Membrane-bound Polyphenol Oxidase Research Articles

Activation mechanism of fatty acids on membrane-bound polyphenol oxidase in peach fruit: Conformational change analysis and molecular docking simulation

Polyphenol oxidase (PPO) is the main enzyme initiating internal browning in peach fruit stored at low temperature. Previous studies showed fatty acids activated PPO, but its specific effect and mechanism of activation on PPO are still not clear. Thus, the effects of fatty acids on the biochemical properties and structural conformation of PPO were studied, with sodium dodecyl sulfate used as comparison. The highest degree of activation (5 folds) of membrane-bound PPO (mPPO) was found with 2.2 mM linoleic acid (LA) and its optimal activity was observed also around pH 7.0. Further structure analysis showed LA led to an increase of α-helix content of mPPO from 23.53% to 52.33%; the maximum emission wavelength of treated mPPO shifted from 338 to 340 nm with the fluorescence intensity increased from 446 to 510. LA also can form hydrogen bonds and hydrophobic interactions with mPPO. These results indicated that LA could contribute to a more stable and open enzyme structure, thus activated mPPO. The results will provide more information for PPO activation that highly probably occurs in postharvest peach fruit and its relationship with internal browning.

Comparing the Effect of HPP on the Structure and Stability of Soluble and Membrane-Bound Polyphenol Oxidase from 'Lijiang Snow' Peach: Multispectroscopic and Molecular Dynamics Simulation.

Polyphenol oxidase (PPO) easily causes fruits and vegetables to lose their color and nutritional value. As a non-thermal process, high-pressure processing (HPP) showed different inactivation effects on endogenous enzymes. In this work, soluble PPO (sPPO) and membrane-bound PPO (mPPO) from 'Lijiang snow' peaches were purified, and then the effect of high pressure on the conformation of sPPO and mPPO was investigated and compared at the molecular level. The maximum activation of sPPO and mPPO by 11.2% and 4.8% was observed after HPP at 200 MPa, while their activities both gradually decreased at 400 MPa and 600 MPa; in particular, the residual activities of sPPO and mPPO at 600 MPa for 50 min were 41.42% and 72.95%, respectively. The spectroscopic results indicated that the secondary structure of PPOs was little affected by HPP, but HPP led to obvious changes in their tertiary structure. The simulations showed that the decreasing distance between the copper ion and His residue in the copper-binding region of two PPOs at 200 MPa was favorable to catalytic activity, while the increasing distance between copper ions and His residues and the disordered movement of the loop region above 400 MPa were unfavorable. In addition, the structure of sPPO was relatively looser than that of mPPO, and high pressure showed a more significant effect on the conformation of sPPO than that of mPPO. This study clarified the effect of HPP on PPO's structure and the relationship between its structure and activity and provided a basis for the prevention of enzymatic browning.

Comparison of Inhibitory Effects of Cinnamic Acid, β-Cyclodextrin, L-Cysteine, and Ascorbic Acid on Soluble and Membrane-Bound Polyphenol Oxidase in Peach Fruit.

There has been considerable interest in controlling polyphenol oxidase (PPO) activity to prevent enzymatic browning in foods. However, studies on inhibitions of different forms of PPO are very limited. Thus, this study focuses on the effects of cinnamic acid, β-cyclodextrin, L-cysteine, and ascorbic acid on soluble PPO (sPPO) and membrane-bound PPO (mPPO) in peach fruit. The activity of partially purified sPPO was 3.17 times higher than that of mPPO. However, mPPO was shown to be more stable than sPPO in the presence of inhibitors with different concentrations (i.e., 1, 3, 5 mM); activation of mPPO was found by 5 mM L-cysteine. Both sPPO and mPPO inhibitions were PPO substrate-dependent. Ascorbic acid showed the highest inhibitory effect on both sPPO and mPPO with all studied inhibitors and substrates. The inhibition of 1 mM ascorbic acid on sPPO and mPPO reached 95.42 ± 0.07% and 65.60 ± 1.16%, respectively. β-Cyclodextrin had a direct inhibitory effect only on sPPO, while the other three inhibitors had direct effects on both sPPO and mPPO. Cinnamic acid exhibited a non-competitive inhibition on sPPO and mPPO, with L-cysteine showing the same, though on sPPO. The inhibition of studied inhibitors on sPPO and mPPO is highly related to the substrate environment, type, and concentration of inhibitors. This study provides a basis for the further prevention of peach fruit browning from the perspective of different enzyme forms.

