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Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency

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.


Results and discussion
Cloning, heterologous expression and purification of pro-DlPPO1 and prepro-DlPPO1. The PCR products on the complementary DNA of D. longan with primers enframing the ORF for prepro-DlPPO1 and pro-DlPPO1 yielded distinct bands of approximately 1800 bp and 1500 bp, respectively ( Figure S4). After the cloning into the pGEX-6P-SG vector 69 , Sanger sequencing confirmed that the prepro-DlPPO1 is composed of an ORF of 1797 nucleotides and encodes for 599 amino acids which are 100% identical to the amino acid sequence deduced from the published DlPPO1 (Uniprot entry: A0A0K0NPU9) 64 . The pro-DlPPO1 contains an ORF of 1512 nucleotides encoding 503 amino acids (Table S1) and has 99.60% identity with the previously published DlPPO1 sequence 64 resulting from five altered amino acids (Pro46Arg, Glu226Asp, Gln451His, Ile455Met, and Asn486Tyr, Figure S5). The pro-DlPPO1 and prepro-DlPPO1 enzymes were heterologously expressed in E. coli with an N-terminal glutathione-S-transferase tag from Schistosoma japonicum 69 (see Materials and Methods). The temperatures upon the isopropyl-ß-D-1-thiogalactopyranoside (IPTG) induction during the expression were varied (37 °C, 28 °C and 17.5 °C) in order to investigate a condition favorable for high level production of soluble protein. Figure S6 shows that the most efficient expression temperature for both pro-DlPPO1 and prepro-DlPPO1 was at 17.5 °C, while 37 °C resulted in the lowest expression level of both soluble and non-soluble forms. Effective productions of soluble PPO enzymes under low temperature expressions were also reported for TYRs from Juglans regia (20 °C) 67 , Agaricus bisporus (20 °C) 3 , Solanum lycopersicum (20 °C) 17 , Malus domestica (20 °C) 66 , Streptomyces avermitilis (18 °C) 70 , Streptomyces sp. ZL-24 (19 °C) 68 and Larrea tridentata (25 °C) 32 .
The C-terminal domain of eukaryotic PPOs has been reported to be necessary for the folding of the catalytic domain 71 . So far, the expression of active grape PPO 72 is the only example of successful production of a plant PPO without its C-terminal domain.
Through the purification by affinity chromatography applying the affinity of glutathione immobilized on cross-linked agarose, the expressions yielded 35.3 and 60.0 mg per liter of expression culture of GST-fused pro-DlPPO1 and GST-fused prepro-DlPPO1, respectively ( Figure S7). Removal of the GST-fusion tag by the HRV3C protease resulted in 5.7 and 7.0 mg of the latent pro-DlPPO1 and prepro-DlPPO1, respectively ( Table 1). The prepro-DlPPO1 was successfully purified with 13.5% recovery of the total protein activity and a specific activity of 7.09 ± 0.05 U mg −1 , while the pro-DlPPO1 was retrieved with 19.6% recovery of the total protein activity and a specific activity of 28.3 ± 0.04 U mg −1 . The copper contents of the DlPPO1 variants were measured photometrically by reducing Cu(II) to Cu(I) with ascorbate and following the complexation of Cu(I) with 2,2′-biquinoline at 546 nm 73 . It was observed that the copper contents of pro-DlPPO1 and prepro-DlPPO1 were 1. 3  www.nature.com/scientificreports/ 2.0 ± 0.030 copper ions per active site, respectively. The freshly purified proteins eluted in 50 mM Tris-HCl and 250 mM NaCl at pH 7.5 and were immediately exchanged to 100 mM MES and 200 mM NaCl at pH 6.5, and glycerol was applied at 30%(v/v) for storage of both forms at − 80 °C until use. The purity of the final pro-DlPPO1 and prepro-DlPPO1 preparations was confirmed by SDS-PAGE ( Fig. 1 and Figure S8 for the full-length gels).

Molecular mass determination of pro-DlPPO1 and prepro-DlPPO1.
