3-DIMENSIONAL STRUCTURE OF AMPC BETA-LACTAMASE FROM ESCHERICHIA-COLI BOUND TO A TRANSITION-STATE ANALOG - POSSIBLE IMPLICATIONS FOR THE OXYANION HYPOTHESIS AND FOR INHIBITOR DESIGN

The structures of AmpC beta-lactamase from Escherichia coli, alone and in complex with a transition-state analogue, have been determined by X-ray crystallography. The native enzyme was determined to 2.0 Angstro m resolution, and the structure with the transition-state analogue m-a minophenylboronic acid was determined to 2.3 Angstrom resolution. The structure of AmpC from E. coil resembles those previously determined f or the class C enzymes from Enterobacter cloacae and Citrobacter freun dii. The transition-state analogue, m-aminophenylboronic acid, makes s everal interactions with AmpC that were unexpected. Perhaps most surpr isingly, the putative ''oxyanion'' of the boronic acid forms what appe ars to be a hydrogen bond with the backbone carbonyl oxygen of Ala318, suggesting that this atom is protonated. Although this interaction ha s not previously been discussed, a carbonyl oxygen contact with the pu tative oxyanion or ligand carbonyl oxygen appears in most complexes in volving a beta-lactam recognizing enzyme. These observations may sugge st that the high-energy intermediate for amide hydrolysis by beta-lact amases and related enzymes involves a hydroxyl and not an oxyanion, al though the oxyanion form certainly cannot be discounted. The involveme nt of the main-chain carbonyl in ligand and transition-state recogniti on is a distinguishing feature between serine beta-lactamases and seri ne proteases, to which they are often compared. AmpC may use the inter action between the carbonyl of Ala318 and the carbonyl of the acylated enzyme to destabilize the ground-state intermediate, this destabiliza tion energy might be relieved in the transition state by a hydroxyl hy drogen bond. The structure of the m-aminophenylboronic acid adduct als o suggests several ways to improve the affinity of this class of inhib itor and points to the existence of several unusual binding-site-like features in the region of the AmpC catalytic site.