Ligand-dependent disorder of the Omega loop observed in extended-spectrum SHV-type beta-lactamase.

ABSTRACT: Among Gram-negative bacteria, resistance to ?-lactams is mediated primarily by ?-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate ?-lactam antibiotics. Substitutions at critical amino acid positions in the class A ?-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an "extended-spectrum" ?-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the ? loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel ?-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 ?-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced Ki 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 ? 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 ? loop flexibility as a mechanism for accommodating and hydrolyzing ?-lactam substrates.

SUBMITTER: Sampson JM 

PROVIDER: S-EPMC3088229 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature