously. Here, we describe the structure and kinetic properties of the first example (SIE-1) of a fourth variant containing the EHH/DHH active site motif (B3-E). SIE-1 was identified in the hexachlorocyclohexane-degrading bacterium Sphingobium indicum , and kinetic analyses demonstrate that although it is active against a wide range of antibiotics its efficiency is lower than that of other B3 MBLs, but with improved efficiency towards cephalosporins relative to other β-lactam substrates. The overall fold of SIE-1 is characteristic of the MBLs; the notable variation is observed in the Zn1 site due to the replacement of the canonical His116 by a glutamate. The unusual preference of SIE-1 for cephalosporins and its occurrence in a widespread environmental organism suggests scope for increased MBL-mediated β-lactam resistance. It is thus relevant to include SIE-1 into MBL inhibitor design studies to widen the therapeutic scope of much needed anti-resistance drugs.

Potent Inhibitors Targeting Metallo-β-lactamases from the B1, B2 and B3 Subgroups: Promising Broad-spectrum Agents to Combat a Major Mechanism of Antibiotic Resistance

“…To obtain accurate inhibition constants and information about the mode of inhibitor binding (competitive, uncompetitive) Michaelis-Menten curves were recorded in the absence of inhibitors and at three different inhibitor concentrations. The data were fit to Equation 2 [7,12,13,15,[19][20][21][28][29][30], where Vmax and Km are the maximum velocity and Michaelis-Menten constant, respectively, and [S] and [I] are the concentration of substrate and inhibitor. Kic and Kiuc are the competitive and uncompetitive inhibition constants.…”

“…B2-type MBLs are rare but have attracted attention due to their unique selectivity for "last line" carbapenem substrates [10]. MBLs from the B3 subgroup have gained less attention, not least because the majority of these enzymes have been identified in environmental microorganisms [7,[11][12][13][14][15]. The catalytic centres in the three MBL subgroups can accommodate up to two metal ions (Zn(II) in most cases) and are characterised by distinct sequence motifs.…”

Structural and Functional Insight into the Mechanism of the Fe−S Cluster‐Dependent Dehydratase from Paralcaligenes ureilyticus

“…The two sites we now refer to as I and II (Figure 8). A probable location for site II is less than 5 Å from site I, suggesting the possibility that a dinuclear metal centre may be formed that resembles those found in metallohydrolases (e. g., purple acid phosphatases, [36,37] pesticide-degrading hydrolases [38][39][40][41] or antibiotic-degrading metalloβ-lactamases [42,43] ), reductases such as ribonucleotide reductase [44] or keto-acid isomeroreductase [45,46] or oxygenases such as methane monooxygenase. [47] The presence of Mn 2 + in site II significantly affects the coordination environment of the Mn 2 + in site I (Figure 8A).…”

Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-β-Lactamases

“…B3 MBLs include SMB-1 ( Wachino et al, 2013 ), AIM-1 ( Leiros et al, 2012 ), L1 ( Spencer et al, 2005 ), GOB-1 ( Moran-Barrio et al, 2016 ), MIM-1 ( Selleck et al, 2020 ), SAM-1 ( Selleck et al, 2020 ), CSR-1 ( Pedroso et al, 2020 ), SIE-1 ( Wilson et al, 2021 ), SPR-1 ( Vella et al, 2013 ), and LRA-8 ( Pedroso et al, 2017 ). When the crystal structures of the eight selected B3 MBLs were superimposed, the RMSD values among the structures were between 0.90 and 1.73 Å, with an average value of 1.51 Å in 231 residues.…”

“…However, the zinc binding sites of AIM-1 were shifted to the lower left position compared to those of NDM-1 due to the change in the core scaffold. Within B3 MBLs, the correlation of the metal-metal distance in the active site was observed ( Wilson et al, 2021 ). Interestingly, even in the shifted or different zinc positions, the interatomic distance between the two zinc ions was almost the same as that observed between NDM-1 and AIM-1 (3.45 Å).…”