Gel electrophoresis demonstrated a prominent band of the final purified pro-DlPPO1 and prepro-DlPPO1 at approximately ~ 57 kDa and ~ 67 kDa, respectively, which suggested the latent (pro-DlPPO1) and precursor (prepro-DlPPO1) forms of longan PPO1 were obtained. The theoretical masses of pro-DlPPO1 and prepro-DlPPO1 were calculated from the respective protein's sum formula using the atomic weight and isotopic composition of the constituent elements 74 considering the presence of two conserved disulfide bonds (− 4H or − 4.032 Da) and one thioether bridge (− 2H or − 2.016 Da) as shown in Table 2. To confirm the actual molecular weights of the enzyme electrospray ionization-mass spectrometry (ESI-MS) was applied. The pro-DlPPO1 yielded 56,845 Da ( Fig. 2) which is in excellent agreement with the calculated mass of the protein starting from the vector-derived GlyProMet (marked as − 3, − 2, − 1 in Figure S5) up to the end of the latent PPO's sequence (Ala 1 → Asp 503). This confirms that pro-DlPPO1 was expressed as a latent PPO form with two disulfide bonds and one thioether bridge. In addition, prepro-DlPPO1 was determined to have a mass of 67,285 Da, containing also one thioether bridge and two disulfide bonds (Fig. 2). The mass matches perfectly with the complete sequence of the prepro-polypeptide (Met 1 → Asp 599) plus the three amino acids GlyProMet (− 3, − 2, − 1) arising from the expression vector.  insoluble fraction of the bacterial cell pellets, (B) total soluble proteins, C) GST-DlPPO1, which is the eluted GST-fusion protein after the glutathione affinity chromatography (first purification step on ÄKTA FPLC), (D) fractions of proteins after GST-DlPPO1 was cut by HRV3C protease and E) final DlPPO1 product (marked with a red frame; see Table 2) from the second round of glutathione affinity chromatography. The first lane of each gel is the molecular weight marker. The full-length gels are shown in Figure S8. www.nature.com/scientificreports/ Thermal shift assay of pro-DlPPO1 and prepro-DlPPO1. The thermal shift assay has been used to assess the thermal stability of recombinant proteins 8,17,75 . Here, the thermal stability of the purified pro-DlPPO1 and prepro-DlPPO1 proteins at pH 6.5 (storage buffer condition) was tested. As shown in Fig. 3, for prepro-DlPPO1 two melting temperatures (T m ) at 69.5 °C and 77.5 °C were measured, while pro-DlPPO1 exhibited three T m values (54.5 °C, 70.5 °C, and 81.5 °C) until the complete denaturation of the enzymes. The unfolding of pro-DlPPO1 began at much lower temperature (~ 40 °C) (Fig. 3, Figure S9) than for prepro-DlPPO1 (~ 65 °C).
The resulting first T m of pro-DlPPO1 is much lower than the other T m values observed for both variants of the enzyme. This strongly indicates that the latent pro-DlPPO1 exhibits some degree of limited unfolding at significantly lower temperature than its precursor prepro-DlPPO1. As the pro-DlPPO1 contains only domains that are also present in the prepro-DlPPO1, it seems that the inclusion of the signal peptides does actually increase the overall thermal stability of DlPPO1 and the signal peptides help to maintain protein stability.
Activation of pro-DlPPO1. Effect of SDS on pro-DlPPO1 activity. The activation of pro-DlPPO1 was monitored via the conversion of tyramine ( Figure S3) to verify the proper SDS concentration for overcoming the enzyme's latency. The reaction was examined using 0.5 µg of pro-DlPPO1 and 8 mM tyramine as substrate in 50 mM MES at pH 7.0 with various SDS concentrations (0 to 1.5 mM). As displayed in Figure S10, an increase in SDS concentration from 0.00 to 0.25 mM caused a significant enhancement of the pro-DlPPO1 activity, where the highest activation efficiency was achieved at 0.25 mM SDS (28.3 ± 0.04 U mg −1 ). At higher SDS concentrations, the enzymatic activity was gradually reduced. The loss in activity by half of the optimum was observed at   www.nature.com/scientificreports/ an SDS concentration of 1.25 mM. Thus, the optimal SDS concentration for the activation of pro-DlPPO1 was defined to be 0.25 mM and this level of added activator was applied in all subsequent experiments. A qualitatively identical activation behavior was seen for prepro-DlPPO1 ( Figure S10). Most enzymes lose their biological activities under treatment with SDS, while PPOs are resistant to SDS to some extent, probably in part due to the existence of disulfide bonds in the structure 55,76,77 . SDS initiates the activation of PPOs by introducing conformational changes of the N-and C-termini of the latent form which allow for exposure of the active site and promote substrate access 78 . PPO activation is generally achieved at low SDS concentrations, while higher concentrations of the anionic detergent significantly inhibit the enzymatic activity 79,80 , which is consistent with the SDS activation profile observed for DlPPO1. The use of SDS for the activation of latent PPOs has been reported to be the best activation method in vitro as it almost completely eliminates the latency of plant PPOs and provides an activity closely resembling mature PPO (after the removal of the C-terminal domain) 66 . The degree of SDS activation varies greatly with plant extracts 78 ; for examples, 0.13 mM SDS was required to activate latent potato PPO (Solanum tuberosum) 33 84 . The necessary concentration of detergent for activation of dandelion PPO has been shown to be determined primarily by the amino acids forming the interface between the catalytic and the C-terminal domain 85 . The use of 0.25 mM SDS for the pro-DlPPO1 activation is at the lower end when compared to other heterologously expressed PPOs such as walnut JrPPO1 (2 mM SDS) 67 , tomato SlPPO1 and SlPPO2 (1.5 mM SDS) 17 , mushroom AbPPO4 (2 mM SDS) 3 and for apple MdPPO1 (3 mM), MdPPO2 (2 mM) and MdPPO3 (4 mM) 66 .