Comparative study of the biochemical properties of membrane-bound and soluble polyphenol oxidase from Prunus mume

Browning occurs during fruit and vegetable processing mainly as a result of oxidation by polyphenol oxidases. We compared the biochemical properties of membrane-bound polyphenol oxidase (mPPO) and soluble polyphenol oxidase (sPPO) isolated and purified from Prunus mume. The sPPO showed a greater affinity for the substrate catechol. The highest activity of sPPO was catechol, while those of the mPPO were catechol and 4-methylcatechol. The optimum pH of sPPO was 5.0, while that of the mPPO were 4.5 and 5.5; the optimum temperatures of sPPO and mPPO were 40 °C and 60 °C, respectively; and the sPPO was more resistant to acidic environments, while the mPPO maintained better stability within temperature changes. The metal ions Mg2+ and Na+ were able to activate both sPPO and mPPO of P. mume, while Al3+ and K+ significantly inhibited their activities, and both Cu2+ and Ca2+ inhibited the activity of sPPO but activated that of mPPO. Different inhibitors were found to have different effects on sPPO and mPPO, however, sodium sulfite, sodium bisulfite, l-cysteine, glutathione and ascorbic acid had obvious inhibitory effects on both forms of polyphenol oxidase. Provide theoretical support for the research on the characteristics and browning mechanism of polyphenol oxidase in fruits and vegetables.

Purification and comparison of soluble and membrane-bound polyphenol oxidase from potato (Solanum tuberosum) tubers

Enzymatic browning greatly affects the quality of potato products. Polyphenol oxidase (PPO) is the enzyme mainly responsible for potato enzymatic browning. PPO has soluble polyphenol oxidase (sPPO) and membrane-bound polyphenol oxidase (mPPO) forms. In this study, the properties of sPPO and mPPO were investigated in potato tubers. The molecular weight of potato sPPO and mPPO were estimated to be 69 kDa in the form of homodimers in vivo. The mass spectrometry results showed that the purified sPPO and mPPO protein in potato tubers was mainly tr|M1BMR6 (Uniprot). The optimum pH for sPPO and mPPO was 6.5, and the optimum temperatures were 20 and 30 °C, respectively. The Michaelis constant (Km) and maximum unit enzyme activity (Vmax) of sPPO were 6.08 mM and 2161 U/S when catechol was used as the substrate, whereas those of mPPO were 2.95 mM and 2129.53 U/S, respectively. The mPPO had stronger affinity to the substrate catechol than sPPO, whereas pyrogallic acid was stronger affinity for sPPO. Ascorbic acid and sodium sulfite were inhibitors of sPPO and mPPO, respectively. After understanding the different binding states of polyphenol oxidase, different inhibitors and treatment methods can be used to treat the enzyme according to different enzymatic properties, so as to achieve a greater degree of Browning control. These results will provide a theoretical basis for regulating PPO activity to reduce enzymatic browning during potato processing.

Comparison of the characterization and the temperature/pressure stability of soluble and membrane-bound polyphenol oxidase from ‘Lijiang’ snow peach

The characterization of soluble polyphenol oxidase (sPPO) and membrane-bound (mPPO) from ‘Lijiang snow’ peach was compared, and the inactivation effects by thermal treatment ranging from 45 to 85 °C and high-pressure processing at 550 MPa for 10–50 min were investigated. sPPO and mPPO both showed optimum activity at pH 7.0, while another optimum pH at 5.0 was observed for mPPO. The optimum temperatures for sPPO and mPPO were 30 °C and 50 °C, respectively. The optimum substrate of sPPO was caffeic acid, while mPPO showed the highest activity against 4-methylcatechol. All inhibitors showed poorer inhibitory effects on mPPO compared with sPPO. According to kinetic parameters, sPPO and mPPO were well described by the first-order model and the fractional-conversion model, respectively. The activity of sPPO was activated after HPP at 550 MPa for 20–50 min, while mPPO was more stable under the treatment conditions investigated.

