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See detailMonitoring the zinc affinity of the metallo-beta-lactamase CphA by automated nanoESI-MS
De Vriendt, K.; Van Driessche, G.; Devreese, B. et al

in Journal of the American Society for Mass Spectrometry (2006), 17(2), 180-188

Metallo-beta-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate beta-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-p-lactamase ... [more ▼]

Metallo-beta-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate beta-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-p-lactamase because it degrades only carbapenems efficiently and is only active when it has one zinc ion bound. A zinc titration experiment was used to study the zinc affinity of the wild-type and of several mutant CphA enzymes. It shows that a second Zn2+ is also bound at high ion concentrations. All samples were analyzed using mass spectrometry in combination with an automated nanoESI source. The metal-free enzyme has a bimodal charge distribution indicative of two conformational states. A completely folded enzyme is detected when the apo-enzyme has bound the first zinc. Intensity ratios of the different enzyme forms were used to deduce the zinc affinities. CphA enzymes mutated in metal ligands show decreased zinc affinity compared to wild-type, especially D120 mutants. [less ▲]

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See detailDramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-beta-lactamase by site-directed mutagenesis
Bebrone, Carine ULg; Anne, C.; De Vriendt, K. et al

in Journal of Biological Chemistry (2005), 280(31), 28195-28202

Among class B beta-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants ... [more ▼]

Among class B beta-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants that possess a strongly broadened activity spectrum when compared with the WT CphA. Strikingly, the N116H/N220G double mutant exhibits a substrate profile close to that observed for the broad spectrum subclass B1 enzymes. The double mutant is significantly activated by the binding of a second zinc ion under conditions where the WT enzyme is non-competitively inhibited by the same ion. [less ▲]

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See detailUpdate of the standard numbering scheme for class B beta-lactamases
Garau, G.; Garcia-Saez, I.; Bebrone, Carine ULg et al

in Antimicrobial Agents and Chemotherapy (2004), 48(7), 2347-2349

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See detailRole of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase.
Vanhove, Marc; Zakhem, M.; Devreese, B. et al

in Cellular and molecular life sciences : CMLS (2003), 60(11), 2501-9

The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results ... [more ▼]

The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196. [less ▲]

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