Structure-Activity Relationship Study of Hydroxycoumarins and Mushroom Tyrosinase

Article abstract of DOI:10.1021/acs.jafc.5b02636

The structure-activity relationships of four hydroxycoumarins, two with the hydroxyl group on the aromatic ring of the molecule and two with the hydroxyl group replacing hydrogen of the pyrone ring, and their interactions with mushroom tyrosinase were studied. These compounds displayed different behaviors upon action of the enzyme. The two compounds, ar-hydroxylated 6-hydroxycoumarin and 7-hydroxycoumarin, were both weak substrates of the enzyme. Interestingly, in both cases, the product of the catalysis was the 6,7-hydroxycoumarin, although 5,6- and 7,8-isomers could also theoretically be formed. Additionally, both were able to reduce the formation of dopachrome when tyrosinase acted on its typical substrate, l-tyrosine. Although none of the compounds that contained a hydroxyl group on the pyrone ring were substrates of tyrosinase, the 3-hydroxycoumarin was a potent inhibitor of the enzyme, and the 4-hydroxycoumarin was not an inhibitor. These results were compared with those obtained by in silico molecular docking predictions to obtain potentially useful information for the synthesis of new coumarin-based inhibitors that resemble the structure of the 3-hydroxycoumarin.

Full text of DOI:10.1021/acs.jafc.5b02636

Article
pubs.acs.org/JAFC
Structure−Activity Relationship Study of Hydroxycoumarins and
Mushroom Tyrosinase
Shailendra Asthana,∥,† Paolo Zucca,∥,§,# Attilio V. Vargiu,⊥ Enrico Sanjust,§ Paolo Ruggerone,⊥
and Antonio Rescigno*,§
†Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), NCR Biotech Science
Cluster, Third Milestone, Faridabad-Gurgaon Expressway, Haryana 121001, India
§Dipartimento di Scienze Biomediche, Universita
̀
degli Studi di Cagliari, Cittadella Universitaria, 09042 Monserrato, Cagliari, Italy
#Consorzio UNO Universita
̀
Oristano, 09170 Oristano, Italy
⊥Dipartimento di Fisica, Universita
̀
degli Studi di Cagliari, Cittadella Universitaria, 09042 Monserrato, Cagliari, Italy
S
* Supporting Information
ABSTRACT: The structure−activity relationships of four hydroxycoumarins, two with the hydroxyl group on the aromatic ring
of the molecule and two with the hydroxyl group replacing hydrogen of the pyrone ring, and their interactions with mushroom
tyrosinase were studied. These compounds displayed different behaviors upon action of the enzyme. The two compounds, ar-
hydroxylated 6-hydroxycoumarin and 7-hydroxycoumarin, were both weak substrates of the enzyme. Interestingly, in both cases,
the product of the catalysis was the 6,7-hydroxycoumarin, although 5,6- and 7,8-isomers could also theoretically be formed.
Additionally, both were able to reduce the formation of dopachrome when tyrosinase acted on its typical substrate, L-tyrosine.
Although none of the compounds that contained a hydroxyl group on the pyrone ring were substrates of tyrosinase, the 3-
hydroxycoumarin was a potent inhibitor of the enzyme, and the 4-hydroxycoumarin was not an inhibitor. These results were
compared with those obtained by in silico molecular docking predictions to obtain potentially useful information for the
synthesis of new coumarin-based inhibitors that resemble the structure of the 3-hydroxycoumarin.
KEYWORDS: tyrosinase, polyphenol oxidase, hydroxycoumarins, docking, structure−activity, inhibition, melanin
bonds and, in the case of His85, on an uncommon thioether
bridge formed by the residue Cys83. The available information
about the detailed 3D structure of the H subunit allows for an
in-depth study of the interactions between the active site and its
substrates/inhibitors. This could result in applications to food
technology (such as vegetable browning),4 skin pathologies
(such as pigmentation upsets),5 technological and pharmaceut-
ical processes,6,7 and other biotechnology fields.
