Functional interaction of diphenols with ... - Wiley Online Library

Functional interaction of diphenols with polyphenol oxidase Molecular determinants of substrate ⁄ inhibitor specificity Santosh R. Kanade1, V. L. Suhas2, Nagasuma Chandra2 and Lalitha R. Gowda1 1 Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore, India 2 Bioinformatics Centre, Indian Institute of Science, Bangalore, India

Keywords autodock; computational modeling; catechol oxidase; enzyme mechanism; field bean (Dolichos lablab) phenolic substrate ⁄ inhibitor Correspondence L. R. Gowda, Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570 020, India Fax: +91 821 251 7233 Tel: +91 821 251 5331 E-mail: [email protected] (Received 22 April 2007, revised 23 May 2007, accepted 15 June 2007) doi:10.1111/j.1742-4658.2007.05944.x

Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones. The quinones autopolymerize to form dark pigments, an undesired effect. PPO is therefore the target for the development of antibrowning and antimelanization agents. A series of phenolic compounds experimentally evaluated for their binding affinity and inhibition constants were computationally docked to the active site of catechol oxidase. Docking studies suggested two distinct modes of binding, dividing the docked ligands into two groups. Remarkably, the first group corresponds to ligands determined to be substrates and the second group corresponds to reversible inhibitors. Analyses of the complexes provide structural explanations for correlating subtle changes in the position and nature of the substitutions on diphenols to their functional properties as substrates and inhibitors. Higher reaction rates and binding are reckoned by additional interactions of the substrates with key residues that line the hydrophobic cavity. The docking results suggest that inhibition of oxidation stems from an interaction between the aromatic carboxylic acid group and the apical His109 of the four coordinates of the trigonal pyramidal coordination polyhedron of CuA. The spatial orientation of the hydroxyl in relation to the carboxylic group either allows a perfect fit in the substrate cavity, leading to inhibition, or because of a steric clash flips the molecule vertically, facilitating oxidation. This is the first study to explain, at the molecular level, the determinants of substrate and inhibitor specificity of a catechol oxidase, thereby providing a platform for the design of selective inhibitors useful to both the food and pharmaceutical industries.

Polyphenol oxidase (PPO; 1,2-benzene: oxygen oxidoreductase, EC 1.10.3.1), also known as polyphenolase, phenolase, catechol oxidase and catecholase, is ubiquitous in nature [1]. PPO, a binuclear copper-containing enzyme, catalyzes a two-electron transfer reaction during the oxidation of a broad range of o-diphenols to

the corresponding quinones at the expense of molecular oxygen. The enzyme is responsible for melanization in animals and browning in plants and fungi [2]. PPO has been the subject of several reviews [3–6]. The most recent comprehensive review by Mayer [7] covered the structure, distribution, location, properties and newly

Abbreviations CAT, catechol; 2,3-DHBA, 2,3-dihydroxybenzoic acid; 3,4-DHBA, 3,4-dihydroxybenzoic acid; DOPAC, 3,4-dihydroxyphenylacetic acid; DPN, dopamine; MBTH, 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate; 4MC, 4-methylcatechol; mHBA, m-hydroxybenzoic acid; oHBA, o-hydroxybenzoic acid; pHBA, p-hydroxybenzoic acid; ibCO, sweet potato catechol oxidase; OdHC, hemocyanin; PPO, field bean polyphenol oxidase; PTU, phenylthiourea; TBC, 4-tertiary butylcatechol; 2,3,4-THBA, 2,3,4-trihydroxybenzoic acid; 3,4,5-THBA, 3,4,5trihydroxybenzoic acid.

FEBS Journal 274 (2007) 4177–4187 ª 2007 The Authors Journal compilation ª 2007 FEBS

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discovered inhibitors. Unfavorable darkening of foods because of enzymatic oxidation alters essential organoleptic characteristics, especially color and taste, which results in loss of nutritional and market value. The first crystal structure of the processed, active form of sweet potato catechol oxidase (ibCO) in complex with a reversible inhibitor phenylthiourea (PTU) was resolved at 2.5 A˚ [8], however, a latent PPO has not been crystallized successfully. Gerdemann et al. [9], using comparative modeling, propose that, prior to its cleavage from the latent enzyme, the tertiary structure of the C-terminal fragment of ibCO is similar to the C-terminal domain of hemocyanin. Sequence alignments and secondary structure predictions provide evidence that the N-terminal domains of all plant and fungal PPOs are likely to be very similar in tertiary structure to ibCO and hemocyanin, and that the C-terminal domains are likely to be similar in tertiary structure to hemocyanin [5]. The crystal structure of a tyrosinase from Streptomyces bound to a ‘caddie protein’ differs significantly from ibCO [10]. New information from the structure of another bacterial tyrosinase reveals the existence of a seventh conserved histidine residue in PPOs contributing to substrate specificity [11]. Despite these advances in structural studies, control of PPO-mediated browning remains a challenge to the food-processing industry [4]. The Food and Drug Administration is re-evaluating the use of chemical agents as food additives and their use in some products is banned, resulting in continued efforts towards identifying naturally occurring inhibitors. Benzoic acid and its sodium salt have long been used to control enzymatic browning [12]. Substitutions around the aromatic nucleus have had varied effects on the degree of PPO inhibition. 2,3-Dihydroxybenzoic acid (2,3-DHBA) showed no inhibition, whereas 2,4-dihydroxybenzoic acid (2,4-DHBA) was a strong PPO inhibitor [13]. Hydroxylation and methylation of the aromatic rings decreased the inhibitory effect of fruit PPOs. The cited literature indicates that the type and degree of inhibition of PPO activity depend on the structure of both the substrate and the inhibitor. In our continuing investigations into the effect of different dihydroxyphenols, trihydroxyphenols, benzoic acid and some of their derivatives on field bean (Dolichos lablab) PPO activity, we noted that subtle structural changes transform PPO substrates into inhibitors and vice versa [14]. Therefore, it is often difficult to predict inhibition based on chemical structure alone. Similar observations reported for other PPOs have been attributed to differences in molecular structure, for example the position of the hydroxyl and carboxyl groups, and the bulkiness and length of the side chain [15], which 4178

