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

• Published in: Scientific reports 2022-11, Vol.12 (1), p.20322-16, Article 20322
• Main Authors: Ruckthong, Leela, Pretzler, Matthias, Kampatsikas, Ioannis, Rompel, Annette
• Format: Article
• Language: English
• Subjects: 631/45; 631/45/49; 631/45/49/1141; 639/638; 639/638/45; 639/638/45/49/1141; Affinity; Affinity chromatography; Caffeic acid; Catechin; Chloroplasts; Dimocarpus longan; E coli; Epicatechin; Flavonoids; Humanities and Social Sciences; Monophenol Monooxygenase - chemistry; multidisciplinary; Peptides; Polyphenol oxidase; Protein Sorting Signals; Protein transport; Pyrogallol; Sapindaceae; Science; Science (multidisciplinary); Signal peptides; Stroma; Substrates; Temperature tolerance; Thermal stress
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-022-20616-7

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- Dl PPO1 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- Dl PPO1 yields pro- Dl PPO1. The prepro- Dl PPO1 exhibited higher thermal tolerance than pro- Dl PPO1 (unfolding at 65 °C vs. 40 °C), suggesting that the signal peptide may stabilize the fold of Dl PPO1. Dl PPO1 can be classified as a tyrosinase because it accepts both monophenolic and diphenolic substrates. The pro- Dl PPO1 exhibited the highest specificity towards the natural diphenol (–)-epicatechin (k cat /K M 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- Dl PPO1 are 23, 36, 1.7 and 4.7-fold lower, respectively, than those observed with pro- Dl PPO1 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 Dl PPO1 and ascertain the affinity of PPOs towards specific flavonoid compounds.