Broad spectrum antibiotic-degrading metallo-β-lactamases are phylogenetically diverse and widespread in the environment

Abstract

Antibiotic resistance has emerged as a major global health threat. The Zn 2+ -dependent metallo-βlactamases (MBLs) are of particular concern as they act on the most widely prescribed class of 25 antibiotics, the β-lactams, and are largely unaffected by commonly used β-lactamase antagonists such as clavulanic acid. MBLs are subdivided into three groups (B1 to B3); despite low overall sequence similarity, their catalytic centers are conserved with two closely spaced Zn 2+ binding sites (α and β site). We recovered almost 1500 B3 MBLs from >100,000 public microbial genomes representing a wide range of habitats including pristine sites not impacted by human activity. 30Although homologs were predominantly identified in members of the bacterial phylum Proteobacteria, the recovered B3 MBLs represent a much broader phylogenetic diversity than is currently appreciated based on the study of model pathogens. This includes three active site variants inferred to have arisen from the ancestral B3 enzyme. One of these variants, B3-RQK, is noteworthy for being broadly sensitive to clavulanic acid. Through targeted mutations we 35 demonstrate that the presence of a lysine residue (Lys263) in the β site of the catalytic center of this variant confers sensitivity to this compound. Replacing this lysine with the canonical histidine (His263) found in all other MBLs restored resistance. Crystallographic and computational data reveal that clavulanic acid inhibits B3-RQK MBLs by displacing the Zn 2+ ion in the β site. Therefore, modifying clavulanic acid to effectively interact with His263 may increase the 40 therapeutic range of this widely used antibiotic resistance drug. SignificanceThis study surveys the environmental and phylogenetic diversity of the B3 subgroup of antibiotic-45 degrading metallo-β-lactamases (MBLs). B3-like MBLs are more widespread in the environment than previously appreciated suggesting multiple unrecognized reservoirs of antibiotic resistance. Three variants of the canonical active site were identified, including B3-RQK, which amongst the B3 MBLs is uniquely inhibited by the antibiotic resistance drug clavulanic acid. We demonstrate that the mode of inhibition involves the displacement of a catalytically essential Zn 2+ ion from the 50 active site. It may thus be possible to modify clavulanic acid so that it can compete with the Zn 2+ ions in other MBLs as well, increasing the therapeutic range of this compound.

Structure and mechanism of potent bifunctional β-lactam- and homoserine lactone-degrading enzymes from marine microorganisms

“…This data interpretation is consistent with considerable the difference in the coordination geometry observed in the α site of these two enzymes. In summary, we previously demonstrated that marine environments harbor a large number of MBLs from the B3 subgroup 43 . Of particular interest among those MBLs are those from the marine organisms N. pentaromativorans (MIM-1) and S. agarivorans (SAM-1, formerly known as MIM-2) as we previously already demonstrated that they are not only potent MBLs, but are also capable to hydrolyze quorum-sensing molecules 23,24 .…”

“…Specifically, the two metal ions in the α and β sites (Zn1 and Zn2) of MIM-1 and SAM-1 are coordinated by His116, His118 and His194 (α site), and Asp120, His121 and His260/259 (β site), respectively. The motif HHH/DHH for the ligands interacting with the metals in the active site is a characteristic feature for MBLs belonging to the B3 subgroup 43 . Additional features in MIM-1 and SAM-1 include the presence of Gln157, located on loop 1 (Figures 2 and 3) and the presence of a disulphide bridge, which locks in the extended N-terminus, thus maintaining the active site pocket in an open conformation (Figures 4 and 5).…”

“…The zinc binding sites in MIM-1 and SAM-1 (Figure 2) are defined by a motif (HHH/DHH), that is characteristic for the majority of MBLs of the B3 subgroup [43][44][45][46][47][48][49] . The Zn-Zn distances in MIM-1 (4.02 Å) and SAM-1 (3.40 Å) are comparable with those observed in other B3 MBLs, i.e.…”

Worldwide distribution and environmental origin of the Adelaide imipenemase (AIM-1), a potent carbapenemase in Pseudomonas aeruginosa

“…Unlike serine β-lactamases, MBLs are not inhibited by commonly used clinical inhibitors, such as clavulanic acid, tazobactam, or sulbactam [18]. Their ability to hydrolyze almost all carbapenems efficiently [19,20] and their rapid dissemination threatens current treatment options.…”