Effect of pH on the pro-DlPPO1 activity. The pH is one of the parameters that affects a PPO's activity 86 . To optimize the pH of the reaction buffer for the pro-DlPPO1 activity, the enzyme reaction was examined through 50 mM MES and TRIS buffers ranging from pH 5.5 to 8.5 using 0.5 µg of pro-DlPPO1, 8 mM tyramine as substrate and 0.25 mM SDS for activation of the enzyme. Figure S11 demonstrates that the enzyme shows different degrees of activity in this pH range. The highest activity was exhibited at pH 7.0 (28.3 ± 0.04 U mg −1 ) in MES buffer whilst the activity slightly decreased at pH 6.5 by 17% and was significantly reduced at pH 6.0 and 5.5 by 61% and 86%, respectively. Likewise, the activity significantly dropped at higher pH (pH 7.5-8.5). A decrease by 43% was observed at pH 7.5 and by 74% at pH 8.0. The activity was almost depleted at pH 8.5 with a 96% activity loss from the optimum. A pH 7.0 optimum was previously reported for the tyrosinases in apple (MdPPO1 and MdPPO3) 66 and mushroom (AbPPO4) 3 . A slightly higher optimal pH at 7.5 was observed in MdPPO3 66 . JrPPO1 and JrPPO2 from walnut had lower pH optima of pH 6.0 67 , whereas the bacterial tyrosinase from a Streptomyces sp. from peatland (SzTYR) revealed an unusually high pH optimum of pH 9.0 as a result of adaptation to its natural pH environment 68 .
Substrate specificity and enzyme kinetics. The catalytic performance of the pro-DlPPO1 was first examined using four standard substrates ( Figure S3): two monophenols (tyramine, d-and l-tyrosine) and two diphenols (dopamine and l-DOPA) were applied at the enzyme's optimal pH 7.0 and an SDS concentration of 0.25 mM. The pro-DlPPO1 does accept all four standard substrates from the monophenolic and diphenolic categories, and consequently can be classified as a TYR (EC 1.14.18.1). K M and k cat values were calculated by nonlinear regression and the substrate specificity was estimated using the k cat /K M ratio. Data reported on the substrate specificity for pro-DlPPO1 are shown in Table 3 and Figure S13. The diphenolic substrate dopamine is processed faster (k cat : www.nature.com/scientificreports/ 260 ± 22 s −1 ) than l-DOPA (k cat : 98 ± 8.5 s −1 ), and the monophenol tyramine (k cat : 35 ± 1.9 s −1 ) is hydroxylated faster than l-or d-tyrosine. Moreover, pro-DlPPO1 has a lower K M value for dopamine (K M : 2.0 ± 0.35 mM), thus resulting in the highest catalytic efficiency (k cat /K M ) among the four tested standard substrates. The k cat / K M ratio for dopamine is 4.6-, 16-, 336-and 504-fold higher than that of l-DOPA (28 ± 3.9 s −1 mM −1 ), tyramine (8.2 ± 0.91 s −1 mM −1 ), d-tyrosine (0.39 ± 0.016 s −1 mM −1 ) and l-tyrosine (0.26 ± 0.016 s −1 mM −1 ), respectively ( Table 3). The prepro-DlPPO1 also accepts both standard monophenols (tyramine and tyrosine) and diphenols (dopamine and l-DOPA), but with different degrees of catalytic efficiency (Table 3 and Figure S14). The prepro-DlPPO1 binds dopamine with a similar K M value (K M : 2.3 ± 0.27 mM) to that observed for the pro form (K M : 2.0 ± 0.35 mM); however, the catalytic efficiency of prepro-DlPPO1 is reduced 7.5-fold compared to pro-DlPPO1 with a k cat /K M of 17 ± 2.4 s −1 mM −1 . The conversion of the diphenol l-DOPA by prepro-DlPPO1 (k cat /K M of 12 ± 1.1 s −1 mM −1 ) was 2.4-fold slower due to the lower catalytic activity (k cat : 40 ± 2.1 s −1 ) compared to pro-DlPPO1. A similar observation was noted for the monophenolic substrates: The hydroxylation of tyramine (k cat / K M of 1.6 ± 0.14 s −1 mM −1 ) by prepro-DlPPO1 is 5.2-fold less efficient than for pro-DlPPO1 with a concomitant increase of K M to 15 ± 1.0 mM and a reduced catalytic activity (k cat : 24 ± 1.4 s −1 ). The kinetic examination towards the monophenolics l-and d-tyrosine demonstrated decreased k cat /K M ratios, which are 1.3-and 1.6-fold lower than those observed for pro-DlPPO.
