Abstract
Metallo-β-lactamases, enzymes which inactivate β-lactam antibiotics, are of increasing biological and clinical significance as a source of antibiotic resistance in pathogenic bacteria. In the present study we describe the high-resolution solution NMR structures of the Bacillus cereus metallo-β-lactamase BcII and of its complex with R-thiomandelic acid, a broad-spectrum inhibitor of metallo-β-lactamases. This is the first reported solution structure of any metallo-β-lactamase. There are differences between the solution structure of the free enzyme and previously reported crystal structures in the loops flanking the active site, which are important for substrate and inhibitor binding and catalysis. The binding of R-thiomandelic acid and the roles of active-site residues are defined in detail. Changes in the enzyme structure upon inhibitor binding clarify the role of the mobile β3–β4 loop. Comparisons with other metallo-β-lactamases highlight the roles of individual amino-acid residues in the active site and the β3–β4 loop in inhibitor binding and provide information on the basis of structure–activity relationships among metallo-β-lactamase inhibitors.
Highlights
The rapid increase in resistance to β-lactam antibiotics is a major clinical and public health concern, as these antibiotics have long been key to the treatment of serious bacterial infections
In the sequence-based Ambler classification of β-lactamases, classes A, C and D are serine enzymes, whereas class B represents the zinc-dependent MBLs [2,3]; the latter are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics
The CphA enzyme functions as a mono-zinc enzyme, and is a strict carbapenemase [2]; we have shown that mercaptocarboxylate inhibitors bind quite differently to this enzyme, with the carboxylate interacting with the zinc atoms [20]
Summary
The rapid increase in resistance to β-lactam antibiotics is a major clinical and public health concern, as these antibiotics have long been key to the treatment of serious bacterial infections. The solution structures were calculated using 7190 (∼31 per residue) non-ambiguous NOE distance restraints for the free enzyme and 6578 (∼29 per residue) for the complex with R-thiomandelate.
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