CRYSTAL STRUCTURE OF THE WIDE-SPECTRUM BINUCLEAR ZINC ß-LACTAMASE FROM BACTEROIDES FRAGILIS. Nestor O. Concha¹, Beth A. Rasmussen², Karen Bush², and Osnat Herzberg¹. ¹Center for Advanced Research in Biotechnology, University of Maryland, Rockville, MD. ²Wyeth-Ayerst Research, Lederle Laboratories, Pearl River, NY

The metallo ß-lactamase from Bacteroides fragilis hydrolyses a wide range of ß-lactam antibiotics, and is not susceptible to any known ß-lactamase inhibitor. The proliferation of the B. fragilis bacteria in nosocomial infections, and the report of plasmid-mediated dissemination of the enzyme, underscore the urgency of acquiring structural information for the development of new therapeutic agents. The crystal structure of the enzyme was determined using multiwavelength anomalous diffraction at the zinc absorption edge, and subsequently refined at 1.85 Å resolution. The enzyme folds into a four layer a/ß/ß/a structure with the active site located at the edge of the ß-sandwich. The molecule contains a binuclear (Zn⁺²-Zn⁺²) center with one zinc in a tetrahedral coordination, and the other in a pentagonal coordination. A water molecule is shared by both zinc ions, indicating that it is a hydroxide. A second water binds only to the pentagonally coordinated zinc. A variety of bound substrates can be modeled in the active site, providing the structural basis for the wide- spectrum profile of the B. fragilis ß-lactamase. The active site architecture and the models of the bound substrates lead to the proposed catalytic mechanism in which the hydroxide is assumed to be the nucleophilic group. An alternative proposal in which the water molecule that binds to the pentagonally coordinated zinc plays the nucleophilic role is less favored, but cannot be ruled out.