Search

Antibiotic resistance has emerged as a major threat to global health care. This is largely due to the fact that many pathogens have developed strategies to acquire resistance to antibiotics. Metallo-β-lactamases (MBL) have evolved to inactivate most of the commonly used β-lactam antibiotics. AIM-1 is one of only a few MBLs from the B3 subgroup that is encoded on a mobile genetic element in a major human pathogen. Here, its mechanism of action was characterised with a combination of spectroscopic and kinetic techniques and compared to that of other MBLs. Unlike other MBLs it appears that AIM-1 has two avenues available for the turnover of the substrate nitrocefin, distinguished by the identity of the rate-limiting step. This observation may be relevant with respect to inhibitor design for this group of enzymes as it demonstrates that at least some MBLs are very flexible in terms of interactions with substrates and possibly inhibitors.

A combination of XAS, UV-vis, NMR, and EPR was used to examine the binding of a series of α-hydroxythiones to CoCA. All three appear to bind preferentially in their neutral, protonated forms. Two of the three clearly bind in a monodentate fashion, through the thione sulfur alone. Thiomaltol (TM) appears to show some orientational preference, on the basis of the NMR, while it appears that thiopyromeconic acid (TPMA) retains rotational freedom. In contrast, allothiomaltol (ATM), after initially binding in its neutral form, presumably through the thione sulfur, forms a final complex that is five-coordinate via bidentate coordination of ATM. On the basis of optical titrations, we speculate that this may be due to the lower initial pKa of ATM (8.3) relative to those of TM (9.0) and TPMA (9.5). Binding through the thione is shown to reduce the hydroxyl pKa by ∼0.7 pH unit on metal binding, bringing only ATM's pKa close to the pH of the experiment, facilitating deprotonation and subsequent coordination of the hydroxyl. The data predict the presence of a solvent-exchangeable proton on TM and TPMA, and Q-band 2-pulse ESEEM experiments on CoCA + TM suggest that the proton is present. ESE-detected EPR also showed a surprising frequency dependence, giving only a subset of the expected resonances at X-band.

Antibiotic resistance is a major global health problem, one that threatens to derail the benefits garnered from arguably the greatest success of modern medicine, the discovery of antibiotics. Among the most potent agents contributing to antibiotic resistance are metallo-β-lactamases (MBLs). The discovery of MBL-like enzymes in microorganisms that are not in contact with the human population is of particular concern as these proteins already have the in-built capacity to inactivate antibiotics, even though they may not need MBL activity for their survival. Here, we demonstrate that a microbiome from a remote and frozen environment in Alaska harbours at least one highly efficient MBL, LRA-8. LRA-8 is homologous to the B3 subgroup of MBLs and has a substrate profile and catalytic properties similar to well-known members of this enzyme family, which are expressed by major human pathogens. LRA-8 is predominantly a penicillinase, but is also active towards carbapenems, but not cephalosporins. Spectroscopic studies indicate that LRA-8 has an active site structure similar to that of other MBLs (in particular B3 subgroup representative AIM-1), and a combination of steady-state and pre-steady-state kinetic data demonstrate that the enzyme is likely to employ a metal ion-bridging hydroxide to initiate catalysis. The rate-limiting step is the decay of a chromophoric, tetrahedral intermediate, as is observed in various other MBLs. Thus, studying the properties of such “pristine” MBL-like proteins may provide insight into the structural plasticity of this family of enzymes that may facilitate functional promiscuity, while important insight into the evolution of MBLs may also be gained.