lead to varied interaction between the active site and the inhibitor. A study of the naturally occurring, highly active inhibitors of tyrosinase, the chalcones and related compounds, showed that the number and position of hydroxyl groups were important to the degree of inhibition exhibited [16–18]. In a very early study, Kermasha & Gonfette [19] concluded that inhibition of PPO by aromatic carboxylic acids and their derivatives was a complex phenomenon. The number of PPO inhibitors with very high inhibitory activity described in the literature with diverse chemical structures related and unrelated to o-diphenol are numerous, yet explanations of why very similar phenolic compounds behave either as inhibitors or substrates remains unclear. Elucidation of the X-ray crystallographic structure of ibCO in the oxidized Cu(II)–Cu(II) state, the reduced Cu(I)–Cu(I) state and in complex with the inhibitor PTU has opened up the way for molecular modeling and a structure-based platform for novel inhibitor design. Because the structural differences between PPO substrates and inhibitors are subtle, their distinct specificity can only be predicted using molecular modeling. Because no structural information is available on field bean PPO, which is also a catecholase, ibCO was used as the model to explain our observations. Consequently, the goal of this study was to identify the molecular determinants of PPO substrate ⁄ inhibitor specificity, by taking advantage of the crystal structure of ibCO (1BUG, 1BT1). In this study, the reversible inhibitor PTU bound at the active site in the ibCO–PTU complex was replaced with various phenolic compounds (Fig. 1), which are either substrates or inhibitors. Ligand docking was carried out to identify the determinants of inhibitor ⁄ substrate selectivity. We show that, in addition to the originally observed stacking interaction of the aromatic ring of a phenyl moiety to His244, the differences in the binding affinities (Km) between catechol- and alkyl-substituted catechols are due to aromatic and van der Waals interactions with the alkyl side chain of Arg245 and b-carbon of His244. Interaction of the carboxyl group of the anchored hydroxybenzoic acids with His109 disrupts the polyhedron of CuA rendering them as inhibitors. The experimental and predicted data on the binding affinity (Km) and dissociation constant (Ki) of the dihydroxyphenolic molecules for ibCO and field bean PPO show the same relative trend. Such information provides a better understanding of the critical interactions responsible for specificity and selectivity, which are vital to the rational design of PPO inhibitors for use in preventing enzymatic browning in foods and melanization in animals.



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OH

OH

OH CH3

OH H3C

1(CAT)

OH H3C 2(4-MC)

HO

OH

O

O

O

OH

CH3 3(TBC)

HO HO

C

OH HO

OH HO

C H2

4(DOPAC)

HO

HO 5(2, 3, 4-THBA)

OH

OH

6(oHBA) HO

Substrate specificity of PPO

HO

HO

OH

OH HO O

O

O 7(2, 3-DHBA)

HO 9(3, 4, 5-THBA)

8(3, 4-DHBA)

Fig. 1. Chemical structures of experimentally tested substrates and inhibitors. 1, CAT; 2, 4MC; 3, TBC; 4, DOPAC; 5, 2,3,4-THBA; 6, oHBA; 7, 2,3-DHBA; 8, 3,4-DHBA; 9, 3,4,5-THBA. Compounds 1–5 are substrates and compounds 6–9 inhibitors of PPO.

Results Enzyme homogeneity The specific activity of the purified ibCO and field bean PPO were 1573.5 and 29 300 UÆmg)1, respectively. Both ibCO and field bean PPO were electrophoretically homogeneous, as revealed by native PAGE (Fig. 2) and MALDI-TOF (results not shown). Native PAGE followed by enzyme staining also revealed the presence of a single isoenzyme (Fig. 2, lanes 2 and 4) 1

as reported previously [20,21]. Homogeneity was further confirmed by the release of a single N-terminal amino acid alanine and asparagine for both native and denatured ibCO and field bean PPO, respectively. SDS ⁄ PAGE indicated that the purified ibCO is a monomer of Mr 38 kDa. The N-terminal sequence obtained after 10 cycles of automated Edman degradation of ibCO and field bean PPO is NH2-APIQAPEI and NH2-NNLISFTMK, respectively. These are identical to that reported previously [20,21].

