X-ray Structure of the Asn276Asp Variant of the Escherichia coli TEM-1 β-Lactamase:  Direct Observation of Electrostatic Modulation in Resistance to Inactivation by Clavulanic Acid^,

Abstract

The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

“…Alternatively, the imine 2 could be directly hydrolyzed to yield the aldehyde 5 and hydrate aldehyde 6. This pathway is entirely consistent with that proposed for the inhibitor-resistant TEM-33 (M69L) and N276D-substituted enzymes (10,11). Our kinetic data for tazobactam and clavulanic acid suggest that the rate that each intermediate is formed and the number of branch points differ for each inhibitor.…”

“…The water molecule may also function with Lys-73 in a coordinated proton shuttle and may substitute for the Ser-130 in protonating the ␤-lactam nitrogen (26,27). In the x-ray structures of the inhibitor resistant TEM-30 (R244S) and TEM-84 (N276D), the relocation of a conserved water plays a crucial role in the binding affinity of inhibitors (10,12). The molecular modeling studies of the E166N amino acid substitution of TEM-1 ␤-lactamase revealed the movement of a catalytic water molecule was essential in modification of the substrate profile (28).…”

“…High resolution atomic structures of the inhibitor resistant ␤-lactamases have also advanced our understanding of this phenotype. The x-ray structure of the clavulanic acid resistant N276D variant of TEM-1 ␤-lactamase revealed a significant movement of Asp-276: the substitution of Asp for Asn displaces three helices (helix 1, helix 10, and helix 11) in the TEM structure (10). The three-dimensional analysis of the M69L TEM ␤-lactamase shows that subtle molecular dynamic changes can also lead to resistance to clavulanic acid (11).…”

Hydrolytic Mechanism of OXA-58 Enzyme, a Carbapenem-hydrolyzing Class D β-Lactamase from Acinetobacter baumannii

“…In classes A and C ␤-lactamases, the catalytic water molecule is anchored through a hydrogen bond network in an optimum position for nucleophilic attack on the carbonyl carbon of the acyl-enzyme species. The position of this water molecule is crystallographically conserved (31,36,41) unlike in class D enzymes (42)(43)(44). Perturbation of the hydrogen bonding network of this water molecule or the physical displacements of the water molecule by the steric bulk of the C-6 side chains of a ␤-lactam are possible mechanisms to render the ␤-lactamase deacylation-deficient (20,(45)(46)(47).…”

Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase,

“…To date, atomic resolution structures of five IRT mutants have been determined (9)(10)(11). The Arg244Ser substitution found in the TEM-30 mutant results in displacement of a water molecule involved in the inactivation chemistry of clavulanate (3,10).…”