Crystal structure of KPC-2: insights into carbapenemase activity in Class A β-lactamases

Carbapenems (imipenem, meropenem, and ertapenem) are the “last resort” β-lactam antibiotics for treating serious infections caused by multi-drug resistant gram-negative bacteria. Unfortunately, the acquisition of carbapenem-hydrolyzing β-lactamases by bacteria has resulted in a major threat to the clinical utility of these compounds (1;2). Currently the Klebsiella pneumoniae carbapenemases (KPC-type β-lactamases), are rapidly emerging as a major threat in the New York area (3-5), Pennsylvania (6), and internationally (7;8). Three KPC variants have been found so far (KPC-1,-2, and -3) of which KPC-1 and KPC-2 are almost indistinguishable, whereas KPC-3 exhibits different hydrolytic properties (9). KPC carbapenemases belong to the Ambler class A β-lactamases (E.C. 3.5.2.6) (10;11). In common with other class A β-lactamase, these highly proficient class A enzymes have an efficient hydrolysis ‘machinery’ involving a catalytic serine residue (S70), which acylates β-lactam substrates, and a well-positioned deacylation water. S70 is involved in the nucleophilic attack at the carbonyl carbon of the substrate leading to cleavage of the bond with the β-lactam ring nitrogen for penicillin and other substrates (Figure 1). The deacylation water molecule is primed by E166 and N170 for nucleophilic attack of the β-lactam-acyl-enzyme intermediate and serves to liberate the β-lactam substrate from the active site by deacylation. Both acylation and deacylation involve a nucleophilic attack which is facilitated by a correctly positioned oxyanion hole (formed by the backbone nitrogens of residues 70 and 237) that attracts the carbonyl oxygen atom. These described active site elements are supported by a complex hydrogen bonding network involving a pair of conserved lysines (K37 and K234), N132, and S130. Figure 1 Schematic diagram of penicillin G, a carbapenem (imipenem), cephamycin (cefoxitin), a cephalosporin (cephalothin), and bicine. The bond that is broken during the acylation step involving S70′s nucleophilic attack (curved grey dotted arrow) is ... Serine carbapenemases such as KPCs are unique among the class A β-lactamases because of their ability to hydrolyze β-lactams containing a substituent at the α position of the carbon atom in the β-lactam ring adjacent to the carbonyl (12;13). Examples of such readily hydrolyzed β-lactams by carbapenemases include imipenem (a carbapenem containing a 6α-hydroxy-ethyl moiety) but also cefoxitin (a cephamycin containing a 7α-O-CH3 group)(Figure 1)(9;12;13). Previous crystallographic studies of the class A serine carbapenemase NMC-A and SME-1 did not yield a unified conclusion regarding their carbapenemase phenotype. Interpretation of the NMC-A structure suggested a role for the slightly shifted position of N132 to perhaps accommodate these α-substituents (12;14) whereas the SME-1 structure suggested a needed conformational change for catalysis and importance for the C69-C238 disulfide, being in close proximity of the active site (13). Mutagenesis studies of the C69-C238 residues in SME-1 confirmed the critical role of this disulfide bond, but its function was not limited to carbapenemase activity since its disruption caused loss of hydrolytic activity towards all β-lactam antibiotics (15). Based upon mutagenesis studies it was concluded that no single residue within the active site of SME-1 is solely responsible for carbapenemase activity and that this activity is likely due to multiple simultaneous substitutions and accompanying conformational rearrangements that prime the active site to accommodate and hydrolyze carbapenems and cephamycins (16). The unresolved structural basis of this carbapenemase activity seen in class A enzymes and the urgent clinical threat presented by KPC β-lactamases found in Klebsiella, Enterobacter, and Salmonella spp. prompted our investigation of KPC-2. We present here the structure of KPC-2 determined to 1.85A resolution. Our analysis of KPC-2 in comparison to other class A β-lactamase enzymes reveals alterations in active site topology with respect to non-carbapenemases such as SHV-1 and TEM-1 that could rationalize the structural basis of carbapenemase activity. Furthermore, the active site of KPC-2 contains a well-resolved bicine molecule with its carboxyl moiety occupying a space that is likely involved in recognizing the carboxyl moiety of β-lactams thus providing insights into substrate recognition.