Still, the enzymatic properties of PPOs are potentially capable of providing important functions in plant specialized metabolism. Are these cases exceptional, or the tip of the iceberg? There are several steps in betalain biosynthesis that might utilize either the tyrosinase and catechol oxidase activities of PPO see Gandia-Herrero and Garcia-Carmona, for a detailed review. The cleavage product spontaneously rearranges to form betalamic acid, which can condense with amino acids or other amine groups to form yellow betaxanthins.
Condensation of betalamic acid with cyclo -DOPA forms the red betacyanin pigments. However, recently a cytochrome P, CYP76AD1, has been identified in beet via a bioinformatic approach that appears to carry out this reaction in vivo and can compliment the R mutant produces yellow, but not red pigment in beet and other species Hatlestad et al. If this is the case in beet, however, the activity would be redundant with another, since silencing of CYP76AD1 results in loss of red, but not yellow pigments whose formation do not require cyclo -DOPA formation Hatlestad et al.
However, transcriptome analysis in beet did not find the abundance of PPO transcripts that might be expected for high betalain production Hatlestad et al. Thus, despite longstanding speculation that PPO is involved in betalain biosynthesis, its role in cyclo -DOPA formation seems unlikely, and definitive demonstration of a role in the initial conversion of tyrosine to L -DOPA in vivo is lacking.
Involvement of polyphenol oxidases PPOs in plant specialized metabolism. Steps where PPO involvement has been demonstrated or proposed are highlighted in red. For simplicity, not all reactants, enzymes, or stereochemistry are shown.
Steps that occur spontaneously not mediated by an enzyme are indicated. A Betalain synthesis as described by Hatlestad et al. B Proposed tyrosine metabolism in walnut Araji et al. Values in parentheses are fold change in metabolite in walnut plants with PPO silenced via RNAi relative to wild type control plants as reported by Araji et al.
D i Aureusidin biosynthesis in A. AS, aurone synthase here, a plastidic PPO. In walnut Juglans regia , PPO is encoded by a single gene and has been demonstrated to have both tyrosinase and catechol oxidase activity Escobar et al.
To examine the in vivo function of PPO in walnut, Araji et al. When placed in soil, these plants had a striking phenotype: they developed disease-like necrotic lesions. Despite the lesions, no pathogens could be identified from the leaves. Levels of salicylic acid, H 2 O 2 , or malondialdehyde an indicator of oxidative damage , previously associated with other lesion-mimic mutants Lorrain et al. Metabolomic analysis of PPO-silenced and wild type leaves did reveal significant differences in many metabolites, however, particularly phenylpropanoids.
Especially striking were changes in levels of compounds associated with tyrosine metabolism Figure 1B ; Araji et al. Conversely, levels of metabolites that would be expected to be derived from the 3-hydroxylation of tyrosine or tyramine both good substrates for the tyrosinase activity of walnut PPO in vitro were markedly reduced in PPO-silenced plants.
Because the enzyme involved in 3-hydroxylation of these compounds had not been previously identified, the authors proposed that the simplest interpretation of the metabolomic results is that walnut PPO is the enzyme that mediates 3-hydroxylation of tyrosine and tyramine Araji et al. Thus, silencing of PPO would be expected to result in increased accumulation of those tyrosine metabolites that do not undergo 3-hydroxylation such as tyramine and the tocopherols and decreased accumulation of metabolites derived from L -DOPA or tyramine.
Further, the authors were able to demonstrate that the necrotic lesion phenotype of the PPO-silenced plants was almost certainly due to the accumulation of tyramine: incubation of petioles of detached wild type leaves in tyramine solution could phenocopy the necrotic lesions Araji et al.
Another metabolite that was dramatically decreased in PPO-silenced plants was esculetin. Although biosynthesis of this compound is not well understood, this observation is consistent with previous suggestion of the involvement of a chloroplast localized phenolase Sato, More definitive demonstration of a central role of walnut PPO in tyrosine metabolism and esculetin biosynthesis in walnut might require approaches such as radioactive pulse labeling.
It will be interesting to see how widespread this role of PPO in tyrosine metabolism is, especially in species whose PPO enzymes have been shown to have tyrosinase activity. Many of these compounds from creosote bush, e. Cho et al. Peptide sequencing of the purified hydroxylase identified fragments with high homology to conserved domains of PPOs from other plant species. The peptide sequence data further allowed cloning and sequencing of a full length cDNA corresponding to the L.