2

3

o-Dihydroxyphenols have long been recognized as PPO substrates. Several phenolic compounds serve as substrates of field bean PPO and ibCO (Table 1). For each of the substrates used, the wavelength at which the corresponding oxidation product exhibited maximum absorption was determined and used to compute the activity. The values of the kinetic constants Km, Vmax and kcat computed from Lineweaver–Burk plots of the kinetic measurements are listed (Table 1). 4-Tertiary butylcatechol (TBC) and 4-methylcatechol (4MC) are the most readily oxidized substrates of field bean PPO, as revealed by the high Vmax values. Field bean PPO obeys Michaelis–Menten kinetics and exhibits the phenomenon of inhibition by excess substrate for catechol (CAT), 4MC and TBC. Among the substrates studied, Km was strongest for dopamine (DPN), although the rate of oxidation was the lowest (Table 1). In the absence of structural information on field bean PPO, we chose to explain these differences by using the available crystal structure of ibCO. Therefore, we evaluated the kinetic constants of ibCO for Table 1. Experimentally determined kinetic constants of field bean (Dolichos lablab) PPO.

4

Compound

NNLISFTMK

APIQAPEI

Fig. 2. Native PAGE (7.5% T, 2.7% C) of the PPO and ibCO. Lane 1, field bean PPO and lane 3, ibCO stained for protein. Lane 2 field bean PPO and lane 4 ibCO stained for PPO activity with CAT and MBTH.

Km (· 10)3

M)

Vmax kcat IC50 (· 103 UÆmg-1) (· 103) (· 10)3

CAT 12.0 ± 0.31 89.7 ± 0.33 4MC 4.5 ± 0.25 120.0 ± 0.25 TBC 4.1 ± 0.41 197.0 ± 0.46 DOPAC 2.9 ± 0.36 33.3 ± 0.32 DPN 1.5 ± 0.25 11.1 ± 0.24 2,3,4-THBA 4.0 ± 0.41 12.2 ± 0.21 BA 0.30 ± 0.02 – oHBA 0.436 ± 0.02 – mHBA 0.40 ± 0.03 – pHBA 0.22 ± 0.01 – 2,3-DHBA 2.2 ± 0.03 – 3,4-DHBA 1.0 ± 0.02 – 3,4,5-THBA 2.4 ± 0.03 – PTU 0.014 ± 0.003 –


7.48 26.66 48.05 11.48 4.9 3.05 – – – – – – – –

1.2 3.6 1.1 1.3 3.7 2.8 3.8 0.07

M)

– – – – – – ± 0.02 ± 0.01 ± 0.02 ± 0.03 ± 0.01 ± 0.01 ± 0.02 ± 0.002

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Table 2. Experimental predicted kinetic constants and predicted free binding energies (DGb) and interactions of phenolic compounds with sweet potato ibCO.

Compound

Experimental Binding constant (· 10)3 M)

CAT 4MC TBC DOPAC 2,3,4-THBA BA oHBA 2,3-DHBA 3,4-DHBA 3,4,5-THBA

9.0 5.2 3.9 6.0 5.6 4.5 3.4 2.3 4.3 2.3

± ± ± ± ± ± ± ± ± ±

0.26 0.25 0.41 0.36 0.41 0.25 0.29 0.42 0.29 0.34

Vmax · 103 (UÆmg)1) 5.6 2.08 12.6 0.86 2.1

± ± ± ± ±

0.5 0.3 0.25 0.29 0.35

Predicted Docking energy DGb (kcalÆmol)1)

Binding constant (· 10)5 M)

Residues in hydrogen bonding

No. of hydrophobic interactions

Aromatic–aromatic interactions

) ) ) ) ) ) ) ) ) )

4.07 1.90 0.43 0.25 0.81 2.2 1.59 1.11 1.12 0.51

– – – – – – His109, Phe261 His109 His109 His109, Asn260

4 5 5 4 2 3 3 2 2 3

His88, 244, Phe261 His88, 244, Phe261 His88, 244, Phe261 His244, Phe261 His244, Phe261 His240, 244, Phe261 His244, Phe261 His88, 244, Phe261 His240,244, Phe261 His240, 244, Phe261

5.97 6.45 7.3 6.92 6.95 6.5 6.54 6.71 6.58 7.09

the phenolic compounds shown in Fig. 1. The rates of oxidation and binding affinities of CAT, 4MC and TBC showed the same trend (Table 2). The best among the substrates evaluated was TBC. Oxidation of TBC to its quinone by ibCO was the highest, with Km ¼ 3.9 ± 0.01 mm. The rate of oxidation of 2,3,4trihydroxybenzoic acid (2,3,4-THBA) to its quinone was much lower than that of TBC and 4MC (Table 1). Pyrogallol a trihydroxyphenol was also oxidized, although slowly, by field bean PPO (Km ¼ 5.9 mm; Vmax ¼ 25.6 · 103 UÆmg)1). Pyragallol was not oxidized by ibCO. Inhibitor studies Benzoic acid and closely related congeners such as o-, m-, p-hydroxy, dihydroxy and trihydroxybenzoic acids were tested as inhibitors of field bean PPO and ibCO. The results are summarized in Tables 1 and 2. The results show that both field bean PPO and ibCO are strongly inhibited by monohydroxybenzoic acids. In most cases the type of inhibition is competitive (Table 2). The close similarities in the Ki values for these inhibitors indicate similar binding affinity to the enzymes. Gallic acid (3,4,5-trihydroxybenzoic acid; 3,4,5-THBA) was as effective as the dihydroxybenzoic acids in inhibiting ibCO. The Ki value was 2.3 mm for both PPO and ibCO. Ligand docking and structural analyses Distinct variations in the binding and inhibitory activity of field bean PPO and ibCO containing a common aromatic ring were evident (Tables 1 and 2). Evidently, the numbers of hydroxyl groups, their position in the aromatic ring with respect to the carboxy group and the length of the side chain have a profound effect on 4180

