Journal
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
Volume 55, Issue 5, Pages 2303-2309Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AAC.01360-10
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Funding
- Robert A. Welch Foundation [N-0871]
- National Institutes of Health [R01 AI062968, RO1 AI063517-01]
- Veterans Affairs Merit Review Program
- Geriatric Research Education and Clinical Care (GRECC)
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Among Gram-negative bacteria, resistance to beta-lactams is mediated primarily by beta-lactamases EC 3.2.6.5), periplasmic enzymes that inactivate beta-lactam antibiotics. Substitutions at critical amino acid positions in the class A beta-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an extended-spectrum beta-lactamase ESBL) phenotype. Using SHV ESBLs with substitutions in the Omega loop R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel beta-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique beta-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced K(i) values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered Omega loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent Omega loop flexibility as a mechanism for accommodating and hydrolyzing beta-lactam substrates.
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