Like most plant PPOs, the L. Unfortunately, in this study, reverse genetics e. Unfortunately, there appears to have been relatively little further work on this pathway or characterization of the L. Does this PPO also form quinones from hydroxylarreatricins and under what conditions? Would such activities have any biological implications? One of the most interesting and well-studied cases of PPO having a role in biosynthesis of specialized metabolites is the biosynthesis of the chalcone-derived yellow aurone pigments in snapdragon Antirrhinum majus flowers.
The enzyme responsible, aureusidin or aurone synthase AS , was purified to homogeneity from yellow snapdragon buds Nakayama et al. Peptide sequencing of the purified enzyme allowed isolation and characterization of a cDNA encoding the enzyme. The predicted protein sequence showed high homology to other plant PPO enzymes.
Expression of the gene corresponded to aurone accumulation e. Further, in vitro , tyrosinase from Neurospora crassa could also convert THC to aureusidin, indicating that the enzymatic activities of PPO are involved in the biosynthetic conversion. Starting with THC, tyrosinase and catechol oxidase activity result in 3-hydroxylation and formation of the corresponding o -quinone. Whether AS PPO carries out the 3-hydroxylation reaction in vivo , or whether a cytochrome P chalcone 3-hydroxylase as described below for Coreopsis grandiflora is also involved has not been definitively established.
AS likely forms the same quinone from PHC without the need for the 3-hydroxylation step. The resulting quinone is predicted to undergo a 2-step non-enzyme mediated rearrangement to form aureusidin Nakayama et al. AS was also unable to oxidize aureusidin to its corresponding quinone, nor could it oxidize several other mono and o -diphenolic compounds, such as tyrosine, p -coumaric acid, L -DOPA, caffeic acid, or eriodictyol, suggesting a relatively strict substrate specificity Nakayama et al.
One of the most novel aspects of the A. Consistent with this, Ono et al. Recently, Kaintz et al. Interestingly, the C. Consistent with the lack of tyrosinase activity, the C.
The plastidic versus vacuolar nature of the C. The above examples could represent the tip of the iceberg with respect to PPO enzymes that have specific roles in biosynthesis of specialized metabolites. Much work on PPOs has focused on their negative impact on food quality due to the browning reactions they promote. In two of the cases above, the specialized roles of the PPOs were identified in the course of research focused on a particular aspect of specialized metabolism.
There, relatively laborious approaches led to the identification of the PPOs involved. This observation indicates that the thioether bridge might have stabilizing effects on the position of the gatekeeper residue in PPOs. In addition and in contrast to the holo-structure, the conserved water molecule, which is believed to deprotonate monophenolic substrates, is present in the apo-structure.
The superimposition of both the holo- and the apo-structure results in a theoretically complete and typical plant PPO structure as it would possess an intact dicopper active site, an intact thioether bridge and a conserved water molecule that is stabilized by the waterkeeper residue Glu To exclude the possibility that the absence of some structural features i.
The results indicated that all apo-structures based on two data sets were identical, whereas the structures of the holo-enzymes based on five data sets differed slightly from one another. Two of the five holo-structures contained the conserved water molecule, whereas the remaining structural features i. This indicates that the conserved water molecule is not absolutely absent in holo- Sl PPO1, whereas the thioether bridge seems indeed to be missing.
The reason for the absence of the Cys97 harboring loop in all holo-structures is not clear and it cannot be excluded that the loop underwent degradation. However, this does not explain thioether bond formation in all apo-forms, which were processed the same way as the holo-form.
Thus, it remains unclear why the thioether bond was not formed in the holo-form and to what extent the X-ray data do reflect the situation in solution.
To gain more information on the PPO-substrate interactions, molecular docking studies have been performed applying the crystal structure of holo- Sl PPO1 and all kinetically tested substrates. All computed docking poses were checked for their reasonableness by comparing them to the binding pose of tyrosine from the crystal structure of tyrosine-bound tyrosinase from Bacillus megaterium Bm TYR, PDB entry 4P6R 1.
Comparison of the docking poses of all Sl PPO1-substrate complexes revealed that phloretin exhibits binding poses that are stabilized much better than those of the other substrates owing to its bulky structure. Phloretin 3- 4-hydroxyphenyl 2,4,6-trihydroxyphenyl propanone is able to approach the dicopper centre with both hydroxyphenyl groups, whereby the approach of the 2,4,6-trihydroxyphenyl ring is favoured.
This pose enables more direct interactions between the substrate and the enzyme than in the binding scenario, where the 4-hydroxyphenyl ring is approaching the copper ions Figs. The para -hydroxy group of the 2,4,6-trihydroxyphenyl moiety exhibits the lowest pK a value and therefore it is most readily deprotonated, which is in accordance to the preferred docking pose. The pose with the 2,4,6-trihydroxyphenyl group approaching the copper ions pose 1 is stabilized by three amino acids, the 1 st activity controller Ser, the CuB coordinating His and Asn Ser hydrogen binds both the carbonyl and the ortho -positioned hydroxy group of the 2,4,6-trihydroxyphenyl moiety of phloretin Fig.