To gain insights into the kinetic behavior of DlPPO1 in its natural environment, a series of natural phenolic substrates ((-)-epicatechin, 4-methylcatechol, caffeic acid, pyrogallol, gallic acid, quercetin, vanillic acid and ferulic acid ( Figure S2)), all of which have been detected in longan peel, pulp and seed [39][40][41] , were assessed. Data reporting the natural substrate specificity of pro-DlPPO1 are shown in Table 3 and Figure S15. Through the kinetic studies it was revealed that pro-DlPPO1 is active towards the polyphenols (-)-epicatechin, 4-metylcatechol, caffeic acid and pyrogallol, while the enzyme was completely inactive with the rest of the tested substrates. The highest efficiency of pro-DlPPO1 was observed towards (-)-epicatechin (k cat : 270 ± 15 s −1 ) with a K M of 0.35 ± 0.052 mM, resulting in k cat /K M of 800 ± 120 s −1 mM −1 . This evidence suggests that (-)-epicatechin is highly likely to be one of the physiological substrates of DlPPO1 in longan. The rates of 4-methylcatechol, pyrogallol and caffeic acid conversions were lower than for (-)-epicatechin with resulting k cat /K M values of 590 ± 99 s −1 mM −1 , 70 ± 9.7 s −1 mM −1 and 4.3 ± 0.72 s −1 mM −1 , respectively. However, the catalytic efficiency towards these natural substrates significantly decreased in the prepro-DlPPO1. The kinetic examination with (-)-epicatechin resulted in a higher K M value (1.1 ± 0.12 mM), indicating that the precursor has lower specificity towards this substrate Table 3. Kinetic parameters of pro-DlPPO1 and prepro-DlPPO1. **The substrate solubilities were too low for a determination of the kinetic parameters using the Michaelis-Menten model. Linearization was subsequently implemented considering substrate concentration < < K M , yielding k cat /K M for l-and d-tyrosine as displayed in the www.nature.com/scientificreports/ than pro-DlPPO1 (K M = 0.35 ± 0.052 mM). Thus, the catalytic efficiency of prepro-DlPPO1 was observed to be only 33 ± 4.1 s −1 mM −1 , which is 23-fold lower than the value of pro-DlPPO1. Likewise, the specificity of the prepro-DlPPO1 to 4-methylcatechol, caffeic acid and pyrogallol is less than the pro-DlPPO1 as indicated by the higher K M values in comparison to the pro-DlPPO1. Data reporting the natural substrate specificity of prepro-DlPPO1 are shown in Table 3 and Figure S16. The resulting k cat /K M values of 17 ± 1.7 s −1 mM −1 (4-methylcatechol), 2.5 ± 0.20 s −1 mM −1 (caffeic acid) and 15 ± 1.2 s −1 mM −1 (pyrogallol) indicate that the catalytic efficiency of the prepro-DlPPO1 decreased 36, 1.7 and 4.7-fold, respectively, from the pro-DlPPO1. Thus, the signal peptide component could be one of the factors responsible for the reduced rate of PPOs in catalyzing the oxidation and hydroxylation of phenolic compounds.
With concentrations of (-)-epicatechin in excess of 2 mM the enzymatic conversion was slower than the rate seen at lower substrate levels for both prepro-DlPPO1 and pro-DlPPO1 (Figures S15A and S16A). Including competitive inhibition by the substrate itself ("substrate inhibition", Equation S3) in the Michaelis-Menten model allowed to expand the modelled concentration range up to 3 mM for pro-DlPPO1 and up to 5 mM for prepro-DlPPO1. Changing the mathematical model of enzymatic conversion causes major changes to the obtained kinetic parameters. For pro-DlPPO1 (K M = 4.6 ± 2.0 mM, k cat = 1500 ± 590 s −1 , K i = 0.56 ± 0.26 mM) as well as for prepro-DlPPO1 (K M = 6.1 ± 3.7 mM, k cat = 140 ± 72 s −1 , K i = 1.2 ± 0.79 mM) both K M and k cat are significantly increased (compare Table 3) to compensate for the newly introduced inhibiting effect of the substrate itself (K i ) while the variances of all fitted parameters are seriously inflated relative to the simple Michaelis-Menten model. For pro-DlPPO1 and dopamine ( Figure S13C; K M = 1.2 ± 0.11 mM, k cat = 250 ± 15 s −1 , K i = 30 ± 7.3 mM), with a much higher K i indicating less efficient substrate inhibition, the inflation of the parameter's variances is less severe and both k cat and K m are slightly lower than with the simple Michaelis-Menten model.
The binuclear copper center of PPOs was reported to function enantioselectively 32 . The enantioselectivity of the catalytic reaction of pro-DlPPO1 on tyrosine was investigated. The enzyme showed a preference for d-tyrosine with a slightly higher catalytic efficiency of 0.39 ± 0.016 s −1 mM −1 over the l-enantiomer (0.26 ± 0.016 s −1 mM −1 ). 1 mM of d-tyrosine is converted at a rate of 0.11 ± 0.01 U mg −1 , which is 1.1-fold faster than the rate of l-tyrosine conversion by pro-DlPPO1. The effect of substrate chirality was also observed in mushroom AbPPO4 where l-tyrosine was slightly more preferable than the d-counterpart 3 . The tyrosinase produced by Streptomyces sp. REN-21 reacted 35.9-fold faster on l-tyrosine than d-tyrosine, which is the strongest enantioselectivity effect on the substrate tyrosine among PPOs that has been reported 87 . The preference towards a d-stereoisomer substrate was also reported in a PPO purified from eggplant (Solanum melongena), in which the conversion of 0.1 mM d-DOPA was 1.8-fold faster than l-DOPA 88 . Another example of the enantiospecific reaction in plant PPOs is found in larreatricin hydroxylase from Larrea tridendata (LtPPO) which exhibits a pronounced preference (23fold) for ( +)-larreatricin in comparison to the (-)-enantiomer, whereas in the same study AbPPO4 reveals an opposite effect by preferring (-)-larreatricin (13-fold) 32 .
