Three-Dimensional Structure of AmpC β-Lactamase from Escherichia coli Bound to a Transition-State Analogue:  Possible Implications for the Oxyanion Hypothesis and for Inhibitor Design

Abstract

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 A resolution, and the structure with the transition-state analogue m-aminophenylboronic acid was determined to 2.3 A resolution. The structure of AmpC from E. coli resembles those previously determined for the class C enzymes from Enterobacter cloacae and Citrobacter freundii. The transition-state analogue, m-aminophenylboronic acid, makes several interactions with AmpC that were unexpected. Perhaps most surprisingly, the putative "oxyanion" of the boronic acid forms what appears to be a hydrogen bond with the backbone carbonyl oxygen of Ala318, suggesting that this atom is protonated. Although this interaction has not previously been discussed, a carbonyl oxygen contact with the putative oxyanion or ligand carbonyl oxygen appears in most complexes involving a beta-lactam recognizing enzyme. These observations may suggest that the high-energy intermediate for amide hydrolysis by beta-lactamases and related enzymes involves a hydroxyl and not an oxyanion, although the oxyanion form certainly cannot be discounted. The involvement of the main-chain carbonyl in ligand and transition-state recognition is a distinguishing feature between serine beta-lactamases and serine proteases, to which they are often compared. AmpC may use the interaction between the carbonyl of Ala318 and the carbonyl of the acylated enzyme to destabilize the ground-state intermediate, this destabilization energy might be relieved in the transition state by a hydroxyl hydrogen bond. The structure of the m-aminophenylboronic acid adduct also suggests several ways to improve the affinity of this class of inhibitor and points to the existence of several unusual binding-site-like features in the region of the AmpC catalytic site.

Practical Methods Using Boronic Acid Compounds for Identification of Class C β-Lactamase-ProducingKlebsiella pneumoniaeandEscherichia coli

“…Boronic acids have been recognized as specific inhibitors of AmpC ␤-lactamases since 1982 (4,8,34,37,41). Using three commercially available boronic acids, APB, NPB, and TPB, in the present study, we evaluated three different methods for the identification of bacteria producing class C ␤-lactamases which would be simple enough for routine use in a clinical microbiology laboratory.…”

“…Serial studies revealed the structure-based mechanism of inhibition of AmpC ␤-lactamases by boronic acids (34,37,41), and novel compounds that inhibit AmpC ␤-lactamases with nano-molar K i values were prepared by stereoselective organic synthesis (23). However, there are only a few reports of studies that applied boronic acids to the identification of class C ␤-lactamase-producing bacteria (19,34).…”

Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

“…AmpC from Escherichia coli was expressed and purified to homogeneity, as described. 6 Compound 1 was obtained from Aldrich Chemical, Milwaukee, WI. Compounds 2-4 were obtained from Lancaster Synthesis, Windham, NH.…”

“…Thus, the structure of AmpC has been determined in complex with inhibitors such as -lactams, 4,[20][21][22] transition state analogues, 23,24 and arylboronic acids, 6,25,26 the leads for which were known before the first structures were determined. 27 There are now 18 inhibitor complex structures with class C -lactamases that have been published.…”

Feasibility Study on a Measurement Method and a Portable Measuring System to Estimate the Concentration of Cloxacillin andβ-Lactamase in Milk

“…Finally, an Escherichia coli K12 AmpC -lactamase [26] was used for all experimental activities. The concentration of AmpC was determined spectrophotometrically in concentrated stock solutions made from lyophilized powder and subsequently diluted; this enzyme had been previously expressed and purified, as described by Powers et al [27].…”