Table 1.
X-ray diffraction data collection and refinement statistics.
Fig 1.
Crystal structure of peracetate-bound EaEST.
(A) Overall structure of monomeric EaEST is shown in front view (left panel) and top view (right panel) rotated at 90°. The structure of EaEST was drawn as a ribbon diagram with α-helices colored slate blue and β-strands colored orange. Bound peracetate molecule (cyan) is shown as a stick model with a 2Fo-Fc electron-density map (green) at 1.0 σ. The catalytic triad residues (Ser96, Asp220, and His248) are shown as stick models. N- and C-termini are labeled in gray letters and indicated with a red dashed arrow. (B) Multiple sequence alignment of EaEST (NCBI reference sequence number WP_014970431.1), aryl esterase (PDB code 3HEA; UniProtKB code P22862), bromoperoxidase (PDB code 3FOB; UniProtKB code Q81NM3), haloperoxidase (PDB code 1A8S; UniProtKB code O31158), chloroperoxidase (PDB code 4DGQ; UniProtKB code B4EA96), and esterase (PDB code 1ZOI; UniProtKB code Q3HWU8). Secondary structural elements in the crystal structure of EaEST are represented above the multiple sequence alignment. The catalytic triad residues (Ser96, Asp220, and His248) are indicated with red circles and the residues involved in the trimer interaction are indicated by black triangles. The multiple sequence alignment was performed with ClustalX and edited with GeneDoc.
Fig 2.
(A) Analytical ultracentrifugation (AUC) experiments using 2 mg/mL EaEST give a mass of 85.2 kDa (sedimentation coefficient of 5.38 S and a frictional ratio of 1.29), indicating that EaEST is a stable trimer in solution. (B) EaEST trimer has a triangular shape. Each protomer has two binding interfaces for trimerization.
Table 2.
Selected structural homologs of EaEST obtained using DALI search (DALI-Lite server).
Fig 3.
(A) The positive electron density (green) was observed in the Fo-Fc omit map (contoured at 2.0 σ). (B) The peracetate model was overlaid on the Fo-Fc omit map (green, contoured at 2.0 σ). (C) The 2Fo-Fc electron-density map at 1.0 σ is shown after peracetate model building and refinement. (D) Peracetate-binding site is shown. Side chains of residues near ligand recognizing hydrophobic pocket (yellow circle) and active site (red circle) are indicated by sticks. (E) Conformational changes between peracetate-bound EaEST and acetate-bound PfEST (PDB code 3HI4). The β6-α4 loop region of EaEST may undergo conformational change to open the entrance hydrophobic channel for ligand exchange.
Fig 4.
Structural comparisons of ligand-binding site between peracetate-bound EaEST and acetate-bound PfEST.
(A) Stereo view of the superimposed structure of peracetate (cyan)-bound EaEST (green) and acetate (yellow)-bound PfEST (PDB code 3HI4, acetate-bound form, salmon). The residues comprising the active and ligand-binding sites are shown in a stick representation. (B) Peracetate-binding mode and its interactions in EaEST structure. (C) Acetate-binding mode and its interactions in PfEST structure.
Fig 5.
(A) The substrate profiles of EaEST were determined using different p-NP esters (A) and naphthyl derivatives (B). (C) The reaction rate of acetic acid perhydrolysis catalyzed by wild-type EaEST. Perhydrolase activities were measured at pH 5.5 at 25°C. Kinetic constants were obtained by varying the concentration of acetic acid. (D) Specific activities for acetic acid perhydrolysis catalyzed by wild-type EaEST and the S96A mutant. (E) pH stability of EaEST. The pH dependence of hydrolysis of p-NA by EaEST was measured at 25°C. (F) Effect of temperature on the residual activity of EaEST. (G) Thermostability of EaEST. Residual activities are expressed relative to the original activity during incubation at 0, 20, 40, 45, and 50°C. The results are the mean of three individual experiments.
Fig 6.
Effect of NaCl (A) and glycerol (B) on the activity of EaEST. Relative activity was determined by incubating the enzyme with different concentrations of NaCl and glycerol (0 to 5 M). The maximum activity value obtained was set to 100%. (C) Chemical stability of EaEST. Residual activity after 1 h of incubation is expressed relative to the original activity obtained without the addition of chemical compounds (100%).
Fig 7.
(A) pH shift assay for enantioselectivity analysis of EaEST and S96A mutant conducted with (R)- and (S)-methyl-3-hydroxy-2-methylpropionate. (B) Absorbance spectra of the reaction mixtures in (A). (C) Hydrolysis of phenyl-substituted substrates: 1, phenyl acetate; 2, 2-phenylethyl acetate; 3, 2-methylbutyl acetate. (D) Absorbance spectra of 1 from (C) were measured. (E) Hydrolysis of glyceryl esters (glyceryl tributyrate [GTB] and glyceryl trioleate [GTO]) and oils (olive oil [OO] and fish oil [FO]) by EaEST was investigated. (F) Absorbance spectra of GTB hydrolysis by wild-type EaEST were measured.
Fig 8.
(A) Effect of concentration of urea on the activity of EaEST. (B) Intrinsic fluorescence spectra were recorded with increasing concentrations of urea (0 to 5 M).
Fig 9.
Analysis of substrate profiles and enantioselectivity of the L27A mutant.
Substrate profiles of L27A were investigated toward different p-nitrophenyl esters (A) and naphthyl derivatives (B). (C) pH shift assay was conducted in the presence of (R)- or (S)-methyl-3-hydroxy-2-methylpropanoate.
Fig 10.
Scanning electron microscope (SEM) image of cross-linked enzyme aggregates (EaEST-CLEAs). Representative images at 50 kX (A) and 100 kX (B) are shown. (C) Thermostability of immobilized EaEST (■) and soluble EaEST (●) at 80°C. Activity was measured every 15 min. (D) Reusability of immobilized EaEST was compared to the soluble enzyme for 20 reaction cycles. Residual activities were expressed relative to those of free Sm23 (100%).