Beta-lactamase-mediated antibiotic resistance continues to challenge the contemporary treatment of serious
bacterial infections. The
KPC-2 beta-lactamase, a rapidly emerging gram-negative resistance determinant, hydrolyzes all commercially available
beta-lactams, including
carbapenems and
beta-lactamase inhibitors; the amino acid sequence requirements responsible for this versatility are not yet known. To explore the bases of
beta-lactamase activity, we conducted site saturation mutagenesis at Ambler position 237. Only the T237S variant of the
KPC-2 beta-lactamase expressed in Escherichia coli DH10B maintained MICs equivalent to those of the wild type (WT) against all of the
beta-lactams tested, including
carbapenems. In contrast, the T237A variant produced in E. coli DH10B exhibited elevated MICs for only
ampicillin,
piperacillin, and the
beta-lactam-
beta-lactamase inhibitor combinations. Residue 237 also plays a novel role in inhibitor discrimination, as 11 of 19 variants exhibit a
clavulanate-resistant,
sulfone-susceptible phenotype. We further showed that the T237S variant displayed substrate kinetics similar to those of the WT KPC-2
enzyme. Consistent with susceptibility testing, the T237A variant demonstrated a lower k(cat)/K(m) for
imipenem,
cephalothin, and
cefotaxime; interestingly, the most dramatic reduction was with
cefotaxime. The decreases in catalytic efficiency were driven by both elevated K(m) values and decreased k(cat) values compared to those of the WT
enzyme. Moreover, the T237A variant manifested increased K(i)s for
clavulanic acid,
sulbactam, and
tazobactam, while the T237S variant displayed K(i)s similar to those of the WT. To explain these findings, a molecular model of T237A was constructed and this model suggested that (i) the
hydroxyl side chain of T237 plays an important role in defining the substrate profile of the
KPC-2 beta-lactamase and (ii) hydrogen bonding between the
hydroxyl side chain of T237 and the sp(2)-hybridized carboxylate of
imipenem may not readily occur in the T237A variant. This stringent requirement for selected
cephalosporinase and
carbapenemase activity and the important role of T237 in inhibitor discrimination in KPC-2 are central considerations in the future design of
beta-lactam antibiotics and inhibitors.