Structural Insights into the Subclass B3 Metallo-β-Lactamase SMB-1 and the Mode of Inhibition by the Common Metallo-β-Lactamase Inhibitor Mercaptoacetate

Abstract

A novel subclass B3 metallo-␤-lactamase (MBL), SMB-1, recently identified from a Serratia marcescens clinical isolate, showed a higher hydrolytic activity against a wide range of ␤-lactams than did the other subclass B3 MBLs, i.e., BJP-1 and FEZ-1, from environmental bacteria. To identify the mechanism underlying the differences in substrate specificity among the subclass B3 MBLs, we determined the structure of SMB-1, using 1.6-Å diffraction data. Consequently, we found that SMB-1 reserves a space in the active site to accommodate ␤-lactam, even with a bulky R1 side chain, due to a loss of amino acid residues corresponding to F31 and L226 of BJP-1, which protrude into the active site to prevent ␤-lactam from binding. The protein also possesses a unique amino acid residue, Q157, which probably plays a role in recognition of ␤-lactams via the hydrogen bond interaction, which is missing in BJP-1 and FEZ-1, whose K m values for ␤-lactams are particularly high. In addition, we determined the mercaptoacetate (MCR)-complexed SMB-1 structure and revealed the mode of its inhibition by MCR: the thiolate group bridges to two zinc ions (Zn1 and Zn2). One of the carboxylate oxygen atoms of MCR makes contact with Zn2 and Ser221, and the other makes contact with T223 and a water molecule. Our results demonstrate the possibility that MCR could be a potent inhibitor for subclass B3 MBLs and that the screening technique using MCR as an inhibitor would be effective for detecting subclass B3 MBL producers.

Crystal Structure of the Metallo-β-Lactamase GOB in the Periplasmic Dizinc Form Reveals an Unusual Metal Site

“…As already described for the MBL fold (1-6), the whole domain of GOB-18 can be depicted as a duplication of two structurally similar ␣/␤ hemidomains. GOB-18 is very similar to other B3 lactamases (20)(21)(22)(23)(24), despite low sequence identity. Structural alignment of GOB-18 (chain A) with available B3 lactamase models allows for similarity quantification (Fig.…”

“…All B3 lactamases crystallized so far are characterized by at least one disulfide bridge, in most cases between Cys residues located in the C-terminal helix and the loop linking elements ␣5 and ␤12 (20)(21)(22)(23)(24). GOB-18 is an exception in this regard within B3 enzymes, with only one Cys residue in its primary structure, i.e., Cys180 (201) (in strand ␤10), which is buried within the protein core (Fig.…”

“…B3 lactamases, distantly related to the B1 and B2 subclasses, are mostly di-Zn(II) enzymes (20)(21)(22)(23)(24). The Zn1 site of B3 enzymes is a 3-H site similar to that of B1 enzymes, whereas two mutations (Cys221Ser and Arg121His) affect the Zn2 coordination geometry (20)(21)(22)(23)(24), and the metal ion is bonded to Asp120, His121, and His263. Ultimately, Ser221 (equivalent to Cys221 in the DCH site) is no longer a metal ligand, so that the Zn2 site is a DHH site in B3 MBLs.…”

“…Ultimately, Ser221 (equivalent to Cys221 in the DCH site) is no longer a metal ligand, so that the Zn2 site is a DHH site in B3 MBLs. In addition, a third water molecule participates as a fifth ligand, giving rise to a square pyramidal coordination sphere of Zn2 (20)(21)(22)(23)(24). Similar to B1 enzymes, the Zn1 ion in B3 lactamases is regarded as responsible for nucleophile activation (20)(21)(22)(23)(24).…”

Evaluation of a Double-Disk Synergy Test with a Common Metallo-^|^beta;-Lactamase Inhibitor, Mercaptoacetate, for Detecting NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii

“…A variety of techniques have already been developed to detect MBL producers (4), including a disk-based synergy test, the sodium mercaptoacetate (SMA) test, which is conventionally used in Japanese clinical microbiology laboratories (5). This test uses a Kirby-Bauer (KB) disk containing a b-lactam antibiotic (ceftazidime [CAZ] recommended) and a disk containing SMA, an MBL inhibitor that can bind to the MBL active site through interactions with zinc ions (6). Although this test works well for the detection of IMPand VIM-type MBL producers, which are the predominant MBL types in Japan (7), preliminary results have indicated that it may fail to detect NDM-1 producers when using the SMA test with the CAZ disk according to general recommendations (8).…”