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.
Table 1.
Minimum inhibitory concentrations (in μg/mL) of recombinant E. coli producing LRA-12 β-lactamase.
Table 2.
Kinetic parameters of LRA-12 metallo-β-lactamase.
Table 3.
Comparative catalytic efficiencies of LRA-12 and other class B3 metallo-β-lactamases.
Fig 2.
Influence of pre-incubation with EDTA on the residual activity of LRA-12.
Table 4.
X-Ray data collection and refinement statistics for LRA-12 β-lactamase.
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].
Table 5.
Composition of the metal-binding sites in LRA-12 and other metallo-β-lactamases.
Fig 4.
Detail of the active site of LRA-12 β-lactamase.
The 2F0 –Fc 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.