GOB, A SECRETED BACTERIAL MONO-Zn(II) METALLO-B-LACTAMASE WITH A NOVEL ACTIVE SITE

MORAN-BARRIO, J.; GONZALEZ, J.; LISA, M.; COSTELLO, A.; DAL-PERARO, M.; CARLONI, P.; BENNET, B.; ADRIANA SARA LIMANSKY; VIALE, A.; VILA, A.

Boston Marriott Copley Place, Boston, MA

Congreso; 21st Annual Symposium of The Protein Society.; 2007

The Protein Society

Metallo-B-lactamases (MBLs) are zinc-dependent enzymes able to hydrolyze and inactivate most â-lactam antibiotics. The large diversity of active site structures and metal content among them has limited so far the design of a pan-MBL inhibitor. We report here the biochemical and biophysical characterization of a novel MBL, GOB-18, from an Elizabethkingia meningoseptica clinical strain. We also characterized the main citoplasmic chaperones involved in the in vivo secretion process of this enzyme. In vitro studies (spectroscopic techniques, 3D modeling and mutagenesis) revealed that the Zn(II) ion in GOB-18 is bound to the canonical Zn2 site of dinuclear MBLs. Contrasting all other related MâLs, GOB-18 is fully active against a broad range of B-lactam substrates using a single Zn(II) ion in this site. Our results confirm that Zn2 is central for MBL-mediated catalysis. In vivo studies showed that GOB-18 is secreted to the E. coli periplasm in a Sec-dependent process, conferring resistance to B-lactams. Genetic studies indicated that main cytoplasmic chaperones including the DnaK system and Trigger Factor were required for GOB secretion. Conversely, GroEL/S chaperones had minor effects on GOB secretion, suggesting minor roles of the chaperonins in this process. The identification of specific cellular components dedicated to the secretion of MBLs could thus be a possible target for the design of new antibiotics.