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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9700842/ https://www.ncbi.nlm.nih.gov/pubmed/36434079 http://dx.doi.org/10.1038/s41598-022-20616-7 |
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author | Ruckthong, Leela Pretzler, Matthias Kampatsikas, Ioannis Rompel, Annette |
author_facet | Ruckthong, Leela Pretzler, Matthias Kampatsikas, Ioannis Rompel, Annette |
author_sort | Ruckthong, Leela |
collection | PubMed |
description | 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 (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-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. |
format | Online Article Text |
id | pubmed-9700842 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-97008422022-11-27 Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency Ruckthong, Leela Pretzler, Matthias Kampatsikas, Ioannis Rompel, Annette Sci Rep Article 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 (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-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. Nature Publishing Group UK 2022-11-25 /pmc/articles/PMC9700842/ /pubmed/36434079 http://dx.doi.org/10.1038/s41598-022-20616-7 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Ruckthong, Leela Pretzler, Matthias Kampatsikas, Ioannis Rompel, Annette Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency |
title | Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency |
title_full | Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency |
title_fullStr | Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency |
title_full_unstemmed | Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency |
title_short | Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency |
title_sort | biochemical characterization of dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9700842/ https://www.ncbi.nlm.nih.gov/pubmed/36434079 http://dx.doi.org/10.1038/s41598-022-20616-7 |
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