For these reasons, we performed a structure−activity
relationship study that included kinetics experiments and
computational docking between MT and some selected
hydroxy-substituted coumarins. Some coumarin derivatives
show bioactive properties.8−10 Hydroxycoumarins (hydroxy-
1,2-benzopyrones) are common among higher plants, including
edible vegetables and fruits, and are well-known to be
bioactive.11,12 We chose and tested some commercially
available monohydroxycoumarins as putative substrates or
inhibitors for MT; their names and chemical structures are
summarized in Scheme 1. For each compound, kinetic data of
their activity as a substrate or inhibitor were collected and
compared with the molecular docking studies. This study aimed
to better understand the mechanism of the interactions
between monohydroxycoumarins and MT, thereby identifying
INTRODUCTION
■
Polyphenol oxidases are copper enzymes that are widespread
among microorganisms, plants, and animals. They catalyze the
ortho-hydroxylation of monophenols, which leads to the
corresponding o-diphenols (catechols), as well as the oxidation
of catechols, which leads to the corresponding o-quinones.1
Under many conditions, these then react further to result in the
formation of various pigments. It is important to note that the
enzyme nomenclature for tyrosinase varies, depending on the
enzyme sources, and includes phenolase, phenol oxidase, and
polyphenol oxidase. Whereas polyphenol oxidase (PPO) is
perhaps the most generic, we use the acronym MT to
specifically differentiate mushroom tyrosinase from Agaricus
bisporus. Within its active site, MT contains two nonidentical
cupric ions, CuA and CuB, that are each coordinated by three
imidazole rings from three histidine residues (see Figure 1A,B).
This dicupric cluster is well-defined as a spectroscopic type III
dicopper center (electron paramagnetic resonance (EPR) silent
in both cuprous and cupric states). Recently, the three-
dimensional (3D) structure of the A. bisporus enzyme has been
reported.2,3 MT, by far the most studied PPO, is a
heterotetrameric protein that is formed by two H subunits
(392 amino acid residues) and two L subunits (150 amino acid
residues) and has a molecular weight of approximately 120 kDa.
The role of the two L subunits is not defined, whereas the two
H subunits contain the two dicupric clusters that are
responsible for catalytic activity. The orientation of the
coordinating histidines depends on the presence of hydrogen
Received: May 28, 2015
Revised: July 20, 2015
Accepted: July 30, 2015
© XXXX American Chemical Society
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The monophenolase activity using L-tyrosine as its substrate was
measured at 468 nm by following the formation of dopachrome with
an Ultrospec 2100 Pro UV−vis (Amersham Biosciences) spectropho-
tometer in the presence of various putative substrates or inhibitors. A
control reaction was also conducted. Diphenolase activity was
monitored spectrophotometrically by tracking the adduct formation
between 4-tert-butyl-1,2-benzoquinone and 4-amino-N,N-diethylani-
line at 625 nm (ε625 = 11120 M−1 cm−1).18,19 The percentage of
inhibition (I %) of the enzyme activity was also calculated according to
the equation I% = (ΔA − ΔB)/ΔA × 100, where ΔA is the difference
in the absorbance of the control sample between two incubation times
when the absorbance increased linearly and ΔB is the difference in the
absorbance of the test sample calculated in the same range of time.20
The results were the average of at least three readings. Other details
are given in the text and in the figure captions.
HPLC Analysis and Sample Preparation. Identification of the
products was performed by HPLC analysis using a Beckman System
Gold apparatus that was equipped with an UV−vis detector module.
An RPC18 column (Phenomenex, Luna, 250 mm × 3 mm i.d., 5 μm),
purchased from Chemtek Analytica (Bologna, Italy), was equilibrated
with 0.085% phosphoric acid and 5% methanol in water, v/v (solvent
A). Solvent B was 0.085% phosphoric acid and 95% methanol in water,
v/v. After an initial isocratic elution (10% B for 10 min), a gradient
phase of 10−90%, using solvent B, was applied for 10 min (1 mL/min
flow rate).
Before the HPLC run was performed, aliquots of the samples were
withdrawn at a fixed time, deproteinized by adding phosphoric acid up
to 0.085% v/v, and centrifuged at 8000g for 10 min. The supernatants
were then filtered using a 0.22 μm pore size membrane.
Molecular Docking. Protein Modeling. The X-ray crystal
structures of MT (PDB entries 2Y9X and 2Y9W)3 were used as
templates for molecular docking studies of the enzyme in complex
with our compounds. Note that the crystal structure denoted with the
PDB entry 2Y9X contained tropolone, a well-known inhibitor that
binds to the active site of the enzyme.
Figure 1. Structural comparison between 2Y9W and 2Y9X: (A)
superimposition of 2Y9W (blue) and 2Y9X (white) (binding site
residues are rendered in licorice and in an atom-wise type: C, white; O,
red; N, blue; S, yellow; whereas tropolone is shown in licorice and in a
cyan color); (B) inset view of tropolone binding site; (C) RMSD per
residue of the two structures under study.
Scheme 1. Chemical Structure of the Hydroxy-Substituted
Coumarins
The protein structures were prepared using the Amber tool Xleap,21
which included all hydrogen atoms. The orientation of polar hydrogen
atoms was optimized, the protein protonation state was adjusted, and
each structure was minimized with harmonic restraints on the heavy
atoms. Successively, all hetero groups and water molecules were
deleted. The structures of the enzyme were compared through root-
mean-square distance (RMSD) analysis by superimposing the 2Y9W
and 2Y9X structures. The RMSD for Cα is low (∼0.3 Å), which
indicates that the structures are similar (Figure 1C). Because no
significant difference was observed in the protein structures,
particularly in the binding site, the prepared cocrystal structure
(after removal of tropolone) was used for docking studies.
