title: Monitoring the zinc affinity of the metallo-beta-lactamase CphA by automated nanoESI-MS.
abstract: 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-beta-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 Zn(2+) 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.
authors.affiliation: Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: De Vriendt
authors.forename: Gonzalez
authors.initials: G
authors.lastname: Van Driessche
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.forename: Carine
authors.initials: C
authors.lastname: Bebrone
authors.forename: Christine
authors.initials: C
authors.lastname: Anne
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Frère
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
mesh_terms: D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry*; D003627:Data Interpretation, Statistical; D007202:Indicators and Reagents; D036103:Nanotechnology; D009862:Online Systems; D021241:Spectrometry, Mass, Electrospray Ionization; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D007202:Indicators and Reagents; D001618:beta-Lactamases; C082063:cphA protein, Aeromonas hydrophila; D015032:Zinc