Kinetic, spectroscopic, and molecular docking studies on the inhibition of membrane-bound polyphenol oxidase from Granny Smith apples (Malus domestica Borkh.)

We investigated the inhibitory effect and binding mechanism of four selected compounds (ascorbic acid, l-cysteine, glutathione, and citric acid) on membrane-bound polyphenol oxidases (mPPO) using spectroscopic and molecular docking techniques. Kinetic analysis demonstrated that these inhibitors reversibly inhibited the mPPO activity. Fluorescence spectroscopy revealed that the intrinsic fluorescence intensity of mPPO was quenched by inhibitors with a single class of the inhibition site on mPPO. Amino acid residues His 180, His 201, His 366, Cys 184, Glu 328, and Asn 333 were the important binding sites in the active center. These sites were identified using molecular docking techniques. Our findings suggested that the inhibitors were allosterically bound to the active center of mPPO through hydrogen bonds and ion contacts. This study provides new insights into the active site residues responsible for catalyzing mPPO and provides applicable information about the design of mPPO inhibitors.

Characterization of membrane-bound polyphenol oxidase from Granny Smith apple (Malus × domestica Borkh.)

Membrane-bound polyphenol oxidase (mPPO) from the Granny Smith apple was purified and characterized. The enzyme was purified by a factor of 20.53 with a recovery of 1.8%. The molecular weight of purified mPPO was determined to be 65 kDa by electrophoresis and nano-electrospray ionization mass spectrometry. mPPO exhibited its highest activity at a temperature of 35 °C and a pH of 7.0 and can be regarded as a diphenol oxidase. A low concentration of SDS (≤0.5 mM) enhanced the enzymatic activity, whereas mPPO was activated at high concentration EDTA (≥2 mM). The thermal transition temperature of mPPO was 76.98 °C. The circular dichroism spectrum showed that mPPO contains high α-helix content, the fluorescence spectroscopy indicated that the tryptophan residues of mPPO are partially buried. The particle size of mPPO was 5–10 nm with a complete structure. The structural characterization of mPPO provided better insights into the regions responsible for its activity.

The Inactivation Kinetics of Soluble and Membrane-Bound Polyphenol Oxidase in Pear during Thermal and High-Pressure Processing

Polyphenol oxidase (PPO) from pear was characterized with catechol as substrate. The Michaelis constant of soluble and membrane-bound PPO were 15.6 and 23.8 mM, respectively, and their optimum pH for activity were 6.0 and 6.5, respectively. The inactivation kinetics of soluble and membrane-bound PPO during thermal (45–75 °C) and high-pressure thermal processing (600 MPa, 40–80 °C) were studied. The inactivation kinetics of pear PPO were described by a first-order model at all processing conditions. Compared to soluble PPO, membrane-bound PPO was more sensitive to thermal and high-pressure inactivation. Both soluble and membrane-bound PPO displayed higher sensitivity towards thermal inactivation at pH 3.5 (pH of pear puree made from pears dipped in citric acid prior to blending) compared to pH 4.4 (pH of non-acidified pear puree). High pressure and temperature had synergistic inactivation effects on pear PPO at pH 4.4 while slight antagonistic effects were observed at pH 3.5.

