Unexpected advanced generation cephalosporinase activity of the M69F variant of SHV β-lactamase

Infections with bacteria that contain hydrolytic beta-lactamase enzymes are becoming a serious problem in the United States. Mutations at Met-69, an amino acid proximal to the active site Ser-70 in the TEM-1 and SHV-1 beta-lactamases, have emerged as a puzzling cause of bacterial resistance to inhibitors of beta-lactamases. Site-saturation mutagenesis of the 69 position in SHV beta-lactamase was performed to determine how mutations of this non-catalytic residue play a role in increasing 50% inhibitory concentrations (IC50 concentrations) for clinically important beta-lactamase enzyme inhibitors. Two distinct phenotypes are evident in the variant beta-lactamases studied: significantly increased minimum inhibitory concentrations (mug/ml) and IC50 concentrations to clavulanic acid for the Met69Ile, Leu, and Val substitutions, and unanticipated increased minimum inhibitory concentrations and hydrolytic activity toward ceftazidime, an advanced generation cephalosporin antibiotic, for the Met69Lys, Tyr- and Phe-substituted enzymes. Molecular modeling studies emphasize the conserved structure of these substitutions despite great variation in substrate specificity. This study demonstrates the key role of Met-69 in defining substrate specificity of SHV beta-lactamases and alerts us to new phenotypes that may emerge clinically.