Molecular Basis for the Catalytic Specificity of the
CTX‑M Extended-Spectrum β‑Lactamases
- Publication date
- Publisher
Abstract
Extended-spectrum β-lactamases
(ESBLs) pose a threat to public
health because of their ability to confer resistance to extended-spectrum
cephalosporins such as cefotaxime. The CTX-M β-lactamases are
the most widespread ESBL enzymes among antibiotic resistant bacteria.
Many of the active site residues are conserved between the CTX-M family
and non-ESBL β-lactamases such as TEM-1, but the residues Ser237
and Arg276 are specific to the CTX-M family, suggesting that they
may help to define the increased specificity for cefotaxime hydrolysis.
To test this hypothesis, site-directed mutagenesis of these positions
was performed in the CTX-M-14 β-lactamase. Substitutions of
Ser237 and Arg276 with their TEM-1 counterparts, Ala237 and Asn276,
had a modest effect on cefotaxime hydrolysis, as did removal of the
Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A
double mutants, however, exhibited 29- and 14-fold losses in catalytic
efficiency for cefotaxime hydrolysis, respectively, while the catalytic
efficiency for benzylpenicillin hydrolysis was unchanged. Therefore,
together, the Ser237 and Arg276 residues are important contributors
to the cefotaximase substrate profile of the enzyme. High-resolution
crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A
variants alone and in complex with cefotaxime show that residues Ser237
and Arg276 in the wild-type enzyme promote the expansion of the active
site to accommodate cefotaxime and favor a conformation of cefotaxime
that allows optimal contacts between the enzyme and substrate. The
conservation of these residues, linked to their effects on structure
and catalysis, imply that their coevolution is an important specificity
determinant in the CTX-M family