Formation of the oxy-form in prepro-DlPPO1 and pro-DlPPO1. The prepro-DlPPO1 and pro-DlPPO1 were spectrophotometrically examined using H 2 O 2 to assess the formation of the oxy-state of the enzyme in the presence of oxygen. Addition of H 2 O 2 to the prepro-DlPPO1 leads to an increase of the specific absorption band (~ 345 nm), which is indicative of the oxygen-induced oxy-form characteristic for type-III copper center enzymes and has been attributed to the charge transfer transition of O 2− 2 π * σ → Cu(II) d x 2 −y 2

89
. A similar observation was made with pro-DlPPO1, where the oxy adduct formation resulted in the formation of a pronounced absorption band at 345 nm (Fig. 4). The absorption at 345 nm became saturated when the titration reached ~ 150 equivalents of H 2 O 2 for prepro-DlPPO1 and ~ 175 equivalents of H 2 O 2 for pro-DlPPO1, respectively. The resulting absorption coefficients are ~ 9000 M −1 cm −1 per mol enzyme for the prepro-DlPPO1 and ~ 10,300 M −1 cm −1 per mol enzyme for the pro-DlPPO1. The formation of the oxy-form by H 2 O 2 has previously been shown for PPOs extracted from natural sources 60,90,91 . Catechol oxidases purified from lemon balm (Melissa officinalis) 86 , sweet potato (Ipomoea batatas) 87 and walnut leaves (Juglans regia) 52 resulted in a full saturation of the oxy form when two equivalents of H 2 O 2 were added. In gypsywort (Lycopus europaeus) catechol oxidase, 6 equivalents of H 2 O 2 were required to saturate the λ 345 band, while in black poplar (Populus nigra) 89  . The present study is the first to investigate the oxy-form formation of a heterologously expressed prepro-PPO which still has its signal peptides attached.
Molecular docking simulations of DlPPO1. Molecular docking studies were performed to provide more insight into the enzyme-substrate interactions of the pro-DlPPO1. The simulation studies were carried out using the AutoDock Vina software 94 . The structural model of pro-DlPPO1 was prepared as described in Materials and Methods and was used for the computational calculations. Binding poses were calculated for all the investigated substrates (Figures S17 and S18) and validated according to the TYR structure of Bacillus megaterium which contains the substrate l-tyrosine bound to the Zn-substituted active site (PDB: 4P6R) 1 . The results provided detailed information about the putative position of the investigated substrates and the binding energies of these poses around the active center (Table S3). The natural substrate (-)-epicatechin exhibited the strongest binding affinity among the investigated substrates as strongly suggested by the lowest measured K M (K M = 0.35 ± 0.052 mM) and the most favorable binding energy ( www.nature.com/scientificreports/ ing weak binding interactions between pro-DlPPO1 and the substrate. The higher catalytic efficiency than the one observed for caffeic acid is due to the much higher k cat value of pyrogallol (Table 3). Further analysis with LiqPlot + of (-)-epicatechin docked to the active center of the DlPPO1 revealed the specific interactions of the substrate with the amino acids around the dicopper active center. Specifically, one of the two hydroxy groups on the (-)-epicatechin's o-diphenolic ring (Fig. 5) establishes two hydrogen bonds; one with the imidazole nitrogen of the conserved copper-coordinating His243 (2.8 Å) and the second one with the carboxylic backbone group of Met257 (2.8 Å). The second hydroxy group of the same ring interacts with the two copper ions and is well-positioned for the oxidative reaction (CuA: 4.1 Å and CuB: 3.7 Å, respectively). Moreover, nine hydrophobic interactions with the residues His87, Asn109, Ala231, Glu235, Asn236, Gly258, Asn259, Phe260 and Ala263 cover the whole body of the substrate (Fig. 5). Similar to other plant PPOs the conserved Phe260 17 , the gatekeeper residue, turns to the phenolic ring of the (-)-epicatechin and forms sandwich type π-π interactions. The high affinity binding of (-)-epicatechin showcases the preference of plant PPOs for phenolic secondary metabolites and strongly suggests (-)-epicatechin to be a physiological substrate of DlPPO1. Similar results have been presented for the tomato PPO (SlPPO1) which has a high affinity towards the dihydrochalcone phloretin 17 and for the aurone synthase from Coreopsis grandiflora for which butein was proposed as its natural substrate 51,95 .