the Vmax and Km ⁄ Ki values of the two enzymes (Tables 1 and 2). Alkyl substitutions by either a methyl or t-butyl group on o-diphenol leads to a considerable increase in the kcat value and tighter binding. As observed, the rate of TBC oxidation was much higher than CAT. The kcat value is also several fold higher (Table 1). The most potent inhibitor activity is exhibited by p-hydroxybenzoic acid (pHBA) with Ki ¼ 0.2 · 10)4 m. The two related phenolic analogs, 2,3-dihydroxybenzoic acid (2,3-DHBA) and 3,4-dihydroxybenzoic acid (3,4-DHBA) did not alter the inhibitory potential. The substitution by a third hydroxy group to form 2,3,4-THBA renders it as a substrate. In direct contrast, a similar addition to form 3,4,5-THBA makes it an inhibitor of the enzymes. These results suggest that the apparent position of key groups (carboxylic and hydroxyl) are important in eliciting either substrate ⁄ inhibitor activity. The compounds shown in Fig. 1 are either substrates or inhibitors of ibCO, as evaluated experimentally (Table 2). To help rationalize and provide an explanation for the experimental data, computational docking studies were performed using autodock 3.0. The availability of the X-ray crystallographic structure of ibCO complexed with PTU (IC50 ¼ 43 lm) paved the way for these molecular modeling studies. In this study, the water molecules and PTU were removed from the ibCO structure. Compounds, which have the same basic phenyl ring, were successfully docked at the active site of ibCO and comparisons were made (Fig. 3). Table 2 lists the results of the docking experiments, calculated free energy of binding (DGb), inhibition ⁄ binding constants for each complex and the interactions with the enzyme. The binding modes of CAT, 4MC and TBC are superimposed (Fig. 3A). Careful inspection of the substrate-binding pocket indicates that the phenyl rings of these compounds are



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A

D

B

C

F

E

Fig. 3. Molecular docking of ibCO. Predicted binding modes of substrates ⁄ inhibitors of ibCO. The best ranked docking is shown. Stereo image of catalytic binding site of ibCO: PDB structure 1BT1 and 1BUG were superimposed and PTU in 1BUG was eliminated. Blue circles represents copper atoms in 1BUG, whereas blue–red sticks represents the copper oxygen bridge in 1BT1. (A) Stick, CAT; ball and stick, 4MC; line, TBC. (B) Stick, oHBA; ball and stick, 2,3-HBA; line, 3,4-DHBA. (C) Ball and stick, 2,3-DHBA; ball and stick (gray color), 2,3,4-THBA; line, 3,4-DHBA. (D) Stick, 3,4,5-THBA; ball and stick, 2,3,4-THBA. (E) Ball and stick, 2,3,4-THBA; line, TBC. (F) Ball and stick, DOPAC; line 3, 4-DHBA.

stacked perfectly against His244. The distance between CuA and CuB are 3.3, 4.2 and 4.2 A˚, respectively. The Cu–N distances are 2.8, 2.9 and 3.0 A˚, respectively. The phenyl moiety of the substrates shows aromatic interactions with Phe261, His244 and His88 (Table 2). These residues are among the highly conserved residues of type-3 dinuclear copper centres of plant PPOs. The hydrophobic interaction between the methyl and t-butyl substituent of 4MC and TBC with the alkyl side chain of Arg245 further stabilizes the respective complex. Further, the t-butyl group of TBC extends down from the phenyl ring and makes additional van der Waals contacts with Ile241 and Cb of His244

(Fig. 3A). Both these residues line the hydrophobic cage of ibCO. These added interactions with the binding-pocket residues complement and improve the fitness of TBC. TBC is locked in with a better fit when compared with either 4MC or CAT. This observation is consistent with the higher binding affinity (lower Km). This result is also reflected in the estimated free energy of docking (Table 2). Among the inhibitors evaluated, the mono- and dihydroxybenzoic acids are potent inhibitors. The binding mode of the reversible inhibitors of ibCO (o-hydroxybenzoic acid [oHBA], 2,3-DHBA, 3,4-DHBA) is shown in Fig. 3B. Docking of these compounds indi-


