Title

Structural basis of activity and mechanism of class C beta-lactamases

Date of Completion

January 2002

Keywords

Biophysics, General

Degree

Ph.D.

Abstract

The bacterium Enterobacter cloacae GC1 produces a 39 kD β-lactamase which is only five amino acids different from that produced by E. cloacae P99 (Nukaga et al., 1995). Although these residues are not near the catalytic residue, the GC1 β-lactamase displays extended-spectrum activity against oxyimino cephalosporins whereas the P99 β-lactamase does not (Nukaga et al., 1995). This extended-spectrum activity has been shown to be due to a three-residue insertion found in the Ω-loop, a flexible loop of the enzyme (Lobkovsky et al., 1993; Nukaga et al., 1998). ^ Crystallographic studies were performed to ascertain the structural basis for the increase in substrate specificity found in the GC1 β-lactamase. The structure of the unliganded GC1 β-lactamase (Crichlow et al., 1999) is similar to that of the P99 β-lactamase (Lobkovsky et al., 1993); however, the Ω-loop extends 1.5 Å farther from the active site nucleophile in the GC1 β-lactamase. Poor Ω-loop electron density in the GC1 β-lactamase demonstrates its greater flexibility. The structure of the GC1 β-lactamase with the novel inhibitor DVR-II-41S (Buynak et al., 2000; Crichlow et al., 2001) reveals a mechanism of inhibition that entails blocking the approach of the water molecule responsible for deacylation, confirming models proposed by Heinze-Krauss et al. (Heinze-Krauss et al., 1998) and Patera et al. (Patera et al., 2000). ^ In spite of the similarity of the unliganded GC1 Ω-loop to that of P99, the conformation of the GC1 Ω-loop changes upon acylation by DVR-II-41S or imipenem. This change in conformation brings Tyr224, which extends into the active site in the unliganded enzyme, 6.0 Å from its previous location as measured by α-carbon displacement (Crichlow et al ., 2001). This motion—which is not detected in the P99 enzyme upon acylation of a phosphonate inhibitor (Lobkovsky et al., 1994)—creates extra space in the binding cavity, which can relieve steric hindrace in large side chain substrates (Patera et al., 2000; Powers et al., 2001). This may allow the substrate to adopt a relaxed conformation in a location in the binding pocket that does not interfere with the deacylating water molecule and permit the hydrolysis of extended-spectrum cephalosporins. ^