Ligand Modeling. The lead molecules were first drawn with ACD
Chem Sketch 11.0 (http://www.acdlabs.com/resources/freeware/
chemsketch/). The resulting molecular geometry was then optimized
at the B3 LYP/6-31G (d, p) level until a convergence, in energy, of
10−5 au using the Gaussian 03 package.22
Docking Protocol. Because no explicit information concerning the
interaction between MT and the compounds under investigation has
been reported previously, we searched for putative binding sites on the
enzyme using two different protocols. These protocols were modified
from our previous works.23,24 First, we exploited the available empirical
data from the guided docking runs concerning the binding of
tropolone to the enzyme.3 To assess the validity of our results, we
performed blind docking calculations. More details about the docking
steps and binding site detection programs are provided in the next
sections.
From the ligand clusters that were obtained from blind docking on
the protein surface, the top five clusters were chosen for further
analysis. We then compared these poses with those obtained from
guided docking. Once consensus sites were identified, we performed
focused docking (“redocking”) calculations that centered the docking
grid on the center of mass of our compounds in the top pose. All
docking runs were performed with AutoDock version 4.2.25 This
program uses a semiempirical, free energy force field to evaluate
lead compounds for the synthesis of new potent PPO inhibitors
that are suitable for biotechnological applications.13−15
MATERIALS AND METHODS
■
Mushroom Tyrosinase and Enzymatic Assay. MT (5771 U/
mg) was purchased from Sigma-Aldrich (Milan, Italy). A stock
solution of the enzyme contained 1 mg/mL. Before use, the enzyme
solution was accurately inspected by means of syringaldazine, a typical
and sensitive laccase substrate, which allowed us to exclude any laccase
contamination. This was necessary because some lots of commercial
tyrosinase contain laccase activity.16,17 This could lead to measure-
ments with ambiguous results as contaminant laccases could share the
same substrates with tyrosinase (i.e., o-diphenols). A tyrosinase unit
was defined according to the manufacturer’s definition and calculated
such that 1 unit will cause an increase in A280 of 0.001 per minute at
pH 6.5 and 25 °C in a 3 mL reaction mix containing L-tyrosine. All
experiments were performed in 100 mM potassium phosphate buffer,
pH 6.5, at 25 °C.
B
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conformations during docking simulations. More details about the
formulation of the scoring function and its testing can be found in the
manual for the program (http://autodock.scripps.edu/faqs-help/
manual/autodock-4-2-user-guide/AutoDock4.2_UserGuide.pdf, pp
8−11). The binding site is composed of two copper ions that are
coordinated by three histidine residues (for CuA, H61, H85, and H94;
and for CuB, H259, H263, and H296). The other residues involved in
the stability of compound bonding are as follows: C83, F90, E256,
N260, H263, M280, G281, S282, and F292 (see Figure 1B).
Therefore, in all docking runs, we modeled the flexibility of these
residues.
hydroxylated on the aromatic ring. Of these, only one has a
reported interaction with PPOs.26−28 In particular, the
inhibitory action of 7-hydroxycoumarin (7-HC, also known as
umbelliferone) toward L-tyrosine hydroxylation was noted.27
The MT-catalyzed transformation of L-tyrosine was monitored
through the detection of dopachrome formation. As shown in
Figure 2, dopachrome is strongly inhibited by 7-HC in a
In an additional protocol for defining the binding sites, we defined
the center of the binding site as the center of mass of the residues
surrounding the tropolone (Figure 1B), that is, H61, H85, H259,
N260, H263, P264, M280, S282, V283, and A286.
Guided Docking. Guided docking is a biased docking procedure, for
which we used previously acquired information about the binding of
the substrate on the target protein (MT). The availability of the
cocrystal structure of MT with tropolone allows us to perform this
guided docking. Thus, to check the reproducibility of our docking
protocol, we first performed the docking with the tropolone molecule
[the complexed structure (PDB-ID 2Y9X)]. In step 1, the tropolone
molecule was removed and then redocked. In step 2, the modeled and
optimized structure of tropolone (drawn by Chemsketch and
optimized through the Gaussian 03 package) was used to perform
the docking. The binding pocket of the tropolone was chosen as the
grid center in both steps.
