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See detailAllosteric inhibition of VIM metallo-beta-lactamase by a camelid nanobody
Sohier, Jean ULg; Laurent, Clémentine ULg; Chevigné, Andy et al

in Biochemical Journal (2013), 450(3), 477-486

Metallo-β-lactamase (MβL) enzymes are usually produced by multiresistant Gram-negative bacterial strains and have spread worldwide. An approach based on phage display was employed to select single-domain ... [more ▼]

Metallo-β-lactamase (MβL) enzymes are usually produced by multiresistant Gram-negative bacterial strains and have spread worldwide. An approach based on phage display was employed to select single-domain antibody fragments (VHHs also called Nanobodies) that would inhibit the clinically relevant VIM-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of NbVIM_38 nanobody were then characterised. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the µM range for all tested β-lactams. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the alpha2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor. [less ▲]

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See detailBiochemical and structural characterization of the subclass B1 metallo-β-lactamase VIM-4.
Lassaux, Patricia ULg; Traoré, Daouda; Loisel, Elodie et al

in Antimicrobial Agents and Chemotherapy (2011)

The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic ... [more ▼]

The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn²+ at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn²+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure. [less ▲]

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See detailcAbVIM4, a nanobody inhibiting the metallo-β-lactamase VIM-4
Sohier, Jean ULg; Laurent, Clémentine ULg; Pardon, Els et al

Poster (2010)

Detailed reference viewed: 14 (11 ULg)
See detailCA1838, A NANOBODY INHIBITING THE METALLO-β-LACTAMASE VIM-4.
Sohier, Jean ULg; Laurent, Clémentine ULg; Pardon, Els et al

Poster (2010)

Detailed reference viewed: 19 (17 ULg)
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See detailCharacterisation of B1 metallo-beta-lactamase inhibition by VHHs
Sohier, Jean ULg; Laurent, Clémentine ULg; Chevigné, Andy et al

Poster (2010)

Detailed reference viewed: 44 (21 ULg)