Tyrosinase is a key enzyme in melanogenesis. Generally, mushroom tyrosinase from
Tyrosinase is a metalloprotein belonging to type 3 copper enzyme family. It is involved in melanin production in a wide range of organisms. The enzyme has a bifunctional catalytic mechanism consisting of the hydroxylation of monophenols to
Tyrosinase is a key enzyme in melanogenesis, which is essential for pigmentation. The catalysis of L-tyrosine to L-dopa is the rate-limiting step of the enzymatic pathway in melanin formation. Tyrosinase is also an important factor in wound healing and cuticle formation in arthropods and browning in plants [
The crystal structure of
In this study, three-dimensional models of tyrosinase were studied focusing on binding structure with four common inhibitors: arbutin, ascorbic acid, kojic acid, and tropolone for screening and prediction of potent inhibitors of tyrosinases. The chelation of copper at the active site of the enzyme explains well the inhibitory effect of kojic acid, Chen et al., 1991 [
Homology modeling is the most reliable method for prediction of three-dimensional structures of unknown protein based on the assumption that the structure of the unknown protein is similar to the known structures of some homologous reference proteins [
To analyze binding scaffold of substrates and inhibitors with tyrosinase, molecular docking and dynamics simulations were carried out. Molecular docking was carried out using AutoDock4.0 software for prediction of binding structures of tyrosinase with inhibitors (ascorbic acid, arbutin, kojic acid, and tropolone). This software employs a semiempirical force field based on a comprehensive thermodynamic model and a Lamarckian genetic algorithm (LGA) for the conformational search [
Molecular dynamics (MD) simulations were performed using the AMBER 12 program [
The crystal structure of
Sequence alignment between human amino acid sequence (AAA61242) and crystal structure of bacterial tyrosinase (3NQ1) with identity of 33.5% and similarity of 50.7%. Six histidine residues, which are provided by a four-helical bundle, coordinate the two copper ions (CuA and CuB) in the active site [
A superposition of the three-dimensional structures of the human homology model and that for
Superimposition of homology model (red) and its template (blue).
The model was validated using PROCHECK and Verify 3D. The Ramachandran plot of human tyrosinase (Figure
Quality of validation of the homology model. (a) Ramachandran plot of human tyrosinase. (b) Residues in disallowed region are Asp59, Lue74, Trp80, Ser152, and Cys174. (c) Verify 3D plot.
Comparison of docking result with experimental data is shown in Table
Docking score and experimental data in terms of binding structure/activity of tyrosinase from mushroom, bacteria, and human.
Inhibitors/substrate | Mushroom tyrosinase | Bacterial tyrosinase | Human tyrosinase | |||
---|---|---|---|---|---|---|
Binding energy |
|
Binding energy |
|
Binding energy |
|
|
(kcal/mol) | (mM) | (kcal/mol) | (mM) | (kcal/mol) | (mM) | |
|
−4.45 | 0.0074–0.68 [ |
−5.06 | — | −6.00 | 0.50–2.73 [ |
|
||||||
|
−4.86 | 0.0004–0.0017 [ |
−4.30 | — | −5.93 | — |
|
||||||
|
−4.63 | ≥0.02 [ |
−5.16 | — | −4.83 | ≥0.1 [ |
|
||||||
|
−4.35 | 0.04–7.3 [ |
−6.09 | — | −4.80 | 1.43–6.50 [ |
|
||||||
|
−10.00 | 0.2 [ |
−11.09 | 0.075 [ |
−11.66 | 0.17 [ |
|
||||||
|
−10.20 | 0.17 [ |
−10.05 | 0.35 [ |
−11.15 | 0.36 [ |
Among these inhibitors tropolone is the best inhibitor of mushroom tyrosinase with the range of the lowest IC50 values of 0.0004–0.0017 mM and binding energy of −4.86 kcal/mol. Ascorbic acid had an IC50 value greater than or equal to 0.02 mM, and the IC50 value for kojic acid was 0.0074–0.68 mM and 0.04–7.3 mM for arbutin. The binding energy is at −4.63, −4.45, and −4.35 kcal/mol for ascorbic acid, kojic acid, and arbutin, respectively. In the case of human tyrosinase, ascorbic acid had the lowest IC50 value and binding energy of ≥0.1 mM and −4.83 kcal/mol. The IC50 value and binding energy for kojic acid were 0.50–2.73 mM and −6.00 kcal/mol, values for arbutin were 1.43–6.50 mM and −4.80 kcal/mol, and tropolone had a binding energy of −5.93 kcal/mol. These results correlate with the higher
To demonstrate attribution of thermal motions on the change of binding site, MD simulations were performed. The binding configuration was rearranged during simulation to observe conformation changes in each time step in comparison with the initial structure. The root mean square deviation of backbone carbon values of the complexes compared with the initial structures is shown in Figure
RMSD of carbon backbone in complexes: (a) mushroom tyrosinase-inhibitor complexes, (b) bacterial tyrosinase-inhibitor complexes, and (c) human tyrosinase-inhibitor complexes: arbutin (blue), ascorbic acid (red), kojic acid (green), and tropolone (purple).
The mushroom tyrosinase-arbutin complex is shown in Figure
The comparison of interaction site found in docked (in parentheses) and MD structures.
Inhibitors | Mushroom tyrosinase | Bacterial tyrosinase | Human tyrosinase | |||
---|---|---|---|---|---|---|
H bonding | Pi interaction | H bonding | Pi interaction | H bonding | Pi interaction | |
Kojic acid | 1:M280 |
H263 |
— |
H208 |
— |
H252 |
|
||||||
Tropolone | — |
H263 |
— |
H60 |
— |
— |
|
||||||
Ascorbic acid | 1:N81 |
— |
2:E195 |
— |
1:E230 |
— |
|
||||||
Arbutin | 1:N60 |
H263 |
— |
H208 |
1:S245 |
H252 |
Binding structure of mushroom tyrosinase and inhibitors: (a) arbutin, (b) ascorbic acid, (c) kojic acid, (d) tropolone, (e) distance measurement of hydrogen bond, and (f) distance measurement of pi interaction.
The binding site of the bacterial tyrosinase-arbutin complex is shown in Figure
Binding structure of bacterial tyrosinase and inhibitors: (a) arbutin, (b) ascorbic acid, (c) kojic acid, (d) tropolone, (e) distance measurement of hydrogen bond, and (f) distance measurement of pi interaction.
The human tyrosinase-arbutin complex is shown in Figure
Binding structure of human tyrosinase and inhibitors: (a) arbutin, (b) ascorbic acid, (c) kojic acid, (d) tropolone, (e) distance measurement of hydrogen bond, and (f) distance measurement of pi interaction.
In our work, from homology modeling, the 3D structure of human tyrosinase was validated and selected for use in simulation. Binding scaffolds were simulated using molecular docking and molecular dynamics simulation. The binding energy estimated from the simulations was found to be correlated well with the
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors gratefully acknowledge the partial financial support from Center of Excellence for Innovation in Chemistry, Center of Excellence in Materials Science and Technology, the Mid-Career Researcher Fund, and co-research grant from International College, Chiang Mai University, Thailand.