Grids with dimensions of 68 × 80 × 80 and a spacing of 0.375 Å
were used. The Lamarckian genetic algorithm was used for
conformational sampling of the compound. For each docking
simulation, 200 runs were carried out with 300 random individuals
in the first population and 2.5 × 106 energy evaluations. For a local
search, the so-called pseudo-Solis and Wets algorithms25 were applied
using the default parameter. The low RMSD difference (0.6 Å)
between the complex docked-pose of tropolone obtained from the
above-described steps 1 and 2 (data not shown) confirmed the
reliability of the docking protocol. Furthermore, the optimized
structures of our compounds were docked in the same binding site
by using the same input parameters. The binding poses of compounds
were ranked and clustered on the basis of their energy scores and
populations.
Blind Docking. This docking protocol is an unbiased docking
approach, whereby a grid covers the whole surface of the protein to
locate the most likely binding site of lead molecules. AutoGrid 4.0 was
employed to build a 126 × 126 × 126 grid map covering the whole
RdRp, with grid spacing of 0.586 Å and a center on the center of mass
of the whole MT. The Lamarckian genetic algorithm was used for
conformational sampling of the compounds. For each docking
simulation, 200 runs were carried out with 300 random individuals
in the first population, 2.5 × 107 energy evaluations, and 2.7 × 107
generation numbers. For each run, the lowest energy conformation of
the compounds was chosen for clustering according to an RMSD
cutoff of 2 Å. Clusters were then ranked and clustered on the basis of
the AutoDock energy scores and populations. The goal of conducting
blind docking was to validate the guided docking outcome: whether
the lead substrate candidate sat only in the tropolone-binding site or in
some other binding location. Indeed, the top cluster representatives
obtained from the blind docking (i.e., the clusters having the lowest
docking energy and maximum number of confirmations/population)
confirmed that each lead occupied the same binding site.
Figure 2. Tyrosinase-catalyzed oxidation of L-tyrosine in the presence
of 7-hydroxycoumarin (7-HC). The reaction mixture contained 3 mM
L-tyrosine, 95 enzyme units, and 7-HC substrate at a concentration
that ranged from 0.3 to 0.9 mM. Other experimental conditions were
100 mM potassium phosphate buffer, pH 6.5, in a 1 mL final volume
and 25 °C temperature.
concentration range of 0.3−0.9 mM. At the maximum 7-HC
concentration, the inhibition reached 88%. This phenomenon is
due to the specific inhibition of L-tyrosine conversion, which is
confirmed by HPLC analysis of the reaction products (see the
Supporting Information, Figure SM1) and not due to any
interference by a putative quinone arising from 7-HC
hydroxylation and oxidation, as previously suggested.26
A
further confirmation of the reduced conversion of L-tyrosine
can be observed in the decreased appearance of L-3,4-
dihyroxyphenylalanine (DOPA), which is the product of the
reaction (see the Supporting Information, Figure SM2).
Moreover, the hydroxylation of 7-HC by MT, which yielded
6,7-dihydroxycoumarin (also known as esculetin), has been
observed; however, this occurred only under particular
experimental conditions and with a very low kcat.26 Although
these observations appear contradictory, they are not surprising.
A substantial reduction of the conversion speed of a certain
substrate is normally observed in the presence of another
substrate, the affinity of which is much higher and having a kcat
much smaller than those observed for the first substrate. The
second substrate then behaves as a “competitive substrate”, as
its enzymatic transformation is too slow to be observed during
the planned reaction time for the first enzymatic reaction.
However, if the favorable substrate is absent from the reaction
mixture, then even the less favorable substrate can be converted
by the enzyme. Recently, a method to discriminate between
alternative substrates and inhibitors of tyrosinase has been
proposed.29
RESULTS AND DISCUSSION
■
In this study, we explored the effects of four hydroxycoumarins
(3-, 4-, 6- and 7 isomers, see Scheme 1) on the activity of MT
under different conditions. This will ultimately provide us with
mechanistic insights from kinetic data.
Hydroxycoumarins with a Hydroxy Substituent on
the Aromatic Ring. Two of the four hydroxy-substituted
coumarins studied in this work (6- and 7-isomers) are
Unlike 7-HC, 6-hydroxycoumarin (6-HC) is rare in nature
and has been found in only a few plants30 and as a secondary
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metabolite of an unsubstituted coumarin in certain animals. Yet,
like 7-HC, it bears its hydroxyl substituent on the aromatic ring;
for this reason, it could be considered a true monophenol
derivative. Therefore, given the monophenolase/hydroxylase
activity of the enzyme, this compound could behave as a
substrate. This was confirmed by the experimental results, with
the consumption of 6-HC in the presence of the enzyme.
