Author:
Ruckthong Leela,Pretzler Matthias,Kampatsikas Ioannis,Rompel Annette
Abstract
AbstractThe “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.
Funder
Österreichische Austauschdienst-Gesellschaft mit beschränkter Haftung
ASEA-UNINET
Skill Development Grant from King Mongkut’s University of Technology, Thonburi
Universität Wien
Austrian Science Fund
Publisher
Springer Science and Business Media LLC
Cited by
2 articles.
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