Structural Basis for Different Substrate Profiles of Two Closely Related Class D β-Lactamases and Their Inhibition by Halogens
| Autor: | Timothy Palzkill, Dar-Chone Chow, Bartlomiej G. Fryszczyn, Liya Hu, Laurent Poirel, B. V. Venkataram Prasad, Patrice Nordmann, V. Stojanoski, Banumathi Sankaran |
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| Rok vydání: | 2015 |
| Předmět: |
Molecular model
Protein Conformation Stereochemistry Ceftazidime Crystal structure Biochemistry beta-Lactamases Article Protein structure Catalytic Domain Enterobacter cloacae Hydrolase polycyclic compounds medicine Humans Enzyme Inhibitors chemistry.chemical_classification integumentary system biology Chemistry Enterobacteriaceae Infections Active site Substrate (chemistry) Iodides biochemical phenomena metabolism and nutrition Anti-Bacterial Agents Cephalosporins Klebsiella Infections Molecular Docking Simulation Klebsiella pneumoniae Enzyme Carbapenems biology.protein bacteria medicine.drug |
| Zdroj: | Biochemistry. 54:3370-3380 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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