Therefore, 6-HC was found to be a substrate for MT (Figure
3A, line a). 6-HC behaved as a monophenol substrate and was
of tyrosinase.31 Indeed, when the reaction was conducted in the
presence of sufficient concentrations of esculetin (Figure 3B,
line b), the disappearance of 6-HC and the formation/
disappearance of esculetin (Figure 3B, line d) accelerated.
This confirmed the ability of this compound to play the dual
role of both a substrate and an activator of tyrosinase.
Hydroxycoumarins with an Unsubstituted Aromatic
Ring. 3-Hydroxycoumarin (3-HC) is also rare in nature, but is
sometimes found in plants.32 Interestingly, 3-HC was recently
demonstrated to protect sea urchin reproductive cells against
ultraviolet B damage.33 It has been chosen for the present study
because MT should not hydroxylate it. This is because the
hydroxyl-bearing ring is not aromatic and the aromatic ring of
the compound is unsubstituted. MT is only able to ortho-
hydroxylate phenols and aromatic amines.31,34,35 Furthermore,
the close proximity of the carbonyl moiety in 3-HC renders the
putative hydroxylation site, the 4-position, relatively electro-
philic and therefore not suitable for hydroxylation by MT,36
which performs ortho-hydroxylation through an electrophilic
mechanism that requires relatively high electron density on the
target carbon atom.37 The HPLC analysis of the products of the
reaction of tyrosinase with 3-HC as a possible substrate shows
that there was no consumption of 3-HC (see the Supporting
Information, Figure SM3). Finally, 3-HC shows the same
structural motif (i.e., an enol−ketone) that is typical of some
well-known PPO inhibitors, such as tropolone, kojic acid, and
mimosine.38 On the basis of these considerations, we tested 3-
HC as a possible inhibitor of MT and compared its
effectiveness with that of tropolone.
Figure 4 shows the trend of catalytic consumption of L-
tyrosine by MT in the presence of 3-HC or tropolone in
comparison to a control. Figure 4A shows that the
consumption of L-tyrosine is negligible when the reaction is
carried out in the presence of tropolone, whereas in the
presence of 3-HC, only approximately 3% of the substrate is
consumed after 30 min of incubation. This behavior of the two
inhibitors was confirmed by the concentration of L-DOPA in
the same samples (Figure 4B), which is below the limit of
measurement in the presence of 3-HC and tropolone.
This sharp inhibitory behavior of 3-HC on MT follows from
the general trend of the structure−activity relationship between
MT and phenolics (and phenolic analogs).
In contrast, nonsubstituted coumarin (1,2-benzopyrone) was
reported to inhibit apple PPO.39 Docking studies performed
with this enzyme revealed that the compound is able to enter
the enzyme active site, where it is anchored to a specific L-
tyrosine residue by a hydrogen bond.39 The alleged complexing
ability of coumarin toward Cu2+ is not presently well evidenced
and, therefore, should not be considered as a driving force for
the observed PPO and coumarin interaction(s). However, we
could not find any interaction between the MT and the
unsubstituted coumarin. This observation highlights the
importance of the hydroxy substituent(s) for a specific
interaction with the enzyme active site to occur.
We can conclude that the chelating ability of tropolone
toward the cupric ions of the dicopper cluster is not important
to explain its inhibitory properties: the molecule lays too far
from the metal center and is most likely connected to one
cupric ion by means of a hydrogen-bond-engaged water
molecule. The kinetics of the inhibition of MT by tropolone
has been already studied,40 and a specific interaction with the
oxy form of the enzyme was proposed to explain the particular
behavior of the inhibitor. To establish the true mechanism of
Figure 3. Tyrosinase-catalyzed oxidation of 6-hydroxycoumarin (6-
HC) and 6,7-dihydroxycoumarin (esculetin). HPLC measurement was
performed in a time course of 6-HC (A) and esculetin (B)
concentration in a reaction mixture containing mushroom tyrosinase
(95 enzyme units), 0.3 M 6-HC (lines a, c), and 0.3 mM 6-HC plus 10
μM esculetin (lines b, d) as the initial concentrations. Other
experimental conditions were 100 mM potassium phosphate buffer,
pH 6.5, in a 1 mL final volume and 25 °C temperature.
therefore o-hydroxylated to yield the corresponding o-diphenol.
