ABSTRACT
The "dragon-eye" fruits produced by the tropical longan tree are rich in nutrients and antioxidants. They suffer from post-harvest enzymatic browning, a process for which mainly the polyphenol oxidase (PPO) family of enzymes is responsible. In this study, two cDNAs encoding the PPO have been cloned from leaves of Dimocarpus longan (Dl), heterologously expressed in Escherichia coli and purified by affinity chromatography. The prepro-DlPPO1 contains two signal peptides at its N-terminal end that facilitate transportation of the protein into the chloroplast stroma and to the thylakoid lumen. Removal of the two signal peptides from prepro-DlPPO1 yields pro-DlPPO1. The prepro-DlPPO1 exhibited higher thermal tolerance than pro-DlPPO1 (unfolding at 65 °C vs. 40 °C), suggesting that the signal peptide may stabilize the fold of DlPPO1. DlPPO1 can be classified as a tyrosinase because it accepts both monophenolic and diphenolic substrates. The pro-DlPPO1 exhibited the highest specificity towards the natural diphenol (-)-epicatechin (kcat/KM of 800 ± 120 s-1 mM-1), which is higher than for 4-methylcatechol (590 ± 99 s-1 mM-1), pyrogallol (70 ± 9.7 s-1 mM-1) and caffeic acid (4.3 ± 0.72 s-1 mM-1). The kinetic efficiencies of prepro-DlPPO1 are 23, 36, 1.7 and 4.7-fold lower, respectively, than those observed with pro-DlPPO1 for the four aforementioned diphenolic substrates. Additionally, docking studies showed that (-)-epicatechin has a lower binding energy than any other investigated substrate. Both kinetic and in-silico studies strongly suggest that (-)-epicatechin is a good substrate of DlPPO1 and ascertain the affinity of PPOs towards specific flavonoid compounds.
Subject(s)
Catechin , Sapindaceae , Monophenol Monooxygenase/chemistry , Protein Sorting SignalsABSTRACT
In this study, we investigate the effect of enzymatic browning on the phenolic composition of apricot in vivo and in vitro. The in vitro browning was caused by the recombinant latent apricot polyphenol oxidase (L-PaPPO). Successful heterologous expression of PaPPO in Escherichia coli yielded substantial amounts of enzyme containing both copper ions in the catalytic active site. The expressed L-PaPPO was characterized with regard to its molecular mass (56531.3 Da), pH optimum (7.0), activation by SDS, and enzyme kinetics. LC-MS/MS was used to compare the phenolic profiles of brown and non-brown apricots. The browning reactions did significantly decrease total phenolics and antioxidant capacity (measured with DPPH and CUPRAC assays). Catechin, epicatechin, and B-type procyanidins were the individual phenolics most affected by browning, followed by chlorogenic and neochlorogenic acid. These phenolics are most likely the main endogenous substrates of L-PaPPO, as they were oxidized much faster than the other identified phenolics.
ABSTRACT
We investigated the bi-colored dahlia cultivar 'Seattle', which exhibits bright yellow petals with white tips, for its potential use as a model system for studies of the anthochlor biosynthesis. The yellow base contained high amounts of the 6'-deoxychalcones and the structurally related 4-deoxyaurones, as well as flavones. In contrast, only traces of anthochlors and flavones were detected in the white tips. No anthocyanins, flavonols, flavanones or dihydroflavonols were found in the petals. Gene expression studies indicated that the absence of anthocyanins in the petals is caused by a lack of flavanone 3-hydroxylase (FHT) expression, which is accompanied by a lack of expression of the bHLH transcription factor IVS. Expression of other genes involved in anthocyanidin biosynthesis such as dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) was not affected. The yellow and white petal parts showed significant differences in the expression of chalcone synthase 2 (CHS2), which is sufficient to explain the absence of yellow pigments in the white tips. Transcriptomes of both petal parts were de novo assembled and three candidate genes for chalcone reductase (CHR) were identified. None of them showed a significantly higher expression in the yellow base compared to the white tips. In summary, it was shown that the bicolouration is most likely caused by a bottleneck in chalcone formation in the white tip. The relative prevalence of flavones compared to the anthochlors in the white tips could be an indication for the presence of a so far unknown differentially expressed CHR.