Conclusion
In this work, prepro-DlPPO1 from D. longan was expressed, purified and characterized in comparison with pro-DlPPO1. This constitutes the first report on the biochemical differences between a PPO expressed in a prokaryotic system both with and without its N-terminal signal sequence. Titration with H 2 O 2 monitored by UV-Vis spectroscopy verified that DlPPO1 contains a type-III copper center. After addition of ~ 150 and ~ 175 equivalents of H 2 O 2 , the absorption band around 345 nm indicating the oxy-form was fully developed in prepro-DlPPO1 and pro-DlPPO1, respectively (Fig. 4). Kinetic experiments showed that both enzymes accept monophenolic substrates, both forms of DlPPO1 were thus classified as tyrosinases (EC. 1.14.18.1). The pro-DlPPO1 exhibited lower K M and higher k cat values, and consequently much higher kinetic efficiencies, towards all tested substrates than prepro-DlPPO1 did ( Table 3). The N-terminal signal peptides seem to provide an efficient barrier for substrate binding to the active site of DlPPO1. Inclusion of the supposedly unfolded signal peptides resulted in a substantial thermal tolerance of prepro-DlPPO1 that even surpasses the thermal stability of pro-DlPPO1 (Fig. 3). In combination with the signal peptides' impeding effect on the catalytic function of DlPPO1, the increased www.nature.com/scientificreports/ thermostability they confer to DlPPO1 may indicate a more ordered and compact structure of these peptides as currently envisioned. (-)-Epicatechin exhibited the lowest K M , the highest catalytic efficiency (Table 3), the most favorable binding to DlPPO1 (Table S3 and Fig. 5) and also the most severe substrate inhibition among all investigated substrates. The results suggest (-)-epicatechin as a physiological substrate of DlPPO1. This work provides insights into the biochemical and catalytic properties of longan PPO that will be useful for future postharvesting interventions for effective management of costly fruit browning reactions.

Materials and methods
Unless specified differently, all used chemicals have been purchased from Sigma-Aldrich (Vienna, Austria) or Carl-Roth (Karlsruhe, Germany) and were at least of analytical grade. Enzymes for the manipulation of nucleic acids were from New England Biolabs (NEB; Frankfurt am Main, Germany) or Thermo Fisher Scientific (Waltham, MA, USA).
Plant material, cloning, and sequencing of DlPPO1. Longan leaves were collected in Lamphun (18.481174° N, 99.176014° E), located in the northern part of Thailand, immediately transported to the laboratory and stored at − 80 °C. Permission to collect longan leaves for research purposes was obtained from the local proprietor. Healthy leaves were ground under liquid nitrogen and the total RNA was isolated using the Rapid CTAB method according to Gambino et al. 96 with an extraction buffer containing 2%(m/v) cetyltrimethylammonium bromide, 2.5%(m/v) polyvinylpyrrolidone, 2 M NaCl, 100 mM Tris-HCl (pH 8.0), 25 mM ethylenediaminetetraacetic acid (EDTA, set to pH 8.0 with NaOH) and 2%(v/v) β-mercaptoethanol. Subsequently, cDNA was synthesized using a poly-T primer (5'-T 25 VN-3') and Moloney Murine Leukemia Virus reverse transcriptase. Specific primers for the longan PPO (prepro-DlPPO1 and pro-DlPPO1) gene were designed (Table S2). The primers were used to amplify the PPO genes from the cDNA template using the Q5 High-Fidelity DNA polymerase (NEB). PCR products were cloned into the pGEX-6P-SG 69   www.nature.com/scientificreports/ the presence of ampicillin (100 mg/L). Overnight cultures were grown at 37 °C. The expression batches were inoculated with the saturated overnight cultures and grown (starting OD 600 of 0.05) at the same temperature for ~ 4 h until the OD 600 reached a value of 0.6-0.8. To optimize the protein quantity and simultaneously to avoid inclusion body formation, the temperature was reduced to 17.5 °C for expression. The cultures were induced with 0.5 mM IPTG, and 2.0 mM CuSO 4 were added. The expression cultures remained at 17.5 °C under shaking for 48 h. When the OD 600 reached a value of 7-10, the cultures were collected by centrifugation at 6500 × g for 25 min at 4 °C.
Cell lysis was carried out implementing the freeze-thaw technique using liquid nitrogen. The pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM EDTA and 50 mM sucrose). Lysozyme (0.5 g/L) and protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 mM benzamidine) were added to lyse the bacterial cell walls and to prevent protein degradation by endogenous proteases, respectively. The suspensions were shaken and incubated on ice for 45 min. The mixtures were then subjected to five cycles of freezing in liquid nitrogen, alternating with thawing in a 25 °C water bath. After the 5th round of the freeze-thaw procedure, 2 mM MgCl 2 and 0.02 g/L DNaseI were added to remove the viscosity of the solutions caused by the DNA that was released from the bacteria cells and to improve protein extraction. The lysates were subsequently centrifuged at 7500 × g for 1 h at 4 °C.
