Hydrolysis Spectrum Extension of CMY-2-Like β-Lactamases Resulting from Structural Alteration in the Y-X-N Loop

Abstract

ABSTRACTTheCitrobacter freundiiisolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) β-lactamase. The chromosome-borneblaAmpC-CHAgene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded theEscherichia coliTOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resultingE. coliTOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 β-lactamase, which is structurally related to the chromosome-borne cephalosporinase ofC. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficientE. colistrain. Moreover, it exhibited increased catalytic efficiency (kcat/Km) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (kcat), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A β-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.