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cates that the stacking of the phenyl ring is exactly like that of the substrates CAT, 4MC and TBC. All the principal aromatic interactions with the residues that line the substrate pocket are similar to the previous group. The carboxylic groups of all the three compounds are hydrogen bonded to His109 more or less like a salt bridge reckoning tighter binding. The metal– metal center distance increased to 5.02 A˚ inhibiting formation of the hydroxo-bridge essential to catalysis, thereby rendering them inhibitors (Fig. 3B,C). The close similarities in the free energies of binding (DGb) and computed Ki values indicate that they inhibit ibCO to the same extent and in an identical manner. A very similar trend was observed when evaluated experimentally (Table 2). The carboxyl group and aromatic ring of 3,4,5THBA are superimposable with those of 2,3-DHBA and 3,4-DHBA, which makes it an inhibitor. In addition to the hydrogen bond with His109 invariant in all plant PPOs, 3,4,5-THBA makes contact with Asn260 via a hydrogen bond. It is therefore expected that 3,4,5THBA will show greater binding affinity. The predicted and experimental values show that it is a more potent inhibitor. The hydroxyl group at position 5 is positioned perfectly in the open cavity of the active site (Fig. 3D). 3,4,5-THBA, as detailed, fits into the substrate cavity with the carboxyl group positioned to interact with His109. This results in inhibition of ibCO. 2,3,4-THBA, a closely related analog, was also docked. Figure 3D shows the final docked position of the two THBAs. The stacking of the phenyl ring and aromatic interactions with the hydrophobic pocket residues remain unchanged. However, there exists a steric clash in the cavity between the 2(OH) of 2,3,4-THBA and Ala264 and Phe261. This steric hindrance does not favor the same binding orientation as that of 3,4,5-THBA. Therefore, the molecule flips vertically mimicking the orientation of a substrate (Fig. 3E). Experimentally, 2,3,4-THBA is an excellent substrate with a binding affinity comparable with TBC. Figure 3E shows that the carboxyl of 2,3,4-THBA is anchored similar to the t-butyl group of TBC, making contacts with the alkyl chain of Arg245. Additional van der Waals contacts with Ile241 and Cb of His244 are also visualized. The distance between the Cu atoms is 4.2 A˚, identical to that observed when TBC is bound. 3,4-Dihydroxyphenylacetic acid (DOPAC) differs from 3,4-DHBA in two aspects. DOPAC is a substrate, whereas 3,4-DHBA is a inhibitor, and DOPAC contains an additional methylene group in the carboxyl side chain. Docking of DOPAC indicates that the orientation, stacking and aromatic interactions in the substrate cavity resemble the other aromatic acids 4182

described above. However, the increase in the length of the side chain leads to several short contacts. This results in a clash with the hydrogen-bonded side chain, which prevents the same conformation as 3,4-DHBA. This causes the molecule to flip like 2,3,4-THBA. Consequently, the carboxylic group does not interact with His109 and the coordination of CuA is undisturbed (Fig. 3F). As a result, DOPAC is oxidized and behaves as a substrate. The predicted Km and DGb indicate that it is a high-affinity substrate, which is in agreement with the experimental Km.

Discussion CAT (o-dihydroxyphenol) the archetype substrate of all plant PPOs is rapidly oxidized to its quinone at the expense of molecular oxygen. The nature of the side chain, number of hydroxyl groups and their positions in the phenyl ring exhibit a profound effect on the binding mode of the substrate. There is a lack of structural information on PPO–inhibitor complexes to explain the intriguing subtle structural differences in the phenolic molecules, which render them as inhibitors or substrates. This motivated us to explore modeling studies with ibCO for which a 3D structure is available. Using a molecular model for the first time, we have shown that the substrate ⁄ inhibitor functions are due to marked changes in the interactions between the compounds and orientation of the molecule at the active site of ibCO. Our results rationalize and support the experimental data and afford a reasonably good explanation for the differential behavior of phenolics. Plant PPOs are capable of oxidizing a wide range of o-diphenols, the primary substrates with varied oxidation rates (Vmax) and affinities of different orders of magnitude. The native concentrations of natural phenols vary from plant to plant. The substrate specificity of a PPO is dependent on species, cultivar and the vegetative part of the plant [22]. We have previously reported that field bean PPO oxidized a number of o-diphenols and phenolic acids. This oxidation was inhibited by related aromatic and phenolic acids [21]. An apparently pure ibCO was used to study the substrate ⁄ inhibitor properties of some phenolic compounds (Fig. 1). Field PPO and ibCO oxidized TBC at a significantly faster rate than 4MC and CAT (Table 1). Furthermore, trihydroxyphenols were also oxidized efficiently. Both field bean PPO and ibCO exhibit a greater affinity for o-diphenols than trihydroxyphenol (pyrogallol). The binding properties (Km) and catalytic power (kcat) of field bean PPO and ibCO increase with an increase in the size of the side chain in the aromatic ring of its substrates (Table 1). Tremo-



