Identification of a Flavin Monooxygenase-Like Flavonoid 8-Hydroxylase with Gossypetin Synthase Activity from Lotus japonicus

Abstract

Lotus japonicus is a model legume, that accumulates 8-hydroxyflavonol derivatives such as gossypetin (8-hydroxyquercetin) 3-O-glycoside, which confer the yellow color to its petals. An enzyme, flavonoid 8-hydroxylase (LjF8H), is assumed to be involved in the biosynthesis, but the specific gene is yet to be identified. The LjF8H cDNA was isolated as an FAD-binding monooxygenase-like protein using flower buds and flower specific EST data of L. japonicus. LjF8H is a single copy gene on chromosome III consisting of six exons. The conserved FAD- and NAD(P)H-dependent oxidase motifs were found in LjF8H. Phylogenetic analysis suggested that LjF8H is a member of the flavin monooxygenase group, but distinctly different from other known flavonoid oxygenases. Analysis of recombinant yeast microsome expressing LjF8H revealed that the enzyme catalyzed the 8-hydroxylation of quercetin. Other flavonoids, such as naringenin, eriodictyol, apigenin, luteolin, taxifolin, and kaempferol also acted as substrates of LjF8H. This broad substrate acceptance was unlike known F8Hs in other plants. Interestingly, flavanone and flavanonol, which have saturated C-C bond at positions 2 and 3 of the flavonoid C-ring, produced 6-hyroxylflavonoids as a by-product of the enzymatic reaction. Furthermore, LjF8H only accepted the 2S-isomer of naringenin, suggesting the conformational state of the substrates might affect product specificity. The overexpression of LjF8H in Arabidopsis thaliana and Petunia hybrida synthesized gossypetin and 8-hydroxykaempferol, respectively, indicating that LjF8H was functional in plant cells. In conclusion, this study represents the first instance of cloning and identification of F8Hs responsible for gossypetin biosynthesis.

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

“…RgF8H shares low sequence identity with fdeE (29.26%) and LjF8H ( Lotus japonicus ) (24.14%). However, a phylogenetic tree constructed among known F8H [ 21 , 24 , 34 , 42 ], and F6H [ 21 , 23 , 43 ] (Fig. 5 ) grouped RgF8H (Rg3610long) under C-8 monooxygenases, suggesting that RgF8H is a flavin-containing monooxygenase (FMO), requiring NADPH and/or FAD for its activity.…”

“…However, the Rg3610long (RgF8H) sequence is devoid of the F motif. Interestingly, the fdeE and LjF8H [ 24 ] sequences also do not contain an F motif, suggesting that all these enzymes may belong to a new FMO group.

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“…However, an in vitro study showed that RgF8H absolutely requires NADPH for its activity, although we do not find an NADP + -binding motif in the sequence. Similarly, LjF8H also showed an absolute requirement for NADPH [ 24 ]. It is debatable whether these enzymes do not require FAD for their activity or whether they bind FAD strictly during expression and do not release it during sample preparation.…”

“…The fdeE from H. seropedicae [ 41 ] shows activity towards naringenin (flavanone), there are no reports of attempts to use this enzyme against other flavonoids. In contrast, LjF8H [ 24 ] shows broad activity against flavanones, flavones, flavanonols, and flavonols. F8H from Chrysanthemum segetum has been characterized as a cytochrome P450 monooxygenase and hydroxylates luteolin and quercetin [ 47 ].…”

“…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].…”