As resistance determinants, KPC ␤-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type ␤-lactamases are often identified as resistant to standard ␤-lactam-␤-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to ␤-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (k cat /k inact ratios, where k inact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (k cat /k inact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (k inact /K m ‫؍‬ 6.5 ؎ 0.6 M ؊1 s ؊1 ). We conclude that KPC-2 is unique among class A ␤-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type ␤-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current ␤-lactamase inhibitors.In Klebsiella pneumoniae, ␤-lactam resistance is mediated predominantly by class A SHV, TEM, and CTX-M ␤-lactamases (7, 35). Single amino acid substitutions in the SHV and TEM ␤-lactamases can drastically alter the substrate profiles of the enzymes and confer resistance to extended-spectrum cephalosporins and ␤-lactamase inhibitors (5,12,34,36). ␤-Lactamases with altered substrate profiles (i.e., extended-spectrum or inhibitor-resistant ␤-lactamases) have significantly challenged the clinician's approach to the treatment of serious infectious diseases (36). Thus, the search for effective mechanism-based inhibitors of novel ␤-lactamases merits significant effort (8,9,32).First identified in K. pneumoniae, KPC class A ␤-lactamases threaten the use of all current ␤-lactam antibiotics (57). These ␤-lactamase enzymes are present in an increasing number of bacterial genera, becoming the major carbapenemase expressed by Gram-negative pathogens (e.g., Enterobacter spp., Escherichia coli, Citrobacter freundii, Pseudomonas spp., Serratia marcescens, Proteus mirabilis, and Salmonella enterica) in the United States (3,10,11,16,17,25,37,45,49,53,59). Moreover, KPC ␤-lactamases are becoming geographically widespread (having been detecte...

Carbapenem antibiotics are often the “last resort” in the treatment of infections caused by bacteria resistant to penicillins and cephalosporins. To understand why meropenem is resistant to hydrolysis by the SHV-1 class A β-lactamase, the atomic structure of meropenem inactivated SHV-1 was solved to 1.05 Å resolution. Two conformations of the Ser70 acylated intermediate are observed in the SHV-1-meropenem complex; the meropenem carbonyl oxygen atom of the acyl-enzyme is in the oxyanion hole in one conformation, while in the other conformation it is not. Although the structures of the SHV-1 apoenzyme and the SHV-1-meropenem complex are very similar (0.29 Å rmsd for Cα atoms), the orientation of the conserved Ser130 is different. Notably, the Ser130-OH group of the SHV-1-meropenem complex is directed toward Lys234Nz, while the Ser130-OH of the apo enzyme is oriented toward the Lys73 amino group. This altered position may affect proton transfer via Ser130 and the rate of hydrolysis. A most intriguing finding is the crystallographic detection of protonation of the Glu166 known to be involved in the deacylation mechanism. The critical deacylation water molecule has an additional hydrogen-bonding interaction with the OH group of meropenem’s 6α-1R-hydroxyethyl substituent. This interaction may weaken the nucleophilicity and/or change the direction of the lone pair of electrons of the water molecule and result in poor turnover of meropenem by the SHV-1 β-lactamase. Using timed mass spectrometry, we further show that meropenem is covalently attached to SHV-1 β-lactamase for at least 60 min. These observations explain key properties of meropenem’s ability to resist hydrolysis by SHV-1 and lead to important insights regarding future carbapenem and β-lactamase inhibitor design.

Inhibitor-resistant class A ␤-lactamases are an emerging threat to the use of ␤-lactam/␤-lactamase inhibitor combinations (e.g. amoxicillin/clavulanate) in the treatment of serious bacterial infections. In the TEM family of Class A ␤-lactamases, single amino acid substitutions at Arg-244 confer resistance to clavulanate inactivation. To understand the amino acid sequence requirements in class A ␤-lactamases that confer resistance to clavulanate, we performed site-saturation mutagenesis of Arg-244 in SHV-1, a related class A ␤-lactamase found in Klebsiella pneumoniae. Twelve SHV enzymes with amino acid substitutions at Arg-244 resulted in significant increases in minimal inhibitory concentrations to ampicillin/ clavulanate when expressed in Escherichia coli. Kinetic analyses of SHV-1, R244S, R244Q, R244L, and R244E ␤-lactamases revealed that the main determinant of clavulanate resistance was reduced inhibitor affinity. In contrast to studies in the highly similar TEM enzyme, we observed increases in clavulanate k inact for all mutants. Electrospray ionization mass spectrometry of clavulanate inhibited SHV-1 and R244S showed nearly identical mass adducts, arguing against a difference in the inactivation mechanism. Testing a wide range of substrates with C 3-4 carboxylates in different stereochemical orientations, we observed impaired affinity for all substrates among inhibitor resistant variants. Lastly, we synthesized two boronic acid transition state analogs that mimic cephalothin and found substitutions at Arg-244 markedly affect both the affinity and kinetics of binding to the chiral, deacylation transition state inhibitor. These data define a role for Arg-244 in substrate and inhibitor binding in the SHV ␤-lactamase.␤-Lactams have been the cornerstone of antibacterial chemotherapy since the introduction of penicillin. Although this family of antibiotics now contains numerous classes (penicillins, cephalosporins, and carbapenems, see Fig. 1), their use is threatened by the expansion and spread of ␤-lactamase enzymes (1). These bacterial enzymes hydrolyze ␤-lactam antibiotics before reaching their target, the penicillin-binding proteins. In an effort to retain the utility of several generations of life-saving ␤-lactam antibiotics, ␤-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) have been developed. These mechanism-based inhibitors are ␤-lactam compounds that experience multistep reactions within the active site of ␤-lactamase enzymes (2-4). Typically the inhibitors lack antimicrobial activity and are formulated with a ␤-lactam antibiotic to act as shields against ␤-lactamase enzymes. This allows the ␤-lactam to bypass the ␤-lactamase and inactivate the penicillin binding proteins.Unfortunately, inhibitor-resistant class A ␤-lactamases are emerging in the clinic and undermining the use of ␤-lactam/␤-lactamase inhibitor therapy (5-7). Interest has been renewed in the discovery of novel ␤-lactamase inactivators to circumvent these inhibitor-resistant enzymes (8 -11). In this context, detailed analy...