The same ortho -hydroxy group of phloretin forms a further hydrogen bond with the H-atom of the ND1 nitrogen atom of His, which is not involved in the coordination of CuA. Asn exhibits a hydrogen bond with the hydroxy group of the 4-hydroxyphenyl ring, which is pointing away from the active site Fig. In contrast, when the 4-hydroxyphenyl ring is approaching the active site pose 2 , phloretin interacts only with two amino acids, Asn and Ser Asn hydrogen binds the para -hydroxyl group of the 2,4,6-trihydroxyphenyl ring, whereas the activity controller is involved in an H-bond with an ortho -positioned hydroxy group of the same ring Fig.
The docking results of the remaining substrates revealed that they interact only with the activity controller Ser via the functional group at their tail i. Binding of phloretin to Sl PPO1 according to molecular docking. The molecule approaches CuA with its 2,4,6-trihydroxyphenyl ring.
Phloretin is stabilized by three amino acids. The molecule approaches CuA with its 4-hydroxyphenyl ring. The substrate interacts only with two amino acids.
However, the docking experiment failed to explain the absence of monophenolase activity in PPO2 as both enzymes led to similar binding poses owing to the almost identical architecture of their active sites. Nevertheless, in the case of phloretin, the docking experiment provided some valuable structural information on the binding discrepancy between PPO1 and PPO2, which are shown in the Supporting Information Fig. According to a previous theory, a bulky residue at this position was believed to act as an active site blocker by preventing the access of monophenolic substrates into the active site of COs This theory was contradicted by the crystal structure of walnut TYR jr PPO1 that possesses phenylalanine as gatekeeper residue However, during the here presented docking study a series of substrate poses were calculated, where the Phe-gatekeeper residue exhibited positions that indeed suggest a blocking role for this residue.
The docking study revealed that the gatekeeper residue, owing to its high flexibility, can interact with small substrates lacking a long chain at the tail of their structure or an additional ring systems in such a way that prevents them from accessing the active site. This situation is additionally complicated by the activity controller Ser as it forms H-bonds with the substrates, which further stabilize and thus lock them in these unfavorable poses Fig.
The docking results suggest a dual-functionality for the Phe-gatekeeper-residue, which on the one hand is able to stabilize the correct orientation of some substrates within the active site and on the other hand can also block the entrance of other substrates into the active site to some extent.
PPOs from S. The two isoenzymes, sharing a high sequence similarity of One of the main hurdles in the field of PPOs is the identification of natural substrates. Sl PPO1 exhibits a high specificity towards the chalcone phloretin as shown by kinetic and docking data , which might indicate that PPOs could accept flavonoids as their natural substrates and therefore might participate in the synthetic pathways of secondary metabolites Subsequent molecular docking did not only provide highly valuable insights into the binding event of different substrates but also confirmed the highly flexible nature of the gatekeeper residue.
Depending on the flexibility of the Phe-gatekeeper residue, i. According to the heterologous expression, Sl PPO1 is more soluble than Sl PPO2, which is in contradiction to their structural characteristics and expected interaction behaviour with membranes as only the former enzyme is supposed to be membrane associated PPOs are type-III copper enzymes exhibiting significant oxidation reactions in the majority of organisms.
Plenty of PPO enzymes have been purified and biochemically characterized, however, only few have been produced purely and were crystallized in order to characterize their biochemical features accurately.
The identification of natural substrates of PPOs represents a tough challenge in this field. However, Sl PPO1 exhibits a high specificity towards the chalcone phloretin, which might indicate that PPOs could accept flavonoids as natural substrates and therefore might participate in the synthetic pathways of secondary metabolites.
Young healthy leaves from tomato plants S. Two pairs of degenerated primers Table S1 were designed for at least six different PPOs, which have been placed in the genome of the S. The clones were sequenced externally by microsynth GmbH Vienna, Austria.
Escherichia coli was grown in a modified 2xYT medium 1. The cultures were induced with 0. Lysis of the cells was effectuated by the freeze-thaw technique using liquid nitrogen. Lysozyme 0. The activity was determined spectrophotometrically by detecting the appearance of the chromophoric quinones, which are produced by the reaction of the substrates monophenols: tyramine and phloretin; diphenols: dopamine and caffeic acid with the respective enzyme, in order to determine the kinetic parameters of latent Sl PPO1 and Sl PPO2.