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lieres & Bieth [23] attributed similar observations to the electron-donating capacity of a methyl group at the para position of 4MC. Decreased oxidation rates with different substrates were explained by the presence of an electron-attracting carboxyl substituent. These results indicate that the spatial orientation of essential vicinal dihydroxy groups play a vital role in determining the binding and catalysis. Lerner et al. [24], based on evidence that the binding sites for oxygen and phenolic substrate are independent, conclude that binding of oxygen induces a conformational change, which accounts for the differences observed in substrate interaction. The success of docking using autodock, based on the Lamarckian genetic algorithm, has been recently demonstrated [25]. This systematic docking study reproduced crystallographic information of eight different protein–ligand complexes. Therefore, using autodock we set out to explain the functional differences in the behavior of related phenolic compounds. As a starting configuration for the docking studies the bound PTU in the ibCO complex (PDB 1BUG) was replaced with the compounds shown in Fig. 1. It is evident from the docking results (Table 2, Fig. 3) that all compounds possessing a basic phenyl ring bind to ibCO. The phenyl moiety of all phenolic compounds stacks perfectly against His244. The aromatic interactions (Table 2) advocate that these interactions are universal to all phenolic compounds irrespective of whether they are substrates or inhibitors. These interactions occur between the phenyl ring and residues that line the hydrophobic cavity. These results reckon that any aromatic compound will bind to all plant PPOs. This is supported by the premise that the interaction involves His88, His244 and Phe261, which are invariant residues in all plant PPOs [11]. This is supported by the fact that PTU is not a phenol and yet binds very strongly to ibCO (Km ¼ 2.5 mm). Field bean PPO also shows a very similar binding affinity. A structural model of p-nitrophenol in coordination to the tyrosinase of Streptomyces antibioticus, derived based on 1H NMR and 2D NMR data binding to ibCO shows that one His residue is particularly sensitive to binding. This corresponded to His244 of ibCO [26] and is consistent with our results showing the interaction with His244. Among the residues involved in the aromatic interactions, His244 is coordinated to CuB, whereas His88 is coordinated to CuA. Phe261, located above the hydrophobic cavity, has been termed as the ‘gate residue’ because it blocks the entrance to the cavity [9]. To explain the absence of the monophenolase of ibCO, it is suggested that Phe261 blocks the direct approach and reorientation of monophenols to CuA, needed for

its hydroxylation [26]. In fungal PPOs this residue is replaced by Leu or Pro. Streptomyces tyrosinase has a Gly at this position. These residues being small cannot block the entry and as expected monophenolase activity will be very high [11]. The orientation of phenols with their hydroxyl group directed towards CuA appears to be necessary for monophenolase activity [27]. The gate residue shields CuA of ibCO, therefore monophenolase activity is restricted. It can therefore be assumed that the lack of monophenolase activity in field bean PPO is due to this shielding. It may not be unreasonable to state that all PPOs that have a conserved Phe261 will not exhibit monophenolase activity. The kcat and Km of TBC are > 4MC > CAT. The higher affinity of 4MC and TBC can be interpreted by the interaction with the alkyl side chains of Arg245 absent in CAT. The kcat value reported for 4MC is several fold higher than CAT for black poplar PPO [23], which shows an Arg residue corresponding to Arg245 [11]. TBC, 2,3,4-THBA and DOPAC make additional van der Waals contact with the residues of the hydrophobic pocket, increasing the binding affinity. Among them His244 is conserved, whereas Ile241 is not. It is observed that in tomato and potato catechol oxidase, where this residue is Ile241, they exhibit a higher binding affinity. The majority of the reversible inhibitors of plant PPO are substrate analogs. Of these, the aromatic acids are the most studied. The Ki values for these compounds are in the millimolar range. The perfect stacking of the phenyl moiety coupled with the aromatic interactions listed in Table 2, indicates that the bound analog is locked in the substrate cavity. The competitive nature of inhibition for all these compounds is not unexpected because the aromatic rings stack perfectly with the imidazole of His244, in the same fashion as CAT, the substrate used in the inhibition studies. All the inhibitors, oHBA, DHBAs and 3,4,5-THBA docked, superimpose on each other and show identical contacts (Fig. 3B,D). The structural model of tyrosinase with its inhibitor p-nitrophenol showed that the phenyl moiety was tilted toward CuA. In this position, the orientation favored efficient oxygen atom transfer [26]. In ibCO, the two cupric ions CuA and CuB are bridged by a hydroxide ion at a distance of 1.8 A˚. This forms the hydroxobridge essential for catalysis. Docking the substrate analogs (Fig. 3B,D) increased the distance to 5.02 A˚. The distance between the two Cu is 4.2 A˚ when ibCO binds to the substrates TBC and 4MC or PTU. Although this distance is the same, the sulfur atom of PTU replaces the hydroxobridge accounting for the potent inhibition (IC50 ¼ 43 lm). PTU is also a very potent inhibitor of field bean PPO (IC50 ¼ 70 lm)


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suggesting that a similar mechanism is operational. The universal hydrogen bond between the inhibitor compounds and His109, a coordinating ligand of CuA in plant PPOs, further fixes the inhibitor. His109 liganded to CuA is covalently linked to a cysteine residue by an unusual thioether bridge. Molecular modeling showed that the flexibility of the His is sufficiently reduced because of this linkage [28]. Our results show that the flexibility is still sufficiently high in ibCO to allow the His109 to form a salt-bridge-like hydrogen bond with all the aromatic inhibitors. Crystallographic study of a metal-free tyrosinase in complex with ORF378 (a caddie protein) has established that this linkage does not allow a bidentate intermediate, therefore monophenols cannot serve as substrates or inhibitors [10]. Because of the universal interactions between the phenolic inhibitors and ibCO it is expected that the binding affinities for the phenolic compound would be similar if not the same. This is justified by the experimental Ki values listed in Table 2. The predicted inhibition constants are several orders lower than the experimental values although the relative trend is similar. The solvation and entropic effects have not been considered in this model. Moreover, it is well established that the binding affinities depend on several in vitro conditions such as the pH and ionic strength of the buffer, temperature and substrate used. These parameters are not accounted for in these models. The Ki values calculated using autodock or any other docking program are therefore best interpreted in a relative manner and not as absolute values. Only a minor structural change is required to transform a substrate into an inhibitor. Experimentally, 2,3,4-THBA is very effectively oxidized to its quinone. However, 3,4,5-THBA is a competitive inhibitor. The docking study has provided a reasonably good explanation for this differential function which would otherwise have not been possible to explain. The steric hindrance caused between the 2(OH) and Ala264 and Phe261 of the cavity has evidently altered its orientation (Fig. 3C,D). But obviously the phenyl moiety still stacks and the aromatic interaction resemble the substrates. Both Ala264 and Phe261 are conserved in plant PPOs suggesting that all PPOs would oxidize 2,3,4-THBA. As observed, field bean PPO oxidizes 2,3,4-THBA, although less efficiently than TBC. Similarly, the addition of a -CH2 in DOPAC makes it bulky and the molecule is reoriented like the substrate, making the same contacts as TBC. These results offer explanations for the previous observations that phenolics acids with increased side-chain length, e.g. cinnamic acid, p-hydroxyphenyl propionic acid and 3,4-dihydroxyphenyl propionic acid are oxidized more efficiently and with Km values in the mm range [23]. 4184

