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Structural and Functional Studies of Bacterial Enolase, a Potential Target against Gram-Negative Pathogens - PubMed

  • ️Tue Jan 01 2019

Structural and Functional Studies of Bacterial Enolase, a Potential Target against Gram-Negative Pathogens

Jolanta Krucinska et al. Biochemistry. 2019.

Abstract

Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation and RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activities of α-, β-, and γ-substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring play an important role in the biological activity of the investigated compounds. Both α- and β-substituted phenyl derivatives of tropolone were the most active with minimum inhibitory concentrations in the range of 11-14 μg/mL. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography, and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring β-thujaplicin and the α- and β-substituted phenyl derivatives of tropolones with IC50 values in range of 8-11 μM. Ligand binding parameters were assessed by isothermal titration calorimetry and differential scanning calorimetry techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of the position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.

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Figures

Figure 1.
Figure 1.

The natural product hinokitiol (HKT or β-thujaplicin) and key species involved in targeting metalloenzymes.

Figure 2.
Figure 2.

E. coli cells treated with a) DMSO, b) methylated HKT as a negative control, compound 15, c) HKT, d) compound 2

Figure 3.
Figure 3.

DART analysis of E. coli lysate treated with compound 5.

Figure 4.
Figure 4.

(A) Overall structure of E. coli enolase complexed with substrate 2-PGA (PDB ID: 6BFY). Three dimers form a hexameric complex through interaction of the N-terminal T7-tag. (B) 2Fobs-Fcalc electron density of 2-PGA and Mg (II) in the 1.8 Å structure (6BFY) contoured at 2 sigma. (C) Superposition of Chain A (apo-enolase) from 2.2 Å structure (PDB ID: 6BFZ) with 2-PGA-bound enolase (PDB ID: 6BFY). Large conformational changes are seen in three main regions; Loop1 (residues 37–47), Loop2 (residues 151–167) and Loop3 (residues 245–271). (D) The crystal structure of the active site of E. coli enolase (PDB 6BFZ) apo-form (Chain A), (E) with 2-PGA substrate (Chain C) (F) with PEP product (Chain E) (Chain E active site contains a mix between substrate and product, but only the PEP product is shown for clarity). (G) The active site in chain A of PDB 6BFY is fully occupied with 2-PGA substrate as well as two Mg-ions.

Figure 5:
Figure 5:

Binding poses of the tropolone derivatives. (a) Compound 1, (b) 2, (c) 10 and (d) 43. Compounds are shown in different colored sticks. Oxygen is colored red and nitrogen is colored blue. Interactions between the compound and Mg1 are shown as dotted lines.

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