Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

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.