Structural basis for the extended substrate spectrum of CMY‐10, a plasmid‐encoded class C β‐lactamase

Abstract

SummaryThe emergence and dissemination of extended-spectrum (ES) b -lactamases induce therapeutic failure and a lack of eradication of clinical isolates even by thirdgeneration b -lactam antibiotics like ceftazidime. CMY-10 is a plasmid-encoded class C b -lactamase with a wide spectrum of substrates. Unlike the well-studied class C ES b -lactamase from Enterobacter cloacae GC1, the W -loop does not affect the active site conformation and the catalytic activity of CMY-10. Instead, a three-amino-acid deletion in the R2-loop appears to be responsible for the ES activity of CMY-10. According to the crystal structure solved at 1.55 Å resolution, the deletion significantly widens the R2 active site, which accommodates the R2 side-chains of blactam antibiotics. This observation led us to demonstrate the hydrolysing activity of CMY-10 towards imipenem with a long R2 substituent. The forced mutational analyses of P99 b -lactamase reveal that the introduction of deletion mutations into the R2-loop is able to extend the substrate spectrum of class C non-ES b -lactamases, which is compatible with the isolation of natural class C ES enzymes harbouring deletion mutations in the R2-loop. Consequently, the opening of the R2 active site by the deletion of some residues in the R2-loop can be considered as an operative molecular strategy of class C b -lactamases to extend their substrate spectrum.

Phenotypic and Biochemical Comparison of the Carbapenem-Hydrolyzing Activities of Five Plasmid-Borne AmpC β-Lactamases

“…The side chain of the reactive Ser-64, which attacks the carbonyl carbon of the ␤-lactam ring, is shown in boldface. vealed an open gap in the R2 binding site between the helices H-9, H-10, and the adjacent helix H-11 (12). Other structural differences in the R2 binding site also may contribute to the hydrolytic activity discrepancies.…”

“…The residues of CMY-2 and ACT-1 that constitute the short coil located downstream of the helix H-9 at the edge of the R2 binding site (residues 287 to 289) presented a 0.55-to 1.15-Å shift compared to that of the AmpC ␤-lactamase of E. coli K-12. This structural discrepancy might improve the accommodation of the antibiotic inside the catalytic pocket by reducing the steric hindrances between the R2 substituents of the ␤-lactam rings and the top of the R2 binding site, which is constituted by the residues 287 to 289 (12,16). Similarly, the crystallographic and biochemical study of the CMY-10 ␤-lactamase, which was derived from the plasmid-borne CMY-1 enzyme by an Asn-to-Ile substitution at position 346 and which exhibited increased catalytic efficiency against imipenem, re-FIG.…”

Role of the Ser-287-Asn Replacement in the Hydrolysis Spectrum Extension of AmpC β-Lactamases in Escherichia coli

“…These enzymes conferred higher levels of resistance to ESCs than the wild-type AmpC ␤-lactamases of E. coli (5,8). Therefore, Asn-287 may contribute in part to the natural expanded spectrum of these cephalosporinases, although other structural discrepancies, such as a shortened R2 loop for the CMY-1-like ␤-lactamases (5), may also be involved.…”

Extended-spectrumβ-Lactamases: Implications for the Clinical Laboratory and Therapy

“…Although it has been determined that such mutants were mainly located on bacterial chromosome [14][15][16], two plasmid-mediated extended-spectrum cephalosporinases, CMY-19 [17] and CMY-10 [18], have been reported. Some specialists may insist on regarding such enzymes as ESBLs because of their wide spectrum of activity.…”