The plate was sealed with an optically clear film Eppendorf. The resulting melting points of each enzyme were then plotted against the respective pH value for stability analysis. The crystallization of Sl PPO1 was performed by applying the hanging drop vapour-diffusion technique using 15 well EasyXtal plates Qiagen.
Crystals usually appeared after 4 d. The apo-form was produced by the removal of the Cu ions from the dicopper centre. This procedure was repeated three times and the final apo- Sl PPO1 was confirmed by activity assays as the enzyme was unable to react with any of the monophenolic or diphenolic substrates. Data collection statistics are summarized in Table S4. The crystals of the apo-form diffracted to a maximum resolution of 1. The crystals of both the apo- and the holo-form belonged to space group P 1 21 1, the crystal parameters are given in Table S4.
The data sets were processed with the program XDS Initial phases for the holo-enzyme, of which structure was solved first, were obtained by molecular replacement MR using the crystal structure of walnut tyrosinase PDB entry 5CE9 16 as the search model. For the apo-enzyme, the solved structure of the holo-form was used as MR search model to deduce initial phases.
The structure of both the apo- and the holo-enzyme were then solved by the same procedure: After initial phases were derived, Autobuild 54 from the PHENIX suite v. The resulting model was refined until convergence using phenix. Since the apo- and holo-form of Sl PPO1 differed significantly in some structural aspects, five further data sets were evaluated in the same way as described above in order to confirm the absence or presence of specific structural features Table S2.
Docking was performed using Autodock Vina 58 to identify binding poses of monophenolic tyramine and phloretin and diphenolic substrates dopamine and caffeic acid within the active centre of Sl PPO1 holo-form structure in order to structurally analyse substrate binding. The crystal structure of Sl PPO1 was prepared for molecular docking by adding missing side chains using COOT and the removal of the C-terminal domain cleavage after Pro in order to create the active form of the isoenzyme.
The gate residue Phe was defined as flexible residue and the exhaustiveness was set to The docking settings i. The resulting docking poses obtained from Autodock Vina applying our settings resembled almost perfectly the tyrosine pose found in the crystal structure of the Bm TYR-tyrosine complex PDB entry 4P6R 1 indicating that the defined settings were suitable. Docking was performed with all important protonation states of each substrate Fig.
Upon docking the binding poses were evaluated by superimposing the docked substrate position with that of tyrosine from the Bm TYR-tyrosine structure. In this case, Phe was defined as flexible residue. Goldfeder, M. Determination of tyrosinase substrate-binding modes reveals mechanistic differences between type-3 copper proteins. Mauracher, S. High level protein-purification allows the unambiguous polypeptide determination of latent isoform PPO4 of mushroom tyrosinase.
Acta Crystallogr. D Biol. Crystallization and preliminary X-ray crystallographic analysis of latent isoform PPO4 mushroom Agaricus bisporus tyrosinase. F Struct. Pretzler, M.
Heterologous expression and characterization of functional mushroom tyrosinase Ab PPO4. Kim, H. Mayer, A. Polyphenol oxidases in plants and fungi: Going places? A review. Tran, L. The polyphenol oxidase gene family in land plants: Lineage-specific duplication and expansion.
Zekiri, F. Purification and characterization of tyrosinase from walnut leaves Juglans regia. Derardja, A. Food Chem. Li, Y. Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type 3 copper enzymes. Lu, A. Insect prophenoloxidase: the view beyond immunity. Lai, X. D i Aureusidin biosynthesis in A. AS, aurone synthase here, a plastidic PPO. In walnut Juglans regia , PPO is encoded by a single gene and has been demonstrated to have both tyrosinase and catechol oxidase activity Escobar et al.
To examine the in vivo function of PPO in walnut, Araji et al. When placed in soil, these plants had a striking phenotype: they developed disease-like necrotic lesions. Despite the lesions, no pathogens could be identified from the leaves. Levels of salicylic acid, H 2 O 2 , or malondialdehyde an indicator of oxidative damage , previously associated with other lesion-mimic mutants Lorrain et al.
Metabolomic analysis of PPO-silenced and wild type leaves did reveal significant differences in many metabolites, however, particularly phenylpropanoids.
Conversely, levels of metabolites that would be expected to be derived from the 3-hydroxylation of tyrosine or tyramine both good substrates for the tyrosinase activity of walnut PPO in vitro were markedly reduced in PPO-silenced plants. Because the enzyme involved in 3-hydroxylation of these compounds had not been previously identified, the authors proposed that the simplest interpretation of the metabolomic results is that walnut PPO is the enzyme that mediates 3-hydroxylation of tyrosine and tyramine Araji et al.