Two different compounds could be the products of 6-HC o-
hydroxylation: esculetin and 5,6-dihydroxycoumarin. From our
experiments, this latter unnatural compound was not detected
at all, indicating that 6-HC exclusively was converted into
esculetin, (Figure 3B, line c), which is a substrate for the
enzyme.27 Indeed, after 60 min, the esculetin concentration
decreased as it was enzymatically converted to the correspond-
ing o-quinone. It is already known that low concentrations of
diphenols can operate by activating the monophenolase activity
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Figure 5. Tyrosinase-catalyzed oxidation of L-tyrosine in the presence
of 4-hydroxycoumarin (4-HC). The reaction mixture contained 3 mM
L-tyrosine and 95 enzyme units. The 4-HC concentration ranged from
0.3 to 0.6 mM. Other experimental conditions were 100 mM
potassium phosphate buffer, pH 6.5, in a 1 mL final volume and 25 °C
temperature.
not substantially affected by the presence of 4-HC. In its
presence, the formation of dopachrome appears to be slightly
increased between the fourth and sixth minutes and then
persists for a longer period before it decreases because of
nonenzymatic reactions that lead to the formation of melanin.
The origin of this behavior is still unknown, but we can assume
that, at concentrations much higher than the amount of
dopachrome, 4-HC can create an effect of crowding at the
catalytic site of the enzyme, thereby slowing the release of the
product of the reaction. Yet, a reaction between 4-HC and
nonenzymatic transformation products of dopachrome is also
possible.
A comparison of the overall effect of the examined
hydroxycoumarins can be observed in Figure 6. The four
compounds, 3-, 4-, 6-, and 7-hydroxycoumarin, were incubated
in the presence of MT. We then used L-tyrosine as a substrate
and measured the formation of melanin over time. Both 4-HC
and 6-HC had no inhibitory effect on enzymatic activity, as the
formation of melanin did not decrease. The presence of 7-HC,
however, slowed the formation of dopachrome in the early
stages of the reaction but failed to prevent melanin formation
after an adequate period of time. In contrast, only 3-HC
persisted in its inhibitory activity, as the formation of melanin
was not observed in the 2 h following the start of the
experiment. Moreover, the inhibitory activity of 3-HC is strictly
dose-dependent (see the Supporting Information, Figure SM5).
Molecular Docking Study. Prompted by these findings,
we searched for the putative positions of three compounds
within the active site of the enzyme (3-HC, 6-HC, and 7-HC)
via in silico molecular docking (4-HC was not considered in the
molecular docking investigation, being neither a substrate nor
an inhibitor). The results of the docking runs are summarized
in Table 1.
Figure 4. Tyrosinase-catalyzed oxidation of L-tyrosine in the presence
of 3-hydroxycoumarin (3-HC) and tropolone. HPLC measurements of
L-tyrosine (A) and L-DOPA (B) concentrations were performed over
time in the presence of 0.5 mM 3-HC and 0.5 mM tropolone and in
their absence (Ctrl). Other experimental conditions were 2 mM L-
tyrosine, 100 mM potassium phosphate buffer, pH 6.5, in a 1 mL final
volume and 25 °C temperature.
MT inhibition by 3-HC, we conducted spectrophotometric
experiments to compare the kinetics of tropolone and 3-HC as
inhibitors of MT under identical experimental conditions.
Whereas tropolone behaved as a slow-binding inhibitor, which
confirmed the findings of Espin and Wichers,40 3-HC behaved
as a “normal” inhibitor (data not shown). This finding suggests
that the two molecules are accommodated differently within the
enzyme active site. More specifically, 3-HC most likely interacts
with the most abundant form of MT, namely, the met form.1
Remarkably, even the addition of 0.2 mM CuSO4 was not able
to remove the inhibition.40
Due to the bonding pattern described above, 4-hydrox-
ycoumarin (4-HC) also should not behave as a substrate for
MT. HPLC analysis confirmed that 4-HC did not behave as a
substrate for MT (see the Supporting Information, Figure
SM4).
Concerning the comparatively bulky 3-HC, the molecular
modeling investigation resulted in two clusters, of which the
representative Ori1 and Ori2 clusters are shown in Figures
7A,B. The two clusters have very different populations (46% of
the configurations are in Ori1, whereas 14% of the
configurations are in Ori2; see Table 1), and Ori1 has a
In Figure 5, the reaction of MT with L-tyrosine in the
presence of increasing concentrations of 4-HC is reported.
Note that in the first minutes, the formation of dopachrome is
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(flip orientation). In Figure 7C, the two orientations are
superimposed upon one another. The H atoms of the hydroxyl
groups of the two conformations are directed in an opposing
manner, whereas, in both conformations, the oxygen atom of
the OH group occupies a position along the location of the
oxygen atom of a crystallographic water molecule. Therefore, 3-
HC accommodates within the MT active site in a manner quite
similar to that proposed for mimosine41 and kojic acid.42 With
these inhibitors, which are transition state analogues, the
oxygen of the hydroxyl group acted as a bridge between the two
cupric ions, thus displacing the pre-existing water (or
hydroxide) already present in the met form. As a consequence,
a stable adduct between the dicupric cluster and the inhibitor
molecule is formed, preventing the approach of the substrate to
the catalytic site. In principle, both Ori1 and Ori2 poses fit well
with such an inhibitory mechanism.
