Mol. Cells, Vol. 25, No. 2, pp. 312-316
Molecules
and
Cells
Communication
©KSMCB 2008
Flavanone 3β-Hydroxylases from Rice: Key Enzymes for
Favonol and Anthocyanin Biosynthesis
Jeong Ho Kim, Yoon Jung Lee, Bong Gyu Kim, Yoongho Lim, and Joong-Hoon Ahn*
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea.
(Received October 30, 2007; Accepted November 26, 2007)
Flavanone 3β-hydroxylases (F3H) are key enzymes in
the synthesis of flavonol and anthocyanin. In this study,
three F3H cDNAs from Oryza sativa (OsF3H-1 ~3) were
cloned by RT-PCR and expressed in E. coli as
gluthatione S-transferase (GST) fusion proteins. The
purified recombinant OsF3Hs used flavanone, naringenin and eriodictyol as substrates. The reaction products with naringen and eriodictyol were determined by
nuclear magnetic resonance spectroscopy to be dihydrokaempferol and taxifolin, respectively. OsF3H-1 had
the highest enzymatic activity whereas the overall expression of OsF3H-2 was highest in all tissues except
seeds. Flavanone 3β-hydroxylase could be a useful target for flavonoid metabolic engineering in rice.
Keywords: Flavanone 3β-Hydroxylase; Flavonoid, Oryza
sativa.
Introduction
More than 6,000 flavonoids are known to be synthesized
by plants from two simple metabolites, malonyl-CoA and
p-coumaroyl-CoA (Harborne and Baxter, 1999). The
structural diversity of flavonoids derives from various
modification reactions (Tahara, 2007). Most modifications are on hydroxyl groups. For example, the hydroxyl
groups serve as acceptors for methyl groups and sugars.
Thus, it seems that hydroxylation of flavonoids is a prerequisite for most further modifications.
In plants hydroxylations are mediated by two enzymes,
cytochrome P450-dependent monooxygenases (P450) and
2-oxoglutarate-dependent dioxygenases (2-ODD). Both
require iron as cofactor either as part of a heme group
(P450) or in non-heme form [Fe (ІІ) for 2-ODD] for full
* To whom correspondence should be addressed.
Tel: 82-2-450-3764; Fax: 82-2-3437-6106
E-mail: [email protected]
activity (Prescott, 2003; Prescott and John, 1996). For
example, 273 P450 family members are responsible for
various hydroxylation steps in Arabidopsis thaliana and
more than 300 in rice (Nelson et al., 2004). Flavonoid 3′hydroxylases (Brugliera et al., 1999), flavonoid 3′, 5′hydroxylases (Kaltenbach et al., 1999), and flavonoid 6hydroxylases (Latunde-Dada et al., 2001) are P450s involved in flavonoid hydroxylation. On the other hand,
flavanone 3β-hydroxylases (F3Hs) (Britsch et al., 1986),
flavones (FNS) and flavonol synthases (FLS) (Martens
and Mithőfer, 2005), as well as anthocyanidin synthases
(ANS) (Saito et al., 1999) are dioxygenases involved in
flavonoid biosynthesis. Among them, F3Hs, which are 2ODDs, mediate hydroxylation at carbon 3. F3Hs catalyze
the hydroxylation of flavanones to dihydrofavonols
(Britsch and Grisebach, 1986; Lukačin et al., 2000a;
2000b; 2000c). It has been demonstrated that F3H mutants have a white flower phenotype, accumulating only
flavanones (Forkmann et al., 1980). F3Hs have been
cloned from Petunia hybrida (Britsch et al., 1992) and
cDNAs with 70−90% sequence similarity have been reported from at least 15 additional plants. However, only a
few of these cDNAs have been functionally expressed and
the recombinant enzymes have barely been studied. F3Hs
are highly similar to flavone synthases at the amino acid
level so that it is not easy to predict their reaction products without in vitro characterization.
Rice is one of the most important crops in the world
and its genome has been completely sequenced. However,
only a few genes that take part in rice flavonoid metabolism have been characterized (Kim et al., 2006; Ko et al.,
2006). For metabolic engineering of anthocyanin biosynthesis, characterization of F3H is indispensable. Thus, we
searched for F3H homologues and characterized their
Abbreviations: ANS, anthocyanidin synthase; F3H, flavonone
3β-hydroxylase; FLS, flavonol synthase; FNS, flavone synthase;
GST, glutathione S-transferase.
Jeong Ho Kim et al.
313
genes. Here, we report the functional expression and characterization of flavonoid 3 β-hydroxylases (OsF3Hs) from
rice.