Thus, silencing of PPO would be expected to result in increased accumulation of those tyrosine metabolites that do not undergo 3-hydroxylation such as tyramine and the tocopherols and decreased accumulation of metabolites derived from L -DOPA or tyramine.
Further, the authors were able to demonstrate that the necrotic lesion phenotype of the PPO-silenced plants was almost certainly due to the accumulation of tyramine: incubation of petioles of detached wild type leaves in tyramine solution could phenocopy the necrotic lesions Araji et al.
Another metabolite that was dramatically decreased in PPO-silenced plants was esculetin. Although biosynthesis of this compound is not well understood, this observation is consistent with previous suggestion of the involvement of a chloroplast localized phenolase Sato, More definitive demonstration of a central role of walnut PPO in tyrosine metabolism and esculetin biosynthesis in walnut might require approaches such as radioactive pulse labeling. It will be interesting to see how widespread this role of PPO in tyrosine metabolism is, especially in species whose PPO enzymes have been shown to have tyrosinase activity.
Many of these compounds from creosote bush, e. Cho et al. Peptide sequencing of the purified hydroxylase identified fragments with high homology to conserved domains of PPOs from other plant species. The peptide sequence data further allowed cloning and sequencing of a full length cDNA corresponding to the L.
Like most plant PPOs, the L. Unfortunately, in this study, reverse genetics e. Unfortunately, there appears to have been relatively little further work on this pathway or characterization of the L. Does this PPO also form quinones from hydroxylarreatricins and under what conditions? Would such activities have any biological implications? One of the most interesting and well-studied cases of PPO having a role in biosynthesis of specialized metabolites is the biosynthesis of the chalcone-derived yellow aurone pigments in snapdragon Antirrhinum majus flowers.
The enzyme responsible, aureusidin or aurone synthase AS , was purified to homogeneity from yellow snapdragon buds Nakayama et al. Peptide sequencing of the purified enzyme allowed isolation and characterization of a cDNA encoding the enzyme. The predicted protein sequence showed high homology to other plant PPO enzymes. Expression of the gene corresponded to aurone accumulation e. Further, in vitro , tyrosinase from Neurospora crassa could also convert THC to aureusidin, indicating that the enzymatic activities of PPO are involved in the biosynthetic conversion.
Starting with THC, tyrosinase and catechol oxidase activity result in 3-hydroxylation and formation of the corresponding o -quinone. Whether AS PPO carries out the 3-hydroxylation reaction in vivo , or whether a cytochrome P chalcone 3-hydroxylase as described below for Coreopsis grandiflora is also involved has not been definitively established. AS likely forms the same quinone from PHC without the need for the 3-hydroxylation step. The resulting quinone is predicted to undergo a 2-step non-enzyme mediated rearrangement to form aureusidin Nakayama et al.
AS was also unable to oxidize aureusidin to its corresponding quinone, nor could it oxidize several other mono and o -diphenolic compounds, such as tyrosine, p -coumaric acid, L -DOPA, caffeic acid, or eriodictyol, suggesting a relatively strict substrate specificity Nakayama et al. One of the most novel aspects of the A.
Consistent with this, Ono et al. Recently, Kaintz et al. Interestingly, the C. Consistent with the lack of tyrosinase activity, the C. The plastidic versus vacuolar nature of the C. The above examples could represent the tip of the iceberg with respect to PPO enzymes that have specific roles in biosynthesis of specialized metabolites.
Much work on PPOs has focused on their negative impact on food quality due to the browning reactions they promote. In two of the cases above, the specialized roles of the PPOs were identified in the course of research focused on a particular aspect of specialized metabolism.
There, relatively laborious approaches led to the identification of the PPOs involved. Bioinformatics approaches will almost certainly facilitate identifying these specialized PPOs in the future. For example, in an analysis of PPO gene families from land plants whose genomes had been sequenced, Tran et al.
Although some of these could certainly be involved in defensive responses, such as seed defense Anderson et al. As they are becoming more routine, transcriptomic, metabolomic, and proteomic analyses could also provide useful information related to PPO function as was the case for walnut PPO discussed above.
These types of analyses can provide answers to questions such as whether expression of a given PPO is tightly correlated with a phenotype of interest or whether a particular PPO is present in the subcellular compartment where a specific biosynthetic reaction is thought to occur. Thus genomics data, combined with other bioinformatic approaches, will almost certainly facilitate better understanding of PPO function in general, and the roles specific PPOs may play in specialized metabolism.
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Guest Associate Editor Dr.
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