Abstract
Class B metallo-β-lactamases (MBLs) are Zn2+-dependent enzymes that catalyze the hydrolysis of β-lactam antibiotics to confer resistance in bacteria. Several problematic groups of MBLs belong to subclass B1, including the binuclear New Delhi MBL (NDM), Verona integrin-encoded MBL, and imipenemase-type enzymes, which are responsible for widespread antibiotic resistance. Aspergillomarasmine A (AMA) is a natural aminopolycarboxylic acid that functions as an effective inhibitor of class B1 MBLs. The precise mechanism of action of AMA is not thoroughly understood, but it is known to inactivate MBLs by removing one catalytic Zn2+ cofactor. We investigated the kinetics of MBL inactivation in detail and report that AMA is a selective Zn2+ scavenger that indirectly inactivates NDM-1 by encouraging the dissociation of a metal cofactor. To further investigate the mechanism in living bacteria, we used an active site probe and showed that AMA causes the loss of a Zn2+ ion from a low-affinity binding site of NDM-1. Zn2+-depleted NDM-1 is rapidly degraded, contributing to the efficacy of AMA as a β-lactam potentiator. However, MBLs with higher metal affinity and stability such as NDM-6 and imipenemase-7 exhibit greater tolerance to AMA. These results indicate that the mechanism of AMA is broadly applicable to diverse Zn2+ chelators and highlight that leveraging Zn2+ availability can influence the survival of MBL-producing bacteria when they are exposed to β-lactam antibiotics.
Highlights
Pseudomonas sp., and Acinetobacter sp. have emerged in the clinic [4]
New opportunities for the synthesis of Aspergillomarasmine A (AMA) have emerged from recent advances in understanding the biosynthesis of AMA, which revealed that only a single synthase is required for the production of AMA [21]
Reversal of carbapenem resistance by AMA is determined by its binding selectivity toward Zn2+
Summary
Reversal of carbapenem resistance by AMA is determined by its binding selectivity toward Zn2+. The time-course assays showed that L-captopril reduced the rate of Zn2+ dissociation from NDM-1 (Fig. 3E) These observations are consistent with the known mechanism of inhibition where the thiol of Lcaptopril intercalates the two Zn2+ cofactors of NDM-1, competing with metal dissociation [26]. AMA treatment resulted in significant labeling of NDM-1, which was partially blocked by L-captopril (Fig. 4F) These results were consistent with our in vitro data, showing that AMA sequesters Zn2+, which has dissociated from the Zn2 site of NDM-1 in the periplasm. To better understand the mechanism behind this variability, we titrated AMA against live E. coli producing some of these MBLs, quantified changes in protein levels, and probed Zn2+ dissociation with B7–Mal. To facilitate this study, NDM-6 and IMP-7 were engineered with C-terminal FLAG tags, which did not affect the resistance profiles of these MBLs in E. coli (Table S1). The discrepancy between the NDM-6 data could be explained by the time dependence of NDM-1FLAG [AMA] ( g/mL)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
