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Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica
Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8469728/ https://www.ncbi.nlm.nih.gov/pubmed/34579488 http://dx.doi.org/10.3390/plants10091956 |
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author | Weissensteiner, Julia Molitor, Christian Marinovic, Silvija Führer, Lisa Waqas Hassan, Syed Hutabarat, Olly Sanny Spornberger, Andreas Stich, Karl Hausjell, Johanna Spadiut, Oliver Haselmair-Gosch, Christian Halbwirth, Heidi |
author_facet | Weissensteiner, Julia Molitor, Christian Marinovic, Silvija Führer, Lisa Waqas Hassan, Syed Hutabarat, Olly Sanny Spornberger, Andreas Stich, Karl Hausjell, Johanna Spadiut, Oliver Haselmair-Gosch, Christian Halbwirth, Heidi |
author_sort | Weissensteiner, Julia |
collection | PubMed |
description | Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3′-hydroxylase (F3′H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3′H cDNA clones from apple leaves and tested recombinant Malus F3′Hs for their substrate specificity. From the two isolated cDNA clones, only F3′HII encoded a functionally active enzyme. In the F3′HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3′HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely. |
format | Online Article Text |
id | pubmed-8469728 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-84697282021-09-27 Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica Weissensteiner, Julia Molitor, Christian Marinovic, Silvija Führer, Lisa Waqas Hassan, Syed Hutabarat, Olly Sanny Spornberger, Andreas Stich, Karl Hausjell, Johanna Spadiut, Oliver Haselmair-Gosch, Christian Halbwirth, Heidi Plants (Basel) Article Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3′-hydroxylase (F3′H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3′H cDNA clones from apple leaves and tested recombinant Malus F3′Hs for their substrate specificity. From the two isolated cDNA clones, only F3′HII encoded a functionally active enzyme. In the F3′HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3′HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely. MDPI 2021-09-19 /pmc/articles/PMC8469728/ /pubmed/34579488 http://dx.doi.org/10.3390/plants10091956 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Weissensteiner, Julia Molitor, Christian Marinovic, Silvija Führer, Lisa Waqas Hassan, Syed Hutabarat, Olly Sanny Spornberger, Andreas Stich, Karl Hausjell, Johanna Spadiut, Oliver Haselmair-Gosch, Christian Halbwirth, Heidi Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica |
title | Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica |
title_full | Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica |
title_fullStr | Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica |
title_full_unstemmed | Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica |
title_short | Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica |
title_sort | molecular and enzymatic characterization of flavonoid 3′-hydroxylase of malus × domestica |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8469728/ https://www.ncbi.nlm.nih.gov/pubmed/34579488 http://dx.doi.org/10.3390/plants10091956 |
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