However, the distances of the O atom of the OH group from
the Cu ions differ in the two poses. The O atom of Ori1 is
roughly at the same distance from both Cu ions (2.4 vs 2.6 Å),
whereas the O atom of Ori2 is located 2.0 and 3.0 Å away from
CuA and CuB, respectively. Ori1 is characterized by the
position of the O atom of the OH group, which is more
symmetric with respect to the two Cu ions when compared
with Ori2. The different population of the two clusters
associated with Ori1 and Ori2 suggests that the hydroxyl
group interacts with the two Cu ions, thus confirming the
bridging role of the oxygen atom of the hydroxyl group as
depicted above. Both kinetic and docking data confirm the
strict analogy between the inhibitory behavior of mimosine and
kojic acid and that of 3-HC. However, despite the structural
analogy, tropolone behaves differently, due to its peculiar
positioning within the active MT site that follows from the
water molecule bridging the inhibitor to the oxy form of the
enzyme.40 The Ori2 pose is less symmetrical than the Ori1 and
could be less effective as an inhibitor; however, due to the
above-described structural and mechanistic reasons, the
potential substrate role for this pose is not possible.
For the 6-HC substrate, two clusters have been identified
that have the lowest docking scoring functions. Interestingly,
the highest affinity site obtained from the blind docking
protocol is similar to the site obtained from the guided docking
protocol (i.e., Ori1 and Ori2 are representatives of the two
clusters that are reported in Figure 7D,E, respectively, with
Figure 7F displaying both poses). The difference in the scoring
function of the two orientations was small, and also the
populations of the two clusters did not differ remarkably (29.5
and 38.0% over the total population, respectively, as reported in
Table 1). The two poses are characterized by the opposite
orientation of the hydroxyl group. In Ori1, the pose that
belonged to the cluster with a better scoring function but a
slightly lower population, the hydroxyl group is close to the two
Cu ions with the OH group at distances of 1.9 and 2.8 Å from
CuA and CuB, respectively. The asymmetrical positioning of
the hydroxyl group in Ori1 is in agreement with the substrate
nature of 6-HC, which can be ortho-hydroxylated to esculetin
by the oxy-MT. The hydroxyl group of Ori2 points toward the
opening of the active site, and the distances between the CuA
and CuB and the closest oxygen atom of Ori2 are 2.1 and 2.9 Å,
respectively. It is readily apparent that this pose is unproductive
as a MT substrate; however, it could act, in principle, as a slight
inhibitor by obstructing the active site. Interestingly, the
stability of the poses of this compound appears to be unaffected
Figure 6. Effect of the four studied hydroxycoumarins on the
formation of melanin via mushroom tyrosinase in the presence of L-
tyrosine as the substrate. The following enzymatic reactions have been
performed on a porcelain spot plate: Ctrl, incubation of tyrosinase (46
EU) and L-tyrosine (2 mM) as the control; 3-HC, 4-HC, 6-HC, and
7HC, incubation of tyrosinase (46 EU) and L-tyrosine (2 mM) in the
presence of 0.6 mM of each hydroxycoumarin. The enzymatic reaction
was buffered with 0.1 M potassium phosphate, pH 6.5. Pictures were
taken over the reported time.
a
Table 1. Results of Molecular Docking
compound orientation scoring function (kcal/mol) population (%)
3-HC
6-HC
7-HC
Ori1
Ori2
Ori1
Ori2
Ori1
Ori2
−7.88
−7.36
−8.29
−8.15
−6.52
−6.28
46.0
14.0
29.5
38.0
17.5
21.5
a
The population is given over the total population of the run
associated with each compound.
slightly better scoring function. The two configurations are
rotated with respect to one another by an angle of
approximately 180° around the major axis of the molecule
F
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Journal of Agricultural and Food Chemistry
Article
Figure 7. Characterization of the binding site and of representatives of the most populated clusters of the lead molecules (3-HC, upper panels; 6-
HC, lower panels). Panels A and B show the two representatives (Ori1 and Ori2) of 3-HC (in thick licorice), and panel C shows the combination of
the two poses (in solid Ori1, in transparence Ori2). In panel A, the residues of the binding site are shown in licorice and are labeled. In all panels, the
Cu ions are colored dark orange and the residues of the binding site are rendered in an atom type manner, such that C is white, O is red, N is blue,
and S is yellow. Panels D and E contain the two representatives of the most populated clusters of 6-HC, and panel F collects both poses. The binding
site residue’s name and their positions are the same in all of the panels as well as the color and rendering codes.
by the position of the hydroxyl group relative to the active site
groups.
molecule behaving as a weak substrate for the enzyme. This
follows from the direct interaction of the hydroxyl group with
the two Cu ions of the enzyme active site. However, if the
hydroxyl group is located on the pyrone ring of coumarin, the
interaction with the enzyme is only possible if the molecule
enters the active site with a rotation of 180°. Because the
pyrone ring cannot be hydroxylated by tyrosinase, the presence
of a pre-existing hydroxyl group makes this molecule unreactive
with the enzyme (e.g., the 4-HC substrate) or a potent inhibitor
(e.g., the 3-HC substrate).
