2 results
The complexed structure and antimicrobial activity of a non-β-lactam inhibitor of AmpC β-lactamase
- RACHEL A. POWERS, JESÚS BLÁZQUEZ, G. SCOTT WESTON, MARÍA-ISABEL MOROSINI, FERNANDO BAQUERO, BRIAN K. SHOICHET
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- Journal:
- Protein Science / Volume 8 / Issue 11 / November 1999
- Published online by Cambridge University Press:
- 01 November 1999, pp. 2330-2337
- Print publication:
- November 1999
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β-Lactamases are the major resistance mechanism to β-lactam antibiotics and pose a growing threat to public health. Recently, bacteria have become resistant to β-lactamase inhibitors, making this problem pressing. In an effort to overcome this resistance, non-β-lactam inhibitors of β-lactamases were investigated for complementarity to the structure of AmpC β-lactamase from Escherichia coli. This led to the discovery of an inhibitor, benzo(b)thiophene-2-boronic acid (BZBTH2B), which inhibited AmpC with a Ki of 27 nM. This inhibitor is chemically dissimilar to β-lactams, raising the question of what specific interactions are responsible for its activity. To answer this question, the X-ray crystallographic structure of BZBTH2B in complex with AmpC was determined to 2.25 Å resolution. The structure reveals several unexpected interactions. The inhibitor appears to complement the conserved, R1-amide binding region of AmpC, despite lacking an amide group. Interactions between one of the boronic acid oxygen atoms, Tyr150, and an ordered water molecule suggest a mechanism for acid/base catalysis and a direction for hydrolytic attack in the enzyme catalyzed reaction. To investigate how a non-β-lactam inhibitor would perform against resistant bacteria, BZBTH2B was tested in antimicrobial assays. BZBTH2B significantly potentiated the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria. This inhibitor was unaffected by two common resistance mechanisms that often arise against β-lactams in conjunction with β-lactamases. Porin channel mutations did not decrease the efficacy of BZBTH2B against cells expressing AmpC. Also, this inhibitor did not induce expression of AmpC, a problem with many β-lactams. The structure of the BZBTH2B/AmpC complex provides a starting point for the structure-based elaboration of this class of non-β-lactam inhibitors.
Functional analyses of AmpC β-lactamase through differential stability
- BETH M. BEADLE, SUSAN L. McGOVERN, ALEXANDRA PATERA, BRIAN K. SHOICHET
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- Journal:
- Protein Science / Volume 8 / Issue 9 / September 1999
- Published online by Cambridge University Press:
- 01 September 1999, pp. 1816-1824
- Print publication:
- September 1999
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Despite decades of intense study, the complementarity of β-lactams for β-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, β-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free β-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of β-lactams for the β-lactamase AmpC through reversible denaturation of adducts of the enzyme with β-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 °C and a van't Hoff enthalpy of unfolding (ΔHVH) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (ΔGuH2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (ΔTm = +5.8 °C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (ΔTm = +4.9 °C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (ΔTm = −3.2 °C) and 0.7 kcal/mol (ΔTm = −1.2 °C), respectively. These β-lactams, which share nonhydrogen substituents in the 6(7)α position of the β-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (ΔTm = −1.3 °C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the β-lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for β-lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other β-lactam binding proteins as well.