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

Thermodynamic Investigation of Inhibitor Binding to 1-Deoxy-d-Xylulose-5-Phosphate Reductoisomerase

Isothermal titration calorimetry (ITC) was used to investigate the binding of six inhibitors to 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), a target for developing novel anti-infectives. The binding of hydroxamate inhibitors to <i>Escherichia coli</i> DXR is Mg²⁺-dependent, highly endothermic (Δ<i>H</i>, 22.7–24.3 kJ/mol), and entropy-driven, while that of nonhydroxamate compounds is metal ion-independent and exothermic (Δ<i>H</i>, −19.4 to −13.8 kJ/mol), showing that hydration/dehydration of the enzyme metal ion binding pocket account for the drastic Δ<i>H</i> change. However, for DXRs from <i>Plasmodium falciparum</i> and <i>Mycobacterium tuberculosis</i>, the binding of all inhibitors is exothermic (Δ<i>H</i>, −24.9 to −9.2 kJ/mol), suggesting that the metal ion binding sites of these two enzymes are considerably less hydrated. The dissociation constants measured by ITC are well correlated with those obtained by enzyme inhibition assays (<i>R</i>² = 0.75). Given the rapid rise of antibiotic resistance, this work is of interest since it provides novel structural implications for rational development of potent DXR inhibitors

Extreme Sensory Complexity Encoded in the 10-Megabase Draft Genome Sequence of the Chromatically Acclimating Cyanobacterium Tolypothrix sp. PCC 7601

Tolypothrix sp. PCC 7601 is a freshwater filamentous cyanobacterium with complex responses to environmental conditions. Here, we present its 9.96-Mbp draft genome sequence, containing 10,065 putative protein-coding sequences, including 305 predicted two-component system proteins and 27 putative phytochrome-class photoreceptors, the most such proteins in any sequenced genome