Subject(s)
Dahlia , Gene Expression Regulation, Plant , Models, Biological , Pigments, Biological , Anthocyanins/genetics , Dahlia/genetics , Dahlia/metabolism , Flowers/chemistry , Flowers/genetics , Flowers/metabolism , Genes, Plant/genetics , Pigments, Biological/biosynthesisABSTRACT
Type-III copper enzymes like polyphenol oxidases (PPOs) are ubiquitous among organisms and play a significant role in the formation of pigments. PPOs comprise different enzyme groups, including tyrosinases (TYRs) and catechol oxidases (COs). TYRs catalyze the o-hydroxylation of monophenols and the oxidation of o-diphenols to the corresponding o-quinones (ECâ 1.14.18.1). In contrast, COs only catalyze the oxidation of o-diphenols to the corresponding o-quinones (ECâ 1.10.3.1). To date (August 2020), 102 PDB entries encompassing 18 different proteins from 16 organisms and several mutants have been reported, identifying key residues for tyrosinase activity. The structural similarity between TYRs and COs, especially within and around the active center, complicates the elucidation of their modes of action on a structural basis. However, mutagenesis studies illuminate residues that influence the two activities and show that crystallography on its own cannot elucidate the enzymatic activity mode. Several amino acid residues around the dicopper active center have been proposed to play an essential role in the two different activities. Herein, we critically review the role of all residues identified so far that putatively affect the two activities of PPOs.
Subject(s)
Catechol Oxidase/metabolism , Monophenol Monooxygenase/metabolism , Plant Proteins/metabolism , Biocatalysis , Catechol Oxidase/chemistry , Catechol Oxidase/genetics , Monophenol Monooxygenase/chemistry , Monophenol Monooxygenase/genetics , Mutagenesis, Site-Directed , Oxidation-Reduction , Phenols/chemistry , Phenols/metabolism , Plant Proteins/chemistry , Plant Proteins/genetics , Substrate SpecificityABSTRACT
Tyrosinases (TYRs) catalyze the hydroxylation of phenols and the oxidation of the resulting o-diphenols to o-quinones, while catechol oxidases (COs) exhibit only the latter activity. Aurone synthase (AUS) is not able to react with classical tyrosinase substrates, such as tyramine and l-tyrosine, while it can hydroxylate its natural substrate isoliquiritigenin. The structural difference of TYRs, COs, and AUS at the heart of their divergent catalytic activities is still a puzzle. Therefore, a library of 39â mutants of AUS from Coreopsis grandiflora (CgAUS) was generated and the activity studies showed that the reactivity of the three conserved histidines (HisA2 , HisB1 , and HisB2 ) is tuned by their adjacent residues (HisB1 +1, HisB2 +1, and waterkeeper residue) either to react as stronger bases or / and to stabilize a position permissive for substrate proton shuffling. This provides the understanding for C-H activation based on the type-III copper center to be used in future biotechnological processes.
Subject(s)
Amino Acids/analysis , Catechol Oxidase/metabolism , Copper/metabolism , Monophenol Monooxygenase/metabolism , Amino Acids/metabolism , Catechol Oxidase/chemistry , Copper/chemistry , Coreopsis/enzymology , Models, Molecular , Monophenol Monooxygenase/chemistryABSTRACT
Polyphenol oxidases (PPOs) comprise tyrosinases (TYRs) and catechol oxidases (COs), which catalyse the initial reactions in the biosynthesis of melanin. TYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas COs react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono- versus diphenolase activity in the type-III copper enzyme jrPPO1.
