Digging up the hydrolysis mechanism of GOB metallo-b-lactamase and its outcome in â-lactam resistance

MORAN BARRIO, J; VILA, AJ

Salta

Congreso; 3rd Latin American Protein Society Meeting, y XXXIX Reunión Anual de la Sociedad Argentina de Biofísica; 2010

Sociedad Argentina de Biofísica

Metallo-β-lactamases (MβLs) are bacterial zinc-dependent enzymes present in the periplasmic space of gram-negative pathogenic bacteria. These enzymes are capable of hydrolyzing most β-lactam antibiotics and they are resistant to all clinically used inhibitors. Moreover, most of MβL genes have been found in mobile genetic elements raising a vital problem in the clinical settings. The rational design of an inhibitor for MβLs has been limited by an incomplete knowledge of their catalytic mechanism and by the structural diversity of their active sites. We studied the MβL GOB from the gram-negative pathogen Elizabethkingia meningoseptica. This enzyme is unique in being the only broad spectrum mononuclear MβL characterized so far1. Kinetic studies have revealed that, even though GOB is active with different divalent metal ions, only the Zn(II) ion present in the active site is capable of stabilizing an anionic intermediate prior to nitrogen protonation2. Here we present stopped-flow tryptophan fluorescence studies under single turnover and pseudo-first-order conditions to investigate the kinetic mechanism of β-lactam hydrolysis by GOB. In addition, we studied the role of residue Met221, and different mutants in this position, in the in vivo β-lactam resistance of the host bacteria. Since there is no crystal structure of this enzyme, these studies shed light into the comprehension of the molecular basis that determines the activity in broad spectrum MβLs. 1. Morán-Barrio J., Gonzalez J.M., Lisa M.N., Costello A.L., Dal Peraro M., Carloni P., Bennett B., Tierney D.L., Limansky A.S., Viale A.M. and Vila A.J. J Biol Chem. 282(25):18286-93 (2007). 2. Lisa M.N., Hemmingsen L., Vila A.J. J Biol Chem. 285(7):4570-7 (2010).