title: The Zn2 position in metallo-beta-lactamases is critical for activity: a study on chimeric metal sites on a conserved protein scaffold.
abstract: Metallo-beta-lactamases (MbetaLs) are bacterial Zn(II)-dependent hydrolases that confer broad-spectrum resistance to beta-lactam antibiotics. These enzymes can be subdivided into three subclasses (B1, B2 and B3) that differ in their metal binding sites and their characteristic tertiary structure. To date there are no clinically useful pan-MbetaL inhibitors available, mainly due to the unawareness of key catalytic features common to all MbetaL brands. Here we have designed, expressed and characterized two double mutants of BcII, a di-Zn(II) B1-MbetaL from Bacillus cereus, namely BcII-R121H/C221D (BcII-HD) and BcII-R121H/C221S (BcII-HS). These mutants display modified environments at the so-called Zn2 site or DCH site, reproducing the metal coordination environments of structurally related metallohydrolases. Through a combination of structural and functional studies, we found that BcII-HD is an impaired beta-lactamase even as a di-Zn(II) enzyme, whereas BcII-HS exhibits the ability to exist as mono or di-Zn(II) species in solution, with different catalytic performances. We show that these effects result from an altered position of Zn2, which is incapable of providing a productive interaction with the substrate beta-lactam ring. These results indicate that the position of Zn2 is essential for a productive substrate binding and hydrolysis.
authors.affiliation: Departamento de Química Biológica, Instituto de Biología Molecular y Celular de Rosario, IBR-CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, S2002LRK, Argentina.
authors.forename: Javier M
authors.initials: JM
authors.lastname: González
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Medrano Martín
authors.forename: Alison L
authors.initials: AL
authors.lastname: Costello
authors.forename: David L
authors.initials: DL
authors.lastname: Tierney
authors.forename: Alejandro J
authors.initials: AJ
authors.lastname: Vila
mesh_terms: D001409:Bacillus cereus / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D006867:Hydrolases / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008667:Metalloproteins; D006867:Hydrolases; C455979:beta-lactamase BcII; D001618:beta-Lactamases; D015032:Zinc