Chromatographic purifications were carried out using an ÄKTA Purifier (GE Healthcare) placed in a refrigerator at 4 °C. The filtrated lysates were placed in a 50 mL injection loop and applied onto a prepacked 5 mL GSTrap FF column using a solution of 50 mM Tris-HCl, 250 mM NaCl pH 7.5 as the binding buffer. The target GST-fusion proteins were trapped onto the column, whereas the unbound proteins were flushed out by the binding buffer applied at a flowrate of 1 mL/min and chaperone washing buffer (50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl 2 and 5 mM Na-ATP set to pH 8; applied for 15 column volumes) to remove binding chaperones from the target fusion proteins. Eventually, the desired proteins were eluted with 50 mM Tris-HCl, 250 mM NaCl and 15 mM reduced glutathione set to pH 7.5. The GST-fusion protein fractions were pooled and concentrated using a Vivaspin ultracentrifugation device with a 30 kDa molecular weight cut-off (Sartorius; Göttingen, Germany). To remove glutathione, the buffer was then exchanged to 50 mM Tris-HCl pH 7.5 and 250 mM NaCl. Subsequently, the samples were mixed with an in-house 3 prepared GST-HRV3C protease at a mass ratio of 1:50 (protease: GST-fusion protein). The proteolysis was carried out over 48 h at 4 °C. Afterwards, the cleaved proteins were again applied onto a 5 mL GSTrap FF column. At this time, the GST-protein and the GST-tagged protease were trapped in the column while the PPOs were able to flow through the column and were immediately eluted. The protein fractions of prepro-DlPPO1 and pro-DlPPO1 were collected, concentrated and stored in 100 mM MES and 200 mM NaCl pH 6.5 with 30%(v/v) glycerol at -80 °C. The protein concentrations were determined using the Beer-Lambert law 97 based on the absorption at 280 nm and the extinction coefficient provided by ExPASy ProtParam 98 . The predicted molecular mass of the expressed proteins were estimated from the atomic weight and standard isotopic composition of the constituent elements 74 . The copper contents of the prepro-DlPPO1 and pro-DlPPO1 enzymes were assessed photometrically after acidification with acetic acid and reducing Cu(II) to Cu(I) with ascorbate, which allowed for the chelation of Cu(I) by 2,2′-biquinoline according to the method published by Hanna et al. 73 .

Molecular mass determination by ESI-LTQ-Orbitrap-Velos. Mass determination of prepro-DlPPO1
and pro-DlPPO1were performed by an ESI-LTQ-Orbitrap Velos (Thermo Fisher Scientific Bremen, Germany) mass spectrometer with a mass range of 200-4000 m/z and a mass accuracy close to 3 ppm with external calibration. Prior to MS 100 µg of the protein solutions were exchanged into 5 mM sodium acetate (pH 7.0) buffer. The protein solutions were diluted 100-fold in a mixture of 80%(v/v) acetonitrile and 0.1%(v/v) formic acid immediately before being applied to the mass spectrometer.
Gel electrophoresis. Denaturing SDS-PAGE was performed as described by Laemmli et al. 99 in a mini gel apparatus (Mini-PROTEAN Tetra Cell, Bio-Rad). The prepro-DlPPO1 and pro-DlPPO1 were diluted in gel loading buffer that contains 30 g/L SDS, 6% (v/v) glycerol, 75 mM Tris, 2.5%(v/v) 2-mercaptoethanol and 50 mg/L bromophenol blue set to pH 6.8. The samples were heated at 99 °C (Thermomixer comfort, Eppendorf) for 5 mins to aid in the denaturation. The samples were then applied on the 5% stacking and 11% resolving polyacrylamide gels along with a molecular weight standard (Precision Plus Protein Standard Dual Color, Bio-Rad) to provide information on the size and the purity of the proteins. The electrophoresis was run at 120 V. Gels were stained in dye solution (200 mg/L Coomassie brilliant blue G-250, 50 g/L Al 2 (SO 4 ) 3 .

H 2 O, 10%
(v/v) ethanol and 20 g/L H 3 PO 4 ) and were subsequently destained with 10%(v/v) ethanol and 20 g/L ortho-phosphoric acid. Photographs of the gels were taken using the BioRad Gel Doc XR Imaging System. Thermal shift assay of prepro-DlPPO1 and pro-DlPPO1. The thermal shift assay was conducted to measure the melting points of the two isoenzymes in the storage buffer (50 mM MES pH 6.5 and 200 mM NaCl) in order to determine the stability of the two states of the enzyme. The assay was performed thrice using PCR tubes (Axygen, INC Corning) in a real-time PCR instrument (mastercycler ep-realplex, Eppendorf). The reaction solutions contained 10 µM enzyme and 4 × SYPRO Orange (Sigma-Aldrich) in the storage buffer. The samples were gradually heated with an increment of one K per minute from 4 °C to 94 °C in the PCR machine, while the change in fluorescence intensity was monitored at 560 nm following excitation at 470 nm. The resulting fluorescence intensities of the solutions containing each enzyme were then plotted against the temperature for stability analysis. www.nature.com/scientificreports/ Enzyme activity assays. The DlPPO1 activities were spectrophotometrically determined by monitoring the increase in absorbance at 480 nm upon the conversion of tyramine by the enzyme. The standard measurements were performed using 0.5 µg of the enzyme and 8 mM of tyramine in a total reaction volume of 200 µL with the determined optimal SDS concentration (0.25 mM) to activate prepro-DlPPO1 or pro-DlPPO. In the absence of SDS no utilizable absorption-time curves were obtained for the tested substrates ( Figure S12). Absorption curves and spectra were recorded at 25 °C in a 96 well microplate applying a TECAN infinite M200 (Tecan). The activities were determined from the slope of the initial linear part of the experimental curves (absorbance vs. time) and expressed as U/min. One unit of enzymatic activity (U) was defined as the amount of enzyme that catalyzed the formation of 1 μmol of quinones per minute (1 U = 1 μmol/min). All assays were performed in triplicate. Optimizations of the SDS concentration and pH of the reaction buffer towards the pro-DlPPO1 activity are as follows (vide infra). Effect of pH on pro-DlPPO1 activity. A series of MES and Tris buffers over a pH range of 5.5-8.5 were used as the reaction buffer to determine the most favorable pH that provides the best enzyme activity for further kinetics studies. Each reaction (200 µL) was carried out with 50 mM buffer concentration using 0.5 µg pro-DlPPO1, 8 mM tyramine and the optimal SDS concentration obtained from the previous experiment.