Subject(s)
Catechol Oxidase/genetics , Catechol Oxidase/metabolism , Juglans/enzymology , Juglans/genetics , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Amino Acid Substitution , Catalytic Domain/genetics , Catechol Oxidase/chemistry , Cloning, Molecular , Copper/analysis , Enzyme Activation , Genes, Plant , Kinetics , Models, Molecular , Molecular Docking Simulation , Molecular Weight , Monophenol Monooxygenase/chemistry , Mutagenesis, Site-Directed , Plant Proteins/chemistry , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
The present research investigates the long term inhibition of enzymatic browning by inactivating the polyphenol oxidase (PPO) of apricot, using combinations of plant proteases and ascorbic acid (AA). The selected proteases were able to inactivate PPO at pH 4.5, with the degree of inactivation proportional to incubation time and protease concentration. Papain was the most effective protease, with 50⯵g completely inactivating PPO in less than one hour. AA prevented browning reactions that occur before or during PPO inactivation by protease. The combinations of AA/proteases were highly effective in vitro, where 2â¯mM AA/500⯵g proteases inhibited PPO activity completely over 24â¯h. The combination of AA/proteases was also effective in vivo, as treated apricot purees preserved their color (pâ¯<â¯0.0001, compared to untreated samples after 10â¯days of storage). The results demonstrate that AA/proteases combinations constitute a promising practical anti-browning method with feasible application in the food industry.
ABSTRACT
ABSTRACT: The novel iron-substituted Krebs-type polyoxotungstate (C12N4H11)4Na2H5[(Fe(H2O)3)2((FeO2)0.5(WO2)0.5)2(ß-SbW9O33)2] (Fe-1) has been synthesized using ortho-phenylenediamine (opda) as a precursor for the in situ formation of the counter cation 2,3-diaminophenazinium (C12N4H11)+ (2,3-DAP). Fe-1 has been thoroughly characterized in the solid state by single-crystal X-ray diffraction (SXRD), powder X-ray diffraction (PXRD), IR spectroscopy, and elemental analysis as well as in solution by UV-Vis spectroscopy. The crystal structure of Fe-1 reveals π-π-interactions between the aromatic systems of the unconventional 2,3-DAP counter cation. POM-protein interaction studies using SDS-PAGE revealed a non-proteolytic behavior of Fe-1 towards Human Serum Albumin (HSA) as a model protein.
ABSTRACT
The conversion of inactive pro-polyphenol oxidases (pro-PPOs) into the active enzyme results from the proteolytic cleavage of its C-terminal domain. Herein, a peptide-mediated cleavage process that activates pro-MdPPO1 (Malus domestica) is reported. Mass spectrometry, mutagenesis studies, and X-ray crystal-structure analysis of pro-MdPPO1 (1.35â Å) and two separated C-terminal domains, one obtained upon self-cleavage of pro-MdPPO1 and the other one produced independently, were applied to study the observed self-cleavage. The sequence Lys 355-Val 370 located in the linker between the active and the C-terminal domain is indispensable for the self-cleavage. Partial introduction (Lys 352-Ala 360) of this peptide into the sequence of two other PPOs, MdPPO2 and aurone synthase (CgAUS1), triggered self-cleavage in the resulting mutants. This is the first experimental proof of a self-cleavage-inducing peptide in PPOs, unveiling a new mode of activation for this enzyme class that is independent of any external protease.
Subject(s)
Catechol Oxidase/metabolism , Malus/enzymology , Peptide Fragments/metabolism , Plant Proteins/metabolism , Catechol Oxidase/chemistry , Catechol Oxidase/genetics , Crystallography, X-Ray , Dipeptides/chemistry , Dipeptides/genetics , Dipeptides/metabolism , Models, Molecular , Mutation , Plant Proteins/chemistry , Protein ConformationABSTRACT
Polyphenol oxidases (PPOs) contain the structurally similar enzymes tyrosinases (TYRs) and catechol oxidases (COs). Two cDNAs encoding pro-PPOs from tomato (Solanum lycopersicum) were cloned and heterologously expressed in Escherichia coli. The two pro-PPOs (SlPPO1-2) differ remarkably in their activity as SlPPO1 reacts with the monophenols tyramine (kcat = 7.94 s-1) and phloretin (kcat = 2.42 s-1) and was thus characterized as TYR, whereas SlPPO2 accepts only diphenolic substrates like dopamine (kcat = 1.99 s-1) and caffeic acid (kcat = 20.33 s-1) rendering this enzyme a CO. This study, for the first time, characterizes a plant TYR and CO originating from the same organism. Moreover, X-ray structure analysis of the latent holo- and apo-SlPPO1 (PDB: 6HQI and 6HQJ) reveals an unprecedented high flexibility of the gatekeeper residue phenylalanine (Phe270). Docking studies showed that depending on its orientation the gatekeeper residue could either stabilize and correctly position incoming substrates or hinder their entrance into the active site. Furthermore, phloretin, a substrate of SIPPO1 (Km = 0.11 mM), is able to approach the active centre of SlPPO1 with both phenolic rings. Kinetic and structural results indicate that phloretin could act as a natural substrate and connote the participation of PPOs in flavonoid-biosynthesis.
