title: Crystal structures of KPC-2 β-lactamase in complex with 3-nitrophenyl boronic acid and the penam sulfone PSR-3-226.
abstract: Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated a K(m) value of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed a K(m) value of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (k(inact)) was 0.034 ± 0.003 s(-1). When covalently bound to S70, PSR-3-226 forms a trans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.
authors.affiliation: Departments of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Ke
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Papp-Wallace
authors.forename: Sundar Ram Reddy
authors.initials: SR
authors.lastname: Pagadala
authors.forename: Micheal
authors.initials: M
authors.lastname: Nottingham
authors.forename: Daniel
authors.initials: D
authors.lastname: Fernandez
authors.forename: John D
authors.initials: JD
authors.lastname: Buynak
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.forename: Focco
authors.initials: F
authors.lastname: van den Akker
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D001897:Boronic Acids / Q000737:chemistry*; D015780:Carbapenems / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006574:Heterocyclic Compounds, 2-Ring / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008958:Models, Molecular; D013450:Sulfones / Q000737:chemistry*; D053778:Thiazolidines / Q000737:chemistry* / Q000378:metabolism; D018440:beta-Lactam Resistance / Q000502:physiology; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001897:Boronic Acids; D015780:Carbapenems; D006574:Heterocyclic Compounds, 2-Ring; C574280:PSR-3-226; D013450:Sulfones; D053778:Thiazolidines; D065093:beta-Lactamase Inhibitors; C017562:3-nitrobenzeneboronic acid; C491478:beta-lactamase KPC-2; D001618:beta-Lactamases