Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
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
_version_ 1784574010507395072
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
work_keys_str_mv AT weissensteinerjulia molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT molitorchristian molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT marinovicsilvija molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT fuhrerlisa molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT waqashassansyed molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT hutabaratollysanny molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT spornbergerandreas molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT stichkarl molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT hausjelljohanna molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT spadiutoliver molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT haselmairgoschchristian molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica
AT halbwirthheidi molecularandenzymaticcharacterizationofflavonoid3hydroxylaseofmalusdomestica