Figure 1.
Enzymatic conversion of ortho-diphenols to the corresponding quinones by PPOs.
Figure 2.
Superposition of the dandelion PPO models with the modeling template.
3D models of the tyrosinase domains of PPOs 1, 2, 6, and 7 were generated using the I-Tasser online structure prediction tool [31]. The crystal structure of IbCO (PDB 1BT3) [23] was used as the template for structure prediction. The coordinating histidine residues of IbCO (HA1–A3 and HB1–B3) were drawn and labeled accordingly, as well as the bound Cu-ions (CuA and CuB) and the hydroxyl ion (OH−) of the met-form.
Table 1.
Evaluations of the molecular models generated for PPOs [23], [32], [34].
Figure 3.
Structures of the diphenolic substrates used for kinetic analysis.
Table 2.
Molar extinction coefficients (ex nm) of PPO substrates determined at pH 5 [42].
Figure 4.
Sequence analysis of the dandelion PPO family.
Amino acid sequences of all PPOs were aligned using MUSCLE v3.7 [28]. Identical and similar residues are shaded in black and gray, respectively. Domain structure was analyzed [13], [43]–[44] and findings were marked as follows. The predicted transit peptide is labelled and the predicted cleavage site of the stromal processing peptidase is marked with an arrow. The catalytic tyrosinase domain, linker region, C-terminal domain, and copper-binding histidines of the CuA (HA1–HA3) and CuB (HB1–HB3) sites are labelled. Structurally important residues are marked by triangles (Δ) and potentially regulative residues by circles (○). The predicted β-strands of the conserved β-sandwich C-terminal domain are marked by straight underlining ( _ ) and the conserved helix by dotted underlining (…). Cysteines of potential multimerization sites [20] are boxed and their position is marked by a diamond (◊). Cysteines potentially involved in two intramolecular disulfide bonds are labeled S1 and S2. [S, disulfide linkage; *, thioether bridge; h, hydrogen bond; pi, π-cation interaction; g, gate residue; b, blocking residue].
Table 3.
Overview of the PPO family in dandelion.
Figure 5.
Phylogenetic tree of the dandelion PPO family.
PPO amino acid sequences were aligned using MUSCLE v3.7 [28]. The phylogenetic tree was constructed using the maximum likelihood method implemented in Phylemon v2.0 [29]. The reliability of internal branches was assessed using the bootstrap method (1000 replicates). PPOs selected for further characterization are underlined.
Figure 6.
Molecular modeling and docking studies of dandelion PPOs.
PPO-2 and PPO-6 are shown here as representatives of each phylogenetic group. Surfaces and stick models are colored according to atom type (blue, nitrogen; red, oxygen; gray, carbon; white, hydrogen) and the following special designations: * = gate residue (phenylalanine); gold spheres = copper atoms; red sphere = bound oxygen in met-form. (A) Surface contour images of the catalytic pocket, showing the crystallographic structure of IbCO (PDB 1BUG,A) and homology models of PPO-2 (group 1) and PPO-6 (group 2). The substrate analog phenylthiourea (PTU, stick representation) occupies the substrate binding site in the hydrophobic cavity. For comparison, the binding of CAT (stick representation) as the simplest substrate is shown in the active site for the modeled dandelion PPOs. Residues HB1+1 and HB2+1 are labeled at the entrance of the catalytic pocket. (B) Predicted interactions of the substrate in the active site resulting from docking analysis. The position and interaction of IbCO with PTU is shown as a reference. PPO-2 and PPO-6 are shown binding to Dopac (green), DA (yellow) and L-Dopa (purple); only polar hydrogen atoms are drawn for the substrates. For PPO-6, different R254 side chain rotamers resulted from binding of the different substrates. The rotamers are drawn as thin stick and are colored green, yellow or purple according to the corresponding substrate. (C) Sequence alignment of the copper-binding sites (CuA and CuB) of all eleven dandelion PPOs. Identical and similar residues are shaded in black and gray, respectively. The Cu-binding histidine residues (HA1–A3, HB1–B3) are labeled, and arrows indicate the HB1+1 and HB2+1 positions. The gate residue is shaded in yellow. Residues located within 8Å of the copper centers in 3D space are boxed in yellow.
Table 4.
Docking studies.
Figure 7.
The gene sequences for dandelion PPOs 1, 2, 6 and 7 (excluding the transit peptide) were each supplemented with an N-terminal Strep2 tag using the enterokinase recognition site as a spacer. Proteins were purified by StrepTactin affinity chromatography and 3-µg samples were analyzed by SDS-PAGE using a non-reducing loading dye, followed by staining with Coomassie Brilliant Blue.
Figure 8.
Optimal conditions for PPO activity.
The activity of purified recombinant PPOs was monitored by spectrophotometry at 405-methylcatechol. (A) The influence of pH on PPO activity was determined using 100 mM acetate-phosphate buffer with the pH adjusted by adding NaOH. Activities were measured in the presence of 0.75 mM SDS (PPOs 2, 6 and 7) or 1.0 mM SDS (PPO-1). (B) Influence of SDS concentration on PPO activity. All measurements were taken at the optimal pH for each PPO (pH 5.0 for PPO 6 and 7, pH 5.5 for PPO-1, and pH 6.0 for PPO-2). All values are means ± standard deviation from one representative experiment measured in triplicate.
Table 5.
Kinetic parameters for recombinant PPOs.
Figure 9.
Comparison of kinetic parameters for the dandelion PPO groups.
(A) Michaelis constants (Km values). (B) Turnover rates (kcat values). (C) Catalytic efficiencies (kcat/Km ratios). The group values for kinetic parameters were calculated from original data (n = 70 for group 1, n = 84 for group 2) as means ± SEM using error propagation. The two groups were compared in terms of Km, kcat and efficiency, and are indicated on the bar graphs as p-values determined in z-tests.