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

Abstract

f Metallo-beta-lactamases (MBLs) are broad-spectrum, Zn(II)-dependent lactamases able to confer resistance to virtually every ␤-lactam antibiotic currently available. The large diversity of active-site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3 that is expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica. This MBL is atypical, since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here, we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18. This enzyme displays a unique active-site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono-and di-Zn(II) forms of GOB-18 are active against penicillins, cephalosporins, and carbapenems. In silico docking and molecular dynamics simulations indicate that residue Met221 is not involved in substrate binding, in contrast to Ser221, which otherwise is conserved in most B3 enzymes. These distinctive features are conserved in recently reported GOB orthologues in environmental bacteria. These findings provide valuable information for inhibitor design and also posit that GOB enzymes have alternative functions.T he expression of ␤-lactamases is the main mechanism of bacterial resistance against ␤-lactam antibiotics. These enzymes catalyze the hydrolysis of the amide bond in the ␤-lactam ring characteristic of this family of drugs (1-5). MBLs are metal-dependent hydrolases which generally use Zn(II) as a Lewis acid to activate a water molecule for the nucleophilic attack. These enzymes are refractive to clinically employed lactamase inhibitors (1) and have a particular relevance in the clinical setting as they can hydrolyze a broad spectrum of ␤-lactam substrates, being able to inactivate carbapenems, the "last-resort" antibiotics in antibacterial therapy (6).MBLs have been classified into subclasses B1, B2, and B3 based on sequence identity (7). Crystal structures of MBLs from the three subclasses have revealed that these enzymes present a common ␣␤/␤␣ sandwich fold, with the active site located within a groove at the interface between these two halves (1-6). The Zn(II)-binding residues vary among different subclasses, giving rise to diverse metal site architectures and metal contents required for activity (1-6). B1 and B3 MBLs are broad-spectrum enzymes that hydrolyze penicillins, cephalosporins, and carbapenems with a wide variety of in vitro catalytic efficiencies, displaying a broad range of resistance profiles in vivo (1)(2)(3)(4)(5)8). The di-Zn(II) form of B1 MBLs has been shown to be the active form in the bacterial periplasm, despite contradictory data obtained from in vitro studies (8-10). These enzymes display a conserved metal binding m...

Structural and biochemical analysis of the metallo‐β‐lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di‐metal scaffold for catalytic activity

“…S. maltophilia L1 is a subclass B3 MBL, one of two chromosome‐encoded β‐lactamases (L2 belongs to a class A) . Several crystal structures of L1 and other B3 MBLs have been reported in the complexes with hydrolyzed antibiotics and inhibitors, which are supported by multiple biochemical analysis . B3 MBLs share a relatively low sequence identity of 23–35%; however, they are structurally well‐conserved particularly their di‐nuclear zinc active sites.…”

“…16 Several crystal structures of L1 [17][18][19][20][21] and other B3 MBLs have been reported in the complexes with hydrolyzed antibiotics and inhibitors, which are supported by multiple biochemical analysis. [17][18][19][20][21][22][23][24][25][26][27][28][29][30] B3 MBLs share a relatively low sequence identity of 23-35%; however, they are structurally well-conserved particularly their di-nuclear zinc active sites. Here, we extend the L1 studies and report several high-resolution structures of L1, the two zinc bound form (native form), the metal-free form (apo form), the captopril inhibitor bound form, several complexes with hydrolyzed antibiotics (imipenem, moxalactam, meropenem, and penicillin G) and with different metal ions (Zn +2 , Cd +2 , and Cu +2 ).…”

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

“…A total of 1,449 putative B3 MBL proteins were identified in 1,383 genomes (representing 1.2% of all analyzed genomes), of which 1,150 have the canonical B3 active site residues (HHH/DHH) and 47 have the B3-RQK motif with changes in both the αand β-sites (HRH/DQK). Two additional minor variants of the B3 motif with single amino acid 125 changes in their α-sites were also discovered; the QHH/DHH motif (B3-Q) previously noted in GOB-1 (26,27) was present in 162 proteins, while the EHH/DHH motif (B3-E), identified in 90 proteins, has not previously been reported. Phylogenetic inference of a representative subset of 673 of these proteins indicates that each of the three motif variants originate from within the B3 radiation (Figure 1).…”

“…NHH/DCH for the α/β site motifs), members of the B2 subgroup only require one Zn 2+ for 75 catalysis (located in the β-site); the presence of a second metal ion leads to inhibition (1,7,15). Only a small number of B3-type MBLs have been identified, including L1 (16)(17)(18)(19)(20), FEZ-1 (21,22), AIM-1 (23)(24)(25), GOB-1 (26,27), LRA-8 (28), MIM-1 and MIM-2 (29,30). They have a structurally similar active site motif to B1 MBLs (HHH/DHH), and also require a bimetallic Zn 2+ center for optimal catalytic efficiency (1,7,13,31).…”

“…This organism can cause life-threatening meningitis, septicemia and necrotizing enterocolitis with a 95 40-80% mortality rate (38)(39)(40), and resistance to antibiotics amongst various Cronobacter strains has already been reported (38). Another B3 MBL (GOB-1) with a minor motif variant (QHH/DHH; B3-Q) was discovered in the opportunistic pathogen Elizabethkingia meningoseptica, which has broad spectrum activity against a range of β-lactam antibiotics, similar to native B3 MBLs such as AIM-1 (26,27). 100…”