Activation of pro-
Substrate specificity and kinetic efficiency determination. Substrate specificity of prepro-DlPPO1 and pro-DlPPO1 was probed using various phenolic substrates. The substrates that resulted in decent activities were subsequently selected to determine kinetic parameters. Activities were evaluated spectrophotometrically on two standard monophenolic substrates (tyramine, l-and d-tyrosine), two standard diphenolic compounds (dopamine and l-DOPA) and four natural phenolic substrates ((-)-epicatechin, 4-methylcatechol, pyrogallol and caffeic acid). The absorption curves of the colored products were monitored on a TECAN infinite M200 for different molarities of the respective substrate. The reaction mixture (total volume of 200 µL) contained variable amounts of the enzyme (see Table S4, stored in 100 mM MES and 200 mM NaCl at pH 6.5) and 0.25 mM SDS in the reaction solution of 50 mM MES buffer at pH 7. The molar absorption coefficients (ɛ λmax ) of the formed chromophores are reported in Table S4 and Figure S19.  94 . Docking studies were used to identify the binding poses of the investigated natural substrates (-)-epicatechin, 4-methylcatechol, caffeic acid, pyrogallol and the standard substrates dopamine, l-DOPA, tyramine, l-tyrosine and d-tyrosine in the dicopper active center of pro-DlPPO1. The molecular model of the pro-DlPPO1 was prepared for molecular docking as described before for MdPPO1 66 . Specifically, from the putative structure of the pro-DlPPO1 the C-terminal domain (Pro336-Asp503) was removed and only the active domain (Ala1-Val335) was used for the docking studies. Moreover, the gatekeeper residue (Phe260) was defined as a flexible residue, as its flexibility has www.nature.com/scientificreports/ been verified for plant PPOs 17 . The structure of all the substrates were downloaded from the PubChem server 108 , then translated to the 3D pdb format and converted into pdbqt files using AutoDockTools (ADT, v. 1.5.6) 109 . For all substrates the highest possible number of active torsions was chosen. Polar hydrogens were assigned to the structures by ADT and were also saved in the substrate pdbqt files. In all the experiments, the search grid was centered in the middle of the two active site copper ions and spread over a cube (12 × 12 × 12 Å 3 , grid point spacing: 1 Å) with edges parallel to the three coordinate axes of the target DlPPO1 model. Then AutoDock/Vina was used for docking by supplying the investigated enzymes and inhibitors along with the grid box properties in the configuration file. For all the measurements the energy range was set to 5, while the exhaustiveness was set to 50. Upon docking, the binding poses were evaluated by superimposing the co-crystallized substrate l-tyrosine from the BmTYR crystal structure (PDB: 4P6R) 1 . Poses that grossly deviated from the binding pose of l-tyrosine in the BmTYR structure (i.e. substrate poses with the phenolic ring not interacting with the dicopper center) were characterized as 'unreasonable' poses. Reasonable docking configurations (the phenolic ring linked to the copper ions similar to BmTYR (PDB: 4P6R) 1 ) were plotted as a 3D image using the visualization software PyMol Molecular graphic system (Schrödinger, LLC) 107 .
Generation of interaction plots. After the docking study, reasonable binding poses of interest were saved as pdb files and further visualized using the LigPlot + software 110,111 to evaluate the interactions between the ligand (-)-epicatechin and the interacting amino acid residues around the active center. Runtime parameters were defined as suggested by the software. Specifically, hydrogen-bonds were allowed up to a maximal range of 2.70 Å between a hydrogen and a hydrogen acceptor and also up to a maximal range of 3.35 Å between a hydrogen donor and a hydrogen acceptor. Non-bonded contact parameters were defined as maximal distances of 2.90 Å between the hydrophobic carbons of the ligand and the protein and of 3.90 Å between sulfur atoms of the ligand and the protein.
Ethical approval. All plant experiments described in this study complied with the relevant institutional, national, and international guidelines and legislation.

Data availability
The sequences generated and analyzed during the presented study are available from the European Nucleotide Archive (ENA) repository under the accessions OU702517 (partial pro-DlPPO1 gene) and OU702518 (prepro-DlPPO1 gene).