STRUCTURAL STUDIES ON BETA-LACTAMASE ENZYMES

Stanford University, Stanford CA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Beta-lactams are the most common antibiotics in clinical use and represent more than 60% of total world consumption of antimicrobial drugs. They include penicillins, cephalosporins, monobactams, penems and carbapenems, and over 50 antibiotics of this class are available on the market. A major mechanism of bacterial resistance to beta-lactam antibiotics is the production of beta-lactamases, enzymes that hydrolyze the conserved four-membered ring of beta-lactams in a two-step process. Firstly the beta-lactam ring is opened and the adduct binds covalently to an active site serine residue (the acylation step). This adduct is then deacylated by a water molecule held in place by a highly conserved glumate residue (Glu166), releasing the inactive peniciloic acid derivative of the drug. We are currently working on two recently-discovered beta-lactamase enzymes. The first, named GES-1 (Guiana Extended-Spectrum, after the country where it was first isolated) was initially described in 2000. This extended spectrum beta-lactamase (ESBL) is very distantly related to other class A beta-lactamases and produces resistance to penicillins and first-, second-, and some third-generation cephalosporins (e.g. ceftazidime) but not to monobactams and carbapenems. Since 2000, nine GES-type enzymes (GES-1 - GES-9) from different geographical locations have been described. The most alarming characteristic of the GES family of enzymes that distinguish them from the TEM and SHV superfamilies, is their apparent ability to evolve into weak carbapenemases, enzymes capable of hydrolyzing carbapenem antibiotics. We determined the GES-1 structure in 2007 and have recently crystallized and determined the structures of two other members of the GES family, GES-2 and GES-5, both of which a single point mutants of the GES-1 enzyme and which show elevated activity against the carbepenem antibiotic imipenem. In addition, we have also determined the structure of a deacylation deficient mutant (E166N) of GES-5. The second enzyme, Oih-1 was isolated