Metallo-B-lactamase double mutants: two is a crowd

JAVIER MARCELO GONZALEZ; RODOLFO RASIA; JULIA CRICCO; ALEJANDRO J. VILA

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Biofísica; 2003

 Metallo-ß-lactamases (MßLs) are bacterial Zn(II)-dependent hydrolases that confer broad-spectrum resistance to ß-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-MßL inhibitors available, mainly due to the unawareness of key catalytic features common to all MßL brands. Here we have designed, expressed and characterized two double mutants of BcII, a di-Zn(II) B1-MßL 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 ß-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 ß-lactam ring. These results indicate that the position of Zn2 is essential for a productive substrate binding and hydrolysis.