Subject(s)
Catechol Oxidase/chemistry , Plant Proteins/chemistry , Solanum lycopersicum/enzymology , Amino Acid Sequence , Cloning, Molecular , Crystallography, X-Ray/methods , Escherichia coli/genetics , Kinetics , Models, Molecular , Molecular Docking Simulation/methods , Protein Conformation , Substrate SpecificityABSTRACT
A novel synthetic pathway to obtain the first Zn6 hexagon tungstoantimonate [(Zn(H2O))6(B-α-SbW9O33)2]6- ([Zn6-α-SbW9] (1)) via rearrangement of a non-lacunary Krebs-POM precursor (C12N4H11)4K4[(Mn(H2O)3)2((Mn0.5W0.5)O2)2(B-ß-SbW9O33)2] ([Mn-ß-SbW9]) has been developed. Addition of ortho-phenylenediamine (opda) in order to optimize the synthesis of [Mn-ß-SbW9] led to the crystallization of a novel Krebs-type Zn-POM (C12N4H11)4Na5[((Zn0.8W0.2)(H2O)3)2((Zn0.2W0.8)O2)2(B-ß-SbW9O33)2] ([(Zn/W)2-ß-SbW9] (2)) comprising four disordered Zn-centers after replacement of MnCl2 with ZnCl2. The compounds were characterized in the solid state by single-crystal and powder X-ray diffraction (XRD), IR spectroscopy, thermogravimetric analysis (TGA) and elemental analysis and in solution by UV-vis spectroscopy and ESI-mass spectrometry. The Krebs-POM archetype and the Zn6 hexagon tungstoantimonate have been investigated towards their interactions with Human Serum Albumin (HSA) as a model protein using SDS-PAGE and trypthophan fluorescence quenching.
ABSTRACT
A concise and efficient total synthesis of the lignan natural product larreatricin as well as an unambiguous assignment of configuration of its enantiomers are reported, resolving a long-held controversy. Enzyme kinetic studies revealed that different polyphenol oxidases show high and remarkably divergent enantioselective recognition of this secondary metabolite.
Subject(s)
Biological Products/chemical synthesis , Catechol Oxidase/chemistry , Furans/chemical synthesis , Lignans/chemistry , Polyphenols/chemical synthesis , Enzyme Activation , Kinetics , Molecular Structure , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Polyphenol oxidase from apricot (Prunus armeniaca) (PaPPO) was purified in its latent form (L-PaPPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-PaPPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO4 and low concentrations (≤ 2 mM) of SDS. PaPPO has its pH and temperature optimum at pH 4.5 and 45 °C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (KM = 2.0 mM) and chlorogenic acid (KM = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-PaPPO). The mass of A-PaPPO was determined by mass spectrometry as 37â¯455.6 Da (Asp102 â Leu429). Both L-PaPPO and A-PaPPO were identified as polyphenol oxidase corresponding to the known PaPPO sequence (UniProt O81103 ) by means of peptide mass fingerprinting.