Purification and structural analysis of membrane-bound polyphenol oxidase from Fuji apple

Membrane-bound polyphenol oxidase (mPPO) in Fuji apple (Malus domestica Borkh. cv. Red Fuji) was purified and analyzed with a nanoelectrospray ionization mass spectrometer. The three-dimensional model and binding site of mPPO to 4-methyl catechol were also studied using molecular docking. mPPO was purified 54.41-fold using temperature-induced phase partitioning technique and ion exchange chromatography. mPPO had a molecular weight of 67.3kDa. Even though a significant level of homology was observed between mPPO and the soluble polyphenol oxidase in the copper binding sequence, there was another region, rich in histidine residues, which differed in 13 amino acids. The three-dimensional structure of mPPO consisted of six α-helices, two short β-strands, and ten random coils. The putative substrate-binding pocket contained six polar or charged amino acids, His191, His221, Trp224, Trp228, Phe227, and Val190. Trp224 and Trp228 formed hydrogen bonds with 4-methyl-catechol.

Purification and characterization of membrane-bound polyphenoloxidase (mPPO) from Snake fruit [Salacca zalacca (Gaertn.) Voss

Membrane-bound polyphenoloxidase (mPPO) an oxidative enzyme which is responsible for the undesirable browning reaction in Snake fruit (Salacca zalacca (Gaertn.) Voss) was investigated. The enzyme was extracted using a non-ionic detergent (Triton X-114), followed by temperature-induced phase partitioning technique which resulted in two separate layers (detergent-poor phase at the upper layer and detergent-rich phase at the lower layer). The upper detergent-poor phase extract was subsequently fractionated by 40–80% ammonium sulfate and chromatographed on HiTrap Phenyl Sepharose and Superdex 200 HR 10/30. The mPPO was purified to 14.1 folds with a recovery of 12.35%. A single prominent protein band appeared on native-PAGE and SDS–PAGE implying that the mPPO is a monomeric protein with estimated molecular weight of 38kDa. Characterization study showed that mPPO from Snake fruit was optimally active at pH 6.5, temperature 30°C and active towards diphenols as substrates. The Km and Vmax values were calculated to be 5.46mM and 0.98U/ml/min, respectively, when catechol was used as substrate. Among the chemical inhibitors tested, l-cysteine showed the best inhibitory effect, with an IC50 of 1.3±0.002mM followed by ascorbic acid (1.5±0.06mM), glutathione (1.5±0.07mM), EDTA (100±0.02mM) and citric acid (186±0.16mM).

An historical perspective on lignan biosynthesis: Monolignol, allylphenol and hydroxycinnamic acid coupling and downstream metabolism

This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-pinoresinol. In a species dependent manner, (+)- or (−)-pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [13C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling. Abbreviations: CD – circular dichroism; e.e. – enantiomeric excess; DP – dirigent protein; ESI-MS – electrospray ionization mass spectrometry; MALDI -TOF – matrix assisted laser desorption ionization-time of flight; MALLS – multiangle laser light scattering; PLR – pinoresinol lariciresinol reductase; SDH – secoisolariciresinol dehydrogenase.

An ultracentrifugation-chloroplast membrane preparatory method for the fine-structural localization of polyphenol oxidase

