Structural and Functional Aspects of Class A Carbapenemases

Abstract

The fight against infectious diseases is probably one of the greatest public health challenges faced by our society, especially with the emergence of carbapenem-resistant gram-negatives that are in some cases pan-drug resistant. Currently,β-lactamase-mediated resistance does not spare even the newest and most powerful β-lactams (carbapenems), whose activity is challenged by carbapenemases. The worldwide dissemination of carbapenemases in gram-negative organisms threatens to take medicine back into the pre-antibiotic era since the mortality associated with infections caused by these "superbugs" is very high, due to limited treatment options. Clinically-relevant carbapenemases belong either to metallo-β- lactamases (MBLs) of Ambler class B or to serine-β-lactamases (SBLs) of Ambler class A and D enzymes. Class A carbapenemases may be chromosomally-encoded (SME, NmcA, SFC-1, BIC-1, PenA, FPH-1, SHV-38), plasmid-encoded (KPC, GES, FRI-1) or both (IMI). The plasmid-encoded enzymes are often associated with mobile elements responsible for their mobilization. These enzymes, even though weakly related in terms of sequence identities, share structural features and a common mechanism of action. They variably hydrolyse penicillins, cephalosporins, monobactams, carbapenems, and are inhibited by clavulanate and tazobactam. Three-dimensional structures of class A carbapenemases, in the apo form or in complex with substrates/inhibitors, together with site-directed mutagenesis studies, provide essential input for identifying the structural factors and subtle conformational changes that influence the hydrolytic profile and inhibition of these enzymes. Overall, these data represent the building blocks for understanding the structure-function relationships that define the phenotypes of class A carbapenemases and can guide the design of new molecules of therapeutic interest.

Fig. (1)

General catalytic pathway of active-site serine β-lactamases. This mechanism is schematically represented by a three-step model. E is the enzyme.

Fig. (2)

Phylogeny of several chromosomally- and plasmid-encoded class A β-lactamases. For naturally-occurring class A β-lactamases, the bacterial host name is indicated. The carbapenemases are highlighted in bold. The dendrogram was constructed with FigTree [21] using ClustalW [22] aligned protein sequences. The references of all these enzymes can be found in the Beta-Lactamase DataBase at http://www.bldb.eu/BLDB.php?class=A.

Fig. (3)

Sequence alignment of representative class A carbapenemases. The stars highlight the four conserved motifs that are characteristic of class A β-lactamases: ⁷⁰SXX⁷³K, ¹³⁰SD¹³²N, ¹⁶⁶E and ²³⁴KT²³⁶G (ABL numbering scheme [18]). The triangles indicate the positions of cystein residues involved in the ⁶⁹S²³⁸S disulfide bond. The image was generated with ESPript [23].

Fig. (4)

Superposition of all three-dimensional structures of class A carbapenemases available in the PDB (see Table 7 for the corresponding PDB codes): KPC-2 in blue (7), GES-2 in red (3), GES-5 in orange (2), GES-18 in yellow (1), NmcA in green (2), SFC-1 in cyan (3), SME-1 in magenta (1), PenA in gray (1). The image was generated using Chimera [137].

Fig. (5)

Three-dimensional structures of active site residues of class A β-lactamases with and without carbapenem-hydrolyzing activity, colored in green and orange, respectively (see the main text for details).

Fig. (6)

Superposition of X-ray structures of KPC-2 (apo form, blue, PDB 2OV5) and CTX-M-15 (complex with avibactam, orange, PDB 4S2I).