The effect of conformational variability of phosphotriesterase upon N-acyl-L-homoserine lactone and paraoxon binding: insights from molecular dynamics studies

Abstract

The organophosphorous hydrolase (PTE) from Brevundimonas diminuta is capable of degrading extremely toxic organophosphorous compounds with a high catalytic turnover and broad substrate specificity. Although the natural substrate for PTE is unknown, its loop remodeling (loop 7-2/H254R) led to the emergence of a homoserine lactonase (HSL) activity that is undetectable in PTE (kcat/km values of up to 2 × 10(4)), with only a minor decrease in PTE paraoxonase activity. In this study, homology modeling and molecular dynamics simulations have been undertaken seeking to explain the reason for the substrate specificity for the wild-type and the loop 7-2/H254R variant. The cavity volume estimated results showed that the active pocket of the variant was almost two fold larger than that of the wild-type (WT) enzyme. pKa calculations for the enzyme (the WT and the variant) showed a significant pKa shift from WT standard values (ΔpKa = 3.5 units) for the His254 residue (in the Arg254 variant). Molecular dynamics simulations indicated that the displacement of loops 6 and 7 over the active site in loop 7-2/H254R variant is useful for N-acyl-L-homoserine lactone (C4-HSL) with a large aliphatic chain to site in the channels easily. Thence the expanding of the active pocket is beneficial to C4-HSL binding and has a little effect on paraoxon binding. Our results provide a new theoretical contribution of loop remodeling to the rapid divergence of new enzyme functions.

Figure 1

Sequence alignment between the WT PTE and loop7-2/H254R.

Figure 2

(a) The 3D structure alignment between the loop 7-2-H254R (green) and the WT PTE (purple); (b) The protein contact potential of the WT PTE generated with Pymol; (c) The protein contact potential of the loop 7-2-H254R variant; (d) The binding pocket between between the loop 7/H254R (purple) and the WT PTE (blue).

Figure 3

(a) H254 makes three hydrogen bonds with D232, D233, and H257, respectively in WT PTE; (b) R254 makes three hydrogen bonds with D232, D233, and H257, respectively in loop 7-2-H254R variant; (c) Leu271 and F132 function as the entrance gate in WT PTE; (d) Glu263 and F132 function as the entrance gate in loop 7-2-H254R variant.

Figure 4

(a) The 3D structure of paraoxon optimized with Gaussian software at 6-31+G* set; (b) The 3D structure of C4-HSL optimized with Gaussian software at 6-31+G* set.

Figure 5

Paraoxon in the (a) WT PTE; (b) loop 7-2/H254R variant. C4-HSL in the (c) WT PTE; (d) loop 7-2/H254R variant. (e) Calculate and visualize molecular hydrophobic/hydrophilic properties using the concept of “Molecular Hydrophobicity Potential” (MHP). Hydrophobic and stacking interactions in paraoxon-PTE complexes can be used to re-rank the results of molecular docking. Color grey represent for a match and color brown represent for a mismatch; (f) Hydrophobic and stacking interactions in paraoxon-loop 7-2/H254R complexes; (g) Hydrophobic and stacking interactions in C4-HSL-WT PTE; (h) Hydrophobic and stacking interactions in C4-HSL-loop 7-2/H254R variant.

Figure 6

(a) The active residue of WT PTE around paraoxon calculated by the Discovery Studio 3.5 client. Residue interaction: pink: eletrostatic, green: van der Waals, blue: water interaction, black: metal interaction; (b) The active residue of loop 7-2/H254R variant around paraoxon; (c) The active residue of WT PTE around C4-HSL (residue number used PTE); (d) The active residue of loop 7-2/H254R variant around C4-HSL.

Figure 7

(a) Root-mean-square deviation (RMSD) of backbone atoms of PTE from the X-ray structure (1HYZ) for the substrate-bound state: paraoxon (black) and C4-HSL (red). (b) Root-mean-square deviation (RMSD) of backbone atoms of the loop 7-2/H254R variant for the substrate-bound state: paraoxon (black) and C4-HSL (red). Values are averaged for the two monomers.

Figure 8

Atom-positional root-mean-square fluctuations (RMSF) of backbone atoms averaged per residue (amino acid 220 to 280) (a) for PTE (1HYZ) substrate free (black), substrate-bound (1HYZ- paraoxon (red) and 1HYZ-C4-HSL (green); (b) for loop7-2/H254 R variant substrate (black), substrate-bound (loop7-2/H254 R - paraoxon (red) and loo7-2/H254 R-C4-HSL). Values are averaged for the two OPH monomers and over 30 ns of simulations.

Figure 9

Radius of gyration (Rg) for the WT and loop 7-2/H254R variant. (a) WT PTE (red), loop 7-2/H254R (black); (b) Rg_x WT PTE (red), loop 7-2/H254R (black); (c) Rg_y WT PTE (red), loop 7-2/H254R (black); (d) Rg_z WT PTE (red), loop 7-2/H254R (black).

Figure 10

(a) Solvent accessible surface area. PTE (red), loop 7-2/H254R (black); (b) hydrophobic area. PTE (red), loop 7-2/H254R (black); (c) hydrophobic area. PTE (red), loop 7-2/H254R (black).

Figure 11

The distance between the Cα of L271 and F132 in the WT enzyme (black). The distance between the Cα of E263 and F132 in the loop 7-2/H254R (red).