Abstract Polyphenol oxidase (PPO) has been localized at the fine structural level in whole plant chloroplasts (Czaninski and Catesson, 1972; Henry, 1976). It is assumed that the PPO of chloroplast thylakoids and lamellae can be either latent or active depending upon specific cellular metabolism, plant treatment, and exposure conditions. Detergents, such as digitonin, accelerate tthe activation of spinach thylakoid membrane-bound PPO (Lieberei and Biehl, 1976). As any PPO activity localized by ultrastructural procedures, would be expected to possibly exist on the outer surfaces of the thylakoids and lamellae, a comprehensive methodology was defined for the isolation of sorbitol disrupted spinach chloroplast membranes, with subsequent ult ultrastructural localization of PPO (Henry et al., 1981). Spinach ( Spinacia oleracea L.) leaves were homogenized in 0.33M sorbitol-Hepes buffer, pH 7.6, followed by filtration through cheesecloth. 1) The filtrate was centrifuged at 300xg for 5 min. 2) The chloroplast pellet was then recentrifuged at 1,200xg for 10 min. The combined filtrates were layered on a discontinuous sucrose gradient and centrifuged at 20,000xg for 6 . Thirty separate 1-ml fractions were recentrifuged at 60,000xg for 20 min, giving individual pellets, each of which were prepared for ultastructural PPO enzyme localization (Henry et al., 1981). PPO is localized in the grana lamellae membranes of the sorbitol disrupted chloroplasts (Fig. 1), using 3,4-dihydroxyphenylalanine (L-DOPA) as the substrate. In the presence of the L-DOPA substrate, plus the PPO inhibitor, sodium diethyldithiocarbamate (DDC), the grana lamellae do not show a positive staining reaction for PPO (Fig. 2). There is also an absence of positive PPO staining in control tissue, lacking pretreatment with either L-DOPA or DDC (Fig. 3). To summarize, sorbitol-disrupted chlorolast membranes have been isolated via discontinuous sucrose density gradient centrifugaion and subsequently stained for the presence of PPO. The grana lamellae show positive PPO reaction product (Figs. 1–2). It is suggested that PPO is an integral part of the grana lamellae, even after sorbitol treatment; however, there is a release of a small amount of PPO into the soluble fractions (Henry et al., 1981). It is suggested that the grana lamellae may represent the possible sites of origin of PPO biosynthesis.

Effect of rust infection on rates of photochemical polyphenol oxidation and latent polyphenol oxidase activity of Vicia faba chloroplast membranes

Changes in (i) the rate of light-dependent polyphenol oxidation (via the non-cyclic electron transport pathway) and (ii) the activity of latent membrane-bound polyphenol oxidase (PPO) (measured in the dark) were determined following infection with the rust fungus Uromyces fabae . The rate of photochemical polyphenol oxidation was consistently suppressed in chloroplast membranes isolated from infected leaves, relative to their healthy counterparts. This decrease seemed to be due to the block in the non-cyclic electron transport pathway that we described earlier for the photoreduction of NADP. This conclusion was supported by the finding that rust infection effected a reduction in the rate of the photo-oxidation of both water (NADP as acceptor) and dihydroxyphenylalanine (DOPA) (O 2 as acceptor). In contrast to light-dependent oxidation, enzyme-mediated polyphenol oxidation [assayed in the dark in either the absence or presence of sodium dodecyl sulfate (SDS)] was elevated in chloroplasts from infected leaves. In this respect, latent membrane-bound chloroplast PPO from Vicia faba resembled its soluble active counterpart from other plants.

Polyphenol Oxidation by Vicia faba Chloroplast Membranes

The mechanism whereby light effects polyphenol oxidation was examined with Vicia faba chloroplast membranes known to contain a bound latent polyphenol oxidase. Results obtained with the inhibitors 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-idopropyl-p-benzoquinone (DBMIB) indicated an involvement of the non-cyclic electron transport pathway in the light-dependent oxidation of polyphenols, such as dihydroxyphenylalanine (DOPA). Further evidence was provided by experiments in which (a) DOPA replaced H(2)O as electron donor for the photoreduction of NADP, (b) NADP replaced O(2) as electron acceptor in the photochemical oxidation of DOPA, and (c) the variable fluorescence associated with photosystem II was increased by DOPA. The photochemical oxidation of DOPA by V. faba chloroplast membranes was insensitive to KCN and to antibodies against purified latent polyphenol oxidase. The results are consistent with the conclusion that the light-dependent oxidation of polyphenols by V. faba chloroplast membranes is achieved independently of the latent membrane-bound polyphenol oxidase. Electrons derived from polyphenols seem to enter the noncyclic electron transport chain on the oxidizing side of photosystem II and to react with O(2) at an unidentified site on the photosystem I side of the DCMU/DBMIB blocks.The physiological mechanism for the activation of latent polyphenol oxidase remains an unanswered question. Present results suggest that activation could occur through either acidification or the release of free fatty acids.