Klabunde et al. [8] propose that Glu236 hydrogen bonded to a solvent molecule functions as a general base ⁄ acid in the diphenol oxidation. We have also recently shown that a carboxylate group is vital to field bean PPO activity and a glutamate residue corresponding to that of Glu236 of ibCO is invariant in all catechol oxidases [11,29]. Mutation studies on this residue would confirm this observation. Two mutations other than the coordinating His at the CuA site of a mammalian tyrosinase did not alter the tyrosinase activity. The mutation of His390 at the CuB site abolished tyrosinase activity completely. Furthermore, His389 was responsible for the stereospecific recognition of o-diphenols not monophenols [30]. Such a His pair has been implicated in the control of the the preference for carboxylated over decarboxylated substrates [11]. A plot of the predicted binding energies and our experimental data on the binding constants (Km ⁄ Ki) shows a linear correlation (Fig. 4). Our calculated difference in binding free energy between CAT (substrate) and BA (inhibitor) was 0.74 kcalÆmol)1, which is near equivalent to the experimental difference of 0.81 kcalÆ mol)1. A similar trend was observed for CAT and the other inhibitors. The linear correlation between the experimental and predicted data indicates the suitability of the atomic approach to predict the relative binding energies of phenolic compounds that show subtle differences in structure. In conclusion, this study shows that the theoretical method based on flexible docking provides insights into the details of ibCO substrate ⁄ inhibitor interactions and is a valuable complement to the limited PPO substrate ⁄ inhibitor crystal structures available. These docking studies have afforded structural explanations for correlating subtle changes in the

Fig. 4. Correlation between AUTODOCK-predicted free energy and calculated binding constants (Km or Ki) of ibCO.



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position and nature of the substitutions on diphenols to their functional properties as substrates and inhibitors. The results also demonstrate that minor changes in diphenol substrates define their binding constants. It is clear that the computational analyses have provided important links between structure and function that are otherwise difficult to obtain by experimental means. We believe that the data obtained by the autodock studies are important for our continuing research efforts and provide useful hints to guide the development and design of selective, potent PPO inhibitors for use in the food and pharmaceutical industries.

assay mixture consisted 0.8 mL of 0.05 m sodium acetate buffer pH 4.0, 0.1 mL of 0.5 m CAT and 0.1 mL of 20 mm 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH) and 10–100 lg of the enzyme. The MBTH–quinone adduct formed was followed at 25 ± 2 C for 3–5 min at 500 nm (e500 ¼ 32500 m)1 cm)1). One unit of enzyme activity is defined the amount of enzyme that produces one lmol of MBTH–quinone adduct per min under the assay conditions. ibCO was assayed similarly using catechol in 0.1 m NaCl ⁄ Pi buffer pH 6.5. The final concentration of all the other diphenol substrates used (Tables 1 and 2) was 10 mm. The reaction rates were measured at the absorption maxima of the corresponding MBTH–quinone adducts at 25 ± 2 C [32].

Experimental procedures Materials Sweet potato tubers (Ipomea batatas) and field bean (Dolichos lablab) seeds were purchased from the local market. TBC was obtained from Merck (Hohenbrunn, Germany), 4-methylcatechol, catechol, 2,3,4-THBA, benzoic acid, oHBA, mHBA, pHBA, 3,4-HBA, 2,3-HBA, 3,4,5-THBA (gallic acid), 3,4-DHBA, phenyl acetic acid, phenyl Sepharose, DEAE-Sepharose and Sephadex G-150, were purchased from Sigma Chemical Co (St. Louis, MO). DPN was purchased from Hi Media Laboratories Ltd (Mumbai, India). All the other chemicals used were of the purest grade commercially available.

Purification of PPO Monomeric 39 kDa ibCO was isolated from sweet potato tubers and purified to > 95% purity as described by Eicken et al. [20]. The buffer extract of the fresh tubers was subjected to (NH4)2SO4 precipitation. The 35–80% precipitate was separated by size-exclusion chromatography using a Superdex 200 column. The pool of active fractions was applied to a DEAE-Sepharose column. The bound ibCO was eluted using a NaCl gradient. The specific activity of the purified ibCO was 1573 UÆmg)1. Field bean seed PPO was purified to > 99% purity as reported previously [21]. The steps involved extraction of defatted field bean powder in buffer, 40–80% (NH4)2SO4 precipitation, DEAE-Sepharose and size-exclusion chromatography on Sephadex G-200. The protein concentration was determined according to the dye-binding method of Bradford [31]. BSA was used as the standard. The molecular mass of ibCO was found to be 39 kDa, by SDS ⁄ PAGE.