Subject(s)
Catechol Oxidase/chemistry , Catechol Oxidase/isolation & purification , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Prunus armeniaca/enzymology , Catechol Oxidase/genetics , Catechol Oxidase/metabolism , Chlorogenic Acid/metabolism , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Plant Proteins/genetics , Plant Proteins/metabolism , Prunus armeniaca/chemistry , Prunus armeniaca/genetics , Substrate Specificity , TemperatureABSTRACT
Tyrosinases and catechol oxidases belong to the polyphenol oxidase (PPO) enzyme family, which is mainly responsible for the browning of fruits. Three cDNAs encoding PPO pro-enzymes have been cloned from leaves of Malus domestica (apple, MdPPO). The three pro-enzymes MdPPO1-3 were heterologously expressed in E. coli yielding substantial amounts of protein and have been characterized with regard to their optimum of activity resulting from SDS, acidic and proteolytic activation. Significant differences were found in the kinetic characterization of MdPPO1-3 when applying different mono- and diphenolic substrates. All three enzymes have been classified as tyrosinases, where MdPPO1 exhibits the highest activity with tyramine (kcat = 9.5 s-1) while MdPPO2 and MdPPO3 are also clearly active on this monophenolic substrate (kcat = 0.92 s-1 and kcat = 1.0 s-1, respectively). Based on the activity, sequence data and homology modelling it is proposed that the monophenolase and diphenolase activity of PPOs can be manipulated by the appropriate combination of two amino acids, which are located within the active site cleft and were therefore named "activity controllers".
Subject(s)
Amino Acids/chemistry , Catechol Oxidase/chemistry , Catechol Oxidase/metabolism , Malus/enzymology , Monophenol Monooxygenase/chemistry , Monophenol Monooxygenase/metabolism , Recombinant Proteins , Amino Acid Sequence , Catechol Oxidase/genetics , Gene Expression , Malus/genetics , Models, Molecular , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/isolation & purification , Protein Conformation , Protoporphyrinogen Oxidase/chemistry , Protoporphyrinogen Oxidase/metabolism , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate SpecificityABSTRACT
Tyrosinases are type 3 copper enzymes that belong to the polyphenol oxidase (PPO) family and are able to catalyze both the ortho-hydroxylation of monophenols and their subsequent oxidation to o-quinones, which are precursors for the biosynthesis of colouring substances such as melanin. The first plant pro-tyrosinase from Malus domestica (MdPPO1) was recombinantly expressed in its latent form (56.4â kDa) and mutated at four positions around the catalytic pocket which are believed to influence the activity of the enzyme. Mutating the amino acids, which are known as activity controllers, yielded the mutants MdPPO1-Ala239Thr and MdPPO1-Leu243Arg, whereas mutation of the so-called water-keeper and gatekeeper residues resulted in the mutants MdPPO1-Glu234Ala and MdPPO1-Phe259Ala, respectively. The wild-type enzyme and two of the mutants, MdPPO1-Ala239Thr and MdPPO1-Phe259Ala, were successfully crystallized, leading to single crystals that diffracted to 1.35, 1.55 and 1.70â Å resolution, respectively. All crystals belonged to space group P212121, exhibiting similar unit-cell parameters: a = 50.70, b = 80.15, c = 115.96â Å for the wild type, a = 50.58, b = 79.90, c = 115.76â Å for MdPPO1-Ala239Thr and a = 50.53, b = 79.76, c = 116.07â Å for MdPPO1-Phe259Ala. In crystallo activity tests with the crystals of the wild type and the two mutants were performed by adding the monophenolic substrate tyramine and the diphenolic substrate dopamine to crystal-containing drops. The effects of the mutation on the activity of the enzyme were observed by colour changes of the crystals owing to the conversion of the substrates to dark chromophore products.
Subject(s)
Copper/chemistry , Dopamine/chemistry , Malus/chemistry , Monophenol Monooxygenase/chemistry , Plant Proteins/chemistry , Tyramine/chemistry , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Cloning, Molecular , Copper/metabolism , Crystallography, X-Ray , Dopamine/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Hydroxylation , Malus/enzymology , Models, Molecular , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/metabolism , Mutation , Oxidation-Reduction , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Tyramine/metabolismABSTRACT
Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80kb (cgAUS1) and 1.85kb (cgAUS2a, 2b), encoding for proteins of 68-69kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS-PAGE and shot-gun type nanoUHPLC-ESI-MS/MS.