Crystal structure and kinetic analysis of the class B3 di-zinc metallo-β-lactamase LRA-12 from an Alaskan soil metagenome

Abstract

We analyzed the kinetic properties of the metagenomic class B3 β-lactamase LRA-12, and determined its crystallographic structure in order to compare it with prevalent metallo-β-lactamases (MBLs) associated with clinical pathogens. We showed that LRA-12 confers extended-spectrum resistance on E. coli when expressed from recombinant clones, and the MIC values for carbapenems were similar to those observed in enterobacteria expressing plasmid-borne MBLs such as VIM, IMP or NDM. This was in agreement with the strong carbapenemase activity displayed by LRA-12, similar to GOB β-lactamases. Among the chelating agents evaluated, dipicolinic acid inhibited the enzyme more strongly than EDTA, which required pre-incubation with the enzyme to achieve measurable inhibition. Structurally, LRA-12 contains the conserved main structural features of di-zinc class B β-lactamases, and presents unique structural signatures that differentiate this enzyme from others within the family: (i) two loops (α3-β7 and β11-α5) that could influence antibiotic entrance and remodeling of the active site cavity; (ii) a voluminous catalytic cavity probably responsible for the high hydrolytic efficiency of the enzyme; (iii) the absence of disulfide bridges; (iv) a unique Gln116 at metal-binding site 1; (v) a methionine residue at position 221that replaces Cys/Ser found in other B3 β-lactamases in a predominantly hydrophobic environment, likely playing a role in protein stability. The structure of LRA-12 indicates that MBLs exist in wild microbial populations in extreme environments, or environments with low anthropic impact, and under the appropriate antibiotic selective pressure could be captured and disseminated to pathogens.

Fig 1. Sequence analysis on LRA-12 protein.

(a) Multi-alignment of amino acid sequences of LRA-12 and other representative class B3 β-lactamases, using the class B standard numbering scheme. Only the four more conserved segments of the sequences are shown for easier visualization. Location of α-helices and β-sheets is indicated in the upper side (taken from the PDB file), and relative solvent accessibility in the bottom (blue: highly accessible; cyan: poorly accessible; white: hidden or non-accessible). Blue and pink stars indicate the position of conserved residues in metal-binding sites 1 and 2, respectively (see text for further details). The figure was prepared using Espript (http://espript.ibcp.fr/ESPript/ESPript/). (b) Neighbor joining tree constructed using class B β-lactamases sequences from the three different sub-classes.

Fig 2. Influence of pre-incubation with EDTA on the residual activity of LRA-12.

Fig 3. Comparative analysis of overall structures of LRA-12 (central structure) and other MBLs.

Color codes: purple: α-helices; green: β-sheets (for LRA-12); pink spheres: Zn(II) atoms; red loops: elongated α3-β7 and β11-α5 loops in B3 β-lactamases; orange: N-terminal segment of LRA-12, and short mobile loops in BcII and SPM-1; pink α-helices: elongated α-helix and extended α3-α4 helix in CphA and SPM-1, respectively. For further details, see reference [10].

Fig 4. Detail of the active site of LRA-12 β-lactamase.

The 2F₀ –F_c map was contoured at 1.5 σ (in grey) around the most important amino acid residues that are part of the metal-binding sites in the active site cavity: Gln116-His118-His196 (Site 1; QHH), and Asp120-His121-His263 (Site 2; DHH). Zinc ions (Zn1 and Zn2) are shown as magenta spheres.

Fig 5. Comparative view of the metal-binding sites of LRA-12 and other metallo-β-lactamases.

Zinc ions (Zn1 and Zn2) and water molecules are represented as magenta and red spheres, respectively; NW, nucleophilic water. Black dotted lines represent the hydrogen bonding interactions between residues or atoms. For ease of interpretation, Zn(II) and water molecules have the same spatial orientation in all figures.