Enzyme assay Field bean PPO was assayed by the improved continuous spectrophotometric method of Espin et al. [32] using a Shimadzu UV–visible spectrophotometer Model 1601. The

PAGE Native PAGE (7.5% T, 2.7% C) was performed as described by Zhang & Flurkey [33]. Duplicate samples were electrophoresed simultaneously for protein and enzyme staining. Gels were stained for protein with Coomassie Brilliant Blue R-250. PPO activity was visualized by incubating the gel in 0.05 m sodium acetate buffer pH 4.0, containing 0.05 m CAT and 0.002 m MBTH. PPO is detected as a pink to red band against a transparent background. SDS ⁄ PAGE was carried out according to the method of Laemmli [34] on a (12.5% T, 2.7% C) polyacrylamide gel at pH 8.8.

Inhibition studies Inhibition studies were performed by preincubating the field bean PPO and ibCO with inhibitors for 3 min at 25 ± 2 C. Residual activity was determined at varying CAT concentrations. The mode of inhibition was determined from a Lineweaver–Burke plot at constant inhibitor concentrations. The inhibition constant (Ki) was determined from the secondary plots.

Docking studies The possibility of binding, precise location of binding sites and precise mode of binding of each ligand was carried out independently by an automated docking algorithm, autodock 3.0 [35]. Possible binding conformations and orientations were analyzed by clustering methods, embedded in autodock. Following docking, a postdocking energy minimization was carried out using the discover suite, by allowing full freedom to all protein and ligand atoms in order to address protein flexibility. accelrys (INSIGHT-II modules; Accelrys Inc, http://www.accelrys.com) was used to visualize, analyze and manipulate various structures. For docking, the crystal structure of catechol oxidase (ibCO) complexed with the inhibitor phenylthiourea (PTU,


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PDB 1BUG) was used as the template in all cases. All water molecules and the inhibitor PTU was removed, whereas the copper atoms were retained in the active site, fixed to their crystal positions, through out the process. Copper was modeled merely to indicate its presence in the system with a charge of +1.5 and a vdw of 140 pm (as done previously) [8]. The coordinates of both the copper ions were fixed to their crystal positions because accurate solvation and other parameters required for docking are not as yet available. Hydrogens were added using the builder module of Insight II and the atomic partial charges were computed using the CVFF force field. Ligands were built and optimized using the Builder module in Insight II. A grid of 60 A˚3, 0.375 A˚ spacing was first computed such that the binding site was well sampled. Each ligand was then individually docked into this grid using the Lamarckian genetic algorithm, the most efficient search method in autodock [25,35] and its interactions monitored using detailed energy estimates. One hundred cycles of docking with 250 000 energy evaluations in each cycle were carried out, which sampled all possibilities of conformations of the ligand in each cycle. Clustering was performed based on the similarities in binding modes and strengths in these cycles. The most populous clusters with the lowest energies in the crystallographically identified binding pockets were taken as the binding modes in each case. The coordinates of the docked conformations are available as ‘Supplementary material’. PTU was docked into the template structure as a positive control experiment by starting the docking run with different initial positions of the inhibitor. The docking mode obtained from this validation exercise superposed well with that of the crystal structure.

Acknowledgements We are grateful to V. Prakash, Director, CFTRI, Mysore, for advice and useful suggestions during the course of this investigation. The authors acknowledge A. G. Appu Rao for the useful discussions and keen interest in this work. Santosh R. Kanade is the recipient of a Senior Research Fellowship from Council of Scientific and Industrial Research (CSIR), New Delhi, India. This study was carried out under the CSIR Networking project CMM004.

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Supplementary material The following supplementary material is available online. Doc. S1. Coordinates of the complexes of sweet potato catechol oxidase with various substrates and inhibitors used, obtained through computational docking studies are provided as supplementary material. Model S1. Molecular model of: (a) sweet potato catechol oxidase with 2,3,4-trihydroxybenzoic acid (2,3,4-THBA.pdb); (b) sweet potato catechol oxidase with 2,3-dihydroxybenzoic acid (2,3-DHBA.pdb); (c) sweet potato catechol oxidase with 3,4,5-trihydroxybenzoic acid (3,4,5-THBA.pdb); (d) sweet potato catechol oxidase with 3,4-dihydroxybenzoic acid (3,4-DHBA.pdb); (e) sweet potato catechol oxidase with 4-methylcatechol (4MC.pdb); (f) sweet potato catechol oxidase with benzoic acid (BA.pdb); (g) sweet potato catechol oxidase with Catechol (CAT.pdb); (h) sweet potato catechol oxidase with 3,4-dihydroxyphenyl acetic acid (DOPAC.pdb); (i) sweet potato catechol oxidase with m-hydroxybenzoic acid (mHBA.pdb); (j) sweet potato catechol oxidase with o-hydroxybenzoic acid (oHBA.pdb); and (k) sweet potato catechol oxidase with 4-tertiary butylcatechol (TBC.pdb). This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.


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