The docking runs for the 7-HC substrate also yielded two
main clusters with populations that were not dissimilar (17.5
and 21.5% for Ori1 and Ori2, respectively, over the total
population; Table 1). The two clusters were characterized by
the worst scoring functions among the three compounds. This
might suggest a poor affinity of the compound for the enzyme.
Yet, the 7-HC substrate behaves as an effective “competitive
substrate” during MT-catalyzed L-tyrosine conversion to
melanin, even though its affinity for the MT active site appears
from the docking calculations to be comparatively low in
comparison with that of 6-HC. Additionally, the 6-HC
substrate is a distinctly more effective MT substrate than the
7-HC substrate (see the Supporting Information, Figure SM6).
Moreover, it is unable to reduce the L-tyrosine conversion into
melanin (Figure 6). The answer could be that the two top
poses found for the 7-HC substrate account for <40% of the
whole population, whereas the remaining 60% of the
population is distributed over several configurations that are
poorly populated.
The results obtained from both the kinetics experiments and
molecular docking approach studies demonstrate that the
structure of 3-hydroxycoumarin may represent a lead
compound for the development of a new class of inhibitors.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.5b02636.
However, the ranking of the poses might be affected by the
intrinsic limitations of the docking procedure, such as the
absence of the solvent or the limited description of the
flexibility of the enzyme and compounds. The apparent
contradiction between kinetic and docking data indicates that
a variety of factors affect the complicated MT and substrate
interactions. Thus, a more complete picture requires a
molecular dynamics study in which the solvent, temperature,
and dynamical effects are considered. This might be a future
direction of investigation.
In conclusion, our study, which was based on the structure−
activity relationship of four hydroxycoumarins and MT, allowed
us to identify anti-tyrosinase properties for use in developing
molecules that are derived from coumarin. In particular, the
positioning of the hydroxyl group of the hydroxycoumarin on
the aromatic ring determines the substrates’ behavior. For
example, the hydroxyl group at positions 6 or 7 results in the
MT-catalyzed oxidation of L-tyrosine and formation of
L-DOPA in the presence of 7-HC; HPLC analysis of
products of MT activity acting on 3-HC and 4-HC; effect
of 3-HC on the formation of MT-catalyzed melanin;
comparison between MT actions on 6-HC and 7-HC
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*(A.R.) E-mail: rescigno@unica.it.
Author Contributions
∥S.A. and P.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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Journal of Agricultural and Food Chemistry
Article
(17) Rescigno, A.; Zucca, P.; Flurkey, A.; Inlow, J.; Flurkey, W. H.
Identification and discrimination between some contaminant enzyme
activities in commercial preparations of mushroom tyrosinase. Enzyme
Microb. Technol. 2007, 41, 620−627.
(18) Rescigno, A.; Sanjust, E.; Pedulli, G. F.; Valgimigli, L.
Spectrophotometric method for the determination of polyphenol
oxidase activity by coupling of 4-tert-butyl-o-benzoquinone and 4-
amino-N,N-diethylaniline. Anal. Lett. 1999, 32, 2007−2017.
(19) Valgimigli, L.; Pedulli, G. F.; Cabiddu, S.; Sanjust, E.; Rescigno,
A. Formation of a blue adduct between 4-tert-butyl-1,2-benzoquinone
and 4-amino-N,N-diethylaniline. Tetrahedron 2000, 56, 659−662.
(20) Marongiu, B.; Piras, A.; Porcedda, S.; Tuveri, E.; Sanjust, E.;
Meli, M.; Sollai, F.; Zucca, P.; Rescigno, A. Supercritical CO2 extract of
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(21) Case, D. A.; Babin, V.; Berryman, J. T.; Betz, R. M.; Cai, Q.;
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H.; Goetz, A. W.; Gusarov, S.; Homeyer, N.; Janowski, P.; Kaus, Y.;
ABBREVIATIONS USED
■
MT, mushroom tyrosinase; PPO, polyphenol oxidase; 3-HC, 3-
hydroxycoumarin; 4-HC, 4-hydroxycoumarin; 6-HC, 6-hydrox-
ycoumarin; 7-HC, 7-hydroxycoumarin
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