Class B metallo-β-lactamases (MBLs) are Zn-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 NDM, VIM, and IMP-type enzymes, which are responsible for widespread antibiotic resistance. Aspergillomarsmine A (AMA) is a natural aminopolycarboxylic acid that functions as an effective inhibitor of class B1 MBLs. AMA's precise mechanism of action is not thoroughly understood, but it is known to inactivate MBLs by removing one catalytic Zn cofactor. We investigated the kinetics of MBL inactivation in detail and report that AMA is a selective Zn scavenger that indirectly inactivates... More
Class B metallo-β-lactamases (MBLs) are Zn-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 NDM, VIM, and IMP-type enzymes, which are responsible for widespread antibiotic resistance. Aspergillomarsmine A (AMA) is a natural aminopolycarboxylic acid that functions as an effective inhibitor of class B1 MBLs. AMA's precise mechanism of action is not thoroughly understood, but it is known to inactivate MBLs by removing one catalytic Zn cofactor. We investigated the kinetics of MBL inactivation in detail and report that AMA is a selective Zn 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 Zn ion from a low-affinity binding site of NDM-1. Zn-depleted NDM-1 is rapidly degraded, contributing to AMA's efficacy as a β-lactam potentiator. However, MBLs with higher metal-affinity and stability such as NDM-6 and IMP-7 exhibit greater tolerance to AMA. These results indicate that the mechanism of AMA is broadly applicable to diverse Zn chelators, and highlight that leveraging Zn availability can influence the survival of MBL-producing bacteria when they are exposed β-lactam antibiotics.
