Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica

Abstract

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.

De novo transcriptome assembly and functional analysis reveal a dihydrochalcone 3-hydroxylase(DHC3H) of wild Malus species that produces sieboldin in vivo

“…Domesticated apple lines overexpressing CsC3H resulted in the production of 3hydroxyphloridzin, which correlated with reduced susceptibility to apple scab and fire blight, suggesting a beneficial role of 3hydroxylated DHCs (Hutabarat et al, 2016). A recent study attempted to discover genes involved in this key step of sieboldin biosynthesis, although the genes F3′HI and F3′HII cloned and characterised from M. × domestica genome did not accept phloretin, leading the authors to conclude that F3′Hs identified were unlikely involved in the 3-hydroxylation towards sieboldin biosynthesis (Weissensteiner et al, 2021). F3′HI and F3′ HII, however, accepted naringenin, dihydrokaempferol and kaempferol, indicating a role in the well-studied 3′-hydroxylation of the B ring of flavonoids.…”

“…Although the glycosylation of 3-hydroxyphloretin can be carried out at least by PGT2 to produce sieboldin in vitro (Wang et al, 2020), the first committed step to 3hydroxyphloretin has not been yet identified. To date, the only attempt to identify genes involved in this step resulted in the characterisation of two flavonoid 3′-hydroxylases from M. × domestica (F3′HI and F3′HII), which did not accept phloretin and therefore unlikely to be involved in its 3-hydroxylation (Weissensteiner et al, 2021). In this study, we aimed to identify candidate genes that may account for the 3-hydroxylation of phloretin, that could be involved in DHC formation of wild Malus species.…”

First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin

“…Therefore, one can conclude that CH3H is a specialized F3′H as it exhibits an additional functionality compared to F3′H. Furthermore, it was shown that apple plants overexpressing Cs CH3H exhibited an in increased level of 3-hydroxyphloretin 15 , while the in vitro assays of apple F3′Hs did not show dihydrochalcone hydroxylation activity 16 , 17 .…”

Novel flavonoid C-8 hydroxylase from Rhodotorula glutinis: identification, characterization and substrate scope

“…Selective hydroxylation of aromatic compounds is still a challenging chemical reaction in synthetic chemistry and has been gaining increasing interest in recent years, especially due to the use of hydroxylated aromatic compounds in the pharmaceutical industry [5]. The use of isolated enzymes or whole cells for biocatalytic oxygen transfer is an environmental friendly, inexpensive and efficient way of targeted hydroxylation [2,[6][7][8][9][10].…”

“…However, the hydroxylation of flavonoids has not been widely studied to date. Recent literature reports give examples of hydroxylation at C-3ʹ, and C-6 positions mediated by enzymes belonging to the cytochrome P-450 monooxygenase group, requiring interaction with at least one oxidoreductase [8,[19][20][21][22][23]. A few examples of flavonoid hydroxylation at the C-8 position have been also described [21,24].…”