title: Inhibitor resistance in the KPC-2 beta-lactamase, a preeminent property of this class A beta-lactamase.
abstract: As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-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 beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact 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 (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-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 beta-lactamase inhibitors.
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106, USA.
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Papp-Wallace
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.forename: Anne M
authors.initials: AM
authors.lastname: Distler
authors.forename: Courtney
authors.initials: C
authors.lastname: Kasuboski
authors.forename: Magdalena
authors.initials: M
authors.lastname: Taracila
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D019818:Clavulanic Acid / Q000737:chemistry / Q000378:metabolism; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013407:Sulbactam / Q000737:chemistry / Q000378:metabolism; D000078142:Tazobactam; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D019818:Clavulanic Acid; D010397:Penicillanic Acid; C491478:beta-lactamase KPC-2; D001618:beta-Lactamases; D013407:Sulbactam; D000078142:Tazobactam