Structural Basis for Different Substrate Profiles of Two Closely Related Class D β-Lactamases and Their Inhibition by Halogens.
OXA-163 and OXA-48 are closely related class D β-lactamases that exhibit different substrate profiles. OXA-163 hydrolyzes oxyimino-cephalosporins, particularly ceftazidime, while OXA-48 prefers carbapenem substrates. OXA-163 differs from OXA-48 by one substitution (S212D) in the active-site β5 strand and a four-amino acid deletion (214-RIEP-217) in the loop connecting the β5 and β6 strands. Although the structure of OXA-48 has been determined, the structure of OXA-163 is unknown. To further understand the basis for their different substrate specificities, we performed enzyme kinetic analysis, inhibition assays, X-ray crystallography, and molecular modeling. The results confirm the carbapenemase nature of OXA-48 and the ability of OXA-163 to hydrolyze the oxyimino-cephalosporin ceftazidime. The crystal structure of OXA-163 determined at 1.72 Å resolution reveals an expanded active site compared to that of OXA-48, which allows the bulky substrate ceftazidime to be accommodated. The structural differences with OXA-48, which cannot hydrolyze ceftazidime, provide a rationale for the change in substrate specificity between the enzymes. OXA-163 also crystallized under another condition that included iodide. The crystal structure determined at 2.87 Å resolution revealed iodide in the active site accompanied by several significant conformational changes, including a distortion of the β5 strand, decarboxylation of Lys73, and distortion of the substrate-binding site. Further studies showed that both OXA-163 and OXA-48 are inhibited in the presence of iodide. In addition, OXA-10, which is not a member of the OXA-48-like family, is also inhibited by iodide. These findings provide a molecular basis for the hydrolysis of ceftazidime by OXA-163 and, more broadly, show how minor sequence changes can profoundly alter the active-site configuration and thereby affect the substrate profile of an enzyme.
Vlatko
V
Stojanoski
Dar-Chone
DC
Chow
Bartlomiej
B
Fryszczyn
Liya
L
Hu
§Medical and Molecular Microbiology "Emerging Antibiotic Resistance" Unit, Department of Medicine, Faculty of Science, University of Fribourg, 1700 Fribourg, Switzerland.
Patrice
P
Nordmann
§Medical and Molecular Microbiology "Emerging Antibiotic Resistance" Unit, Department of Medicine, Faculty of Science, University of Fribourg, 1700 Fribourg, Switzerland.
Laurent
L
Poirel
∥Berkeley Center for Structural Biology, Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States.
Banumathi
B
Sankaran
B V Venkataram
BV
Prasad
Timothy
T
Palzkill
D000900:Anti-Bacterial Agents / Q000378:metabolism; D015780:Carbapenems / Q000378:metabolism; D020134:Catalytic Domain; D002442:Ceftazidime / Q000378:metabolism; D002511:Cephalosporins / Q000378:metabolism; D016972:Enterobacter cloacae / Q000737:chemistry / Q000201:enzymology*; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007454:Iodides / Q000494:pharmacology*; D007710:Klebsiella Infections / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000201:enzymology*; D062105:Molecular Docking Simulation; D011487:Protein Conformation; D001618:beta-Lactamases / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D015780:Carbapenems; D002511:Cephalosporins; D004791:Enzyme Inhibitors; D007454:Iodides; D002442:Ceftazidime; D001618:beta-Lactamases; C108449:oxacillinase