Materials and Methods
Cloning and expression of OsF3Hs Total RNA from 2-weekold rice seedlings (Oryza sative cultivar Heokjinju) was isolated
with a Qiagen plant total RNA isolation kit (Qiagen, USA), and
cDNA was synthesized as described by Kim et al. (2003). Two
primers spanning the whole open reading frame of OsF3H-1, 5′ATGGCGCCGGTGGCCACGACGTTCCTC-3′ (forward) and
reverse 5′-CGTCCATGGCGATCTAGG-3′ (reverse), were designed based on an OsF3H-1 sequence (GenBank accession
number NM_001060692). Primers for OsF3H-2 (GenBank accession number AAL58118) were 5′-GTCGCCGTCGRGAACATG-3′ (forward) and 5′-GCGATGAGCTAAGTCCTGAACAG-3′ (reverse). OsF3H-3 primers (GenBank accession number
CAE02796) were 5′-ATGTCTGACACGTCGAA-GGGTA-3′
(Forward) and 5′-TGAAGACCGTACCGTGAATG-3′ (reverse).
PCR conditions followed the protocol described by Kim et al.
(2005). The PCR products were subcloned into pGEMT-easy
(Promega, USA) and sequenced.
The open reading frames of OsF3H-1~3 were subcloned into
pGEX 5X-3 (Amersham Biotech, USA), and expression and purification of the recombinant OsF3Hs were carried out as described
by Kim et al. (2006).
For the analysis of expression in different tissues, total RNA
was isolated from leaves, roots, seeds, and stems of rice plants
seven days after flowering. Real time quantitative RT-PCR was
performed as described by Kim et al. (2005). Primers for OsF3H1 were 5′-AGGAGCCCATACTGGAGGAG-3′ (forward) and 5′CTCCTTGGCCTTCTTCTTGA-3′ (reverse), primers for OsF3H2 were 5′-AGAAGCTCATCACCGACGAC-3′ (forward) and 5′CAGTGCTCCTGGTCAAGGTT-3′ (reverse), and primers for
OsF3H-3 were 5′-GCAGCAGCAACAGCTGGAAA-3′ (forward)
and GCGCGGTGCACAACAGTCTT-3′ (reverse).
Enzyme assay The reaction mixture contained 1 mM ascorbate, 2
mg catalase (bovine, Sigma, USA), 100 μM FeSO4, 160 μM 2oxoglutaric acid, 40 μM flavonoid substrate and 40 μg of the
purified recombinant OsF3H in 500 μl 10 mM Tris/HCl (pH 8.0).
The mixture was incubated at 37°C for 1 h, extracted with ethyl
acetate and evaporated by speed vacuum. Analysis of reaction
products was carried out as described by Kim et al. (2006). The
two enantiomers of naringenin were separated by HPLC equipped
with a chiral column (Chiralpack® AD-RH column, 150 × 4.6 mm,
Daicel, USA) as described by Miyahisa et al. (2006).
The structures of the reaction products were determined by
nuclear magnetic resonance spectroscopy (NMR). The reaction
products of eriodictyol and naringenin were dissolved in
CD3OD. All NMR experiments were performed on a Bruker
Avance 400 NMR spectrometer using the XWINNMR software
package.
Fig. 1. The phylogenetic relationships of the flavanone 3β-hydroxylases from several plants. The tree was constructed by aligning the sequences in the Clustal X program and visualizing with
the Treeview program. OsF3H-1~3, flavanone 3β-hydroxylases
from Oryza sativa; CF3H, flavanone 3-hydroxylase from Citrus
sinensis (BAA36553); AF3H, flavanone 3-hydroxylase from
Arabidopsis thaliana (NP_190692); EF3H, flavanone 3-hydroxylase from Eustoma grandiflorum (BAD34459); GF3H, flavanone 3-hydroxylase from Ginkgo biloba (AAU93347); PeF3H,
flavanone 3β-hydroxylase from Petunia x hybrida (AAC 49929);
PF3H, flavanone 3β-hydroxylase from Petroselinum crispum
(AAP57394).
Results and Discussion
We searched the rice genome using Petunia hybrida flavanone 3β-hydroxylase (GenBank accession number
X60512) as reference, and identified three genes, OsF3H1~3 belonging to the 2-oxoglutaric acid-Fe2+ superfamily.
Full length cDNAs of the OsF3Hs were cloned from
Oryza sativa by RT-PCR and sequenced. OsF3H-1, -2,
and -3 consisted of 1167-bp, 1026-bp, and 978-bp, which
encoded 42.7, 38.7, and 36.8-kDa proteins, respectively.
In addition, phylogenetic analysis of the F3Hs from several plants revealed two groups (Fig. 1). As shown in Fig. 1,
OsF3H-2 is closer to the F3H of Petunia hybrida than
OsF3H-1. OsF3H-3 forms a subgroup. Four residues (his at
residue 77, his at residue 217, asp at residue 219, and his at
residue 287 of OsF3H-1; his at residue 76, his at residue
218, asp at residue 220, and his at residue 274 of OsF3H-2;
his at residue 62, his at residue 200, asp at residue 202, and
his at residue 256 of OsF3H-3) that are known to bind Fe2+
are conserved in OsF3H-1~3 (Jin et al., 2005). Likewise,
the oxo-glutarate binding residues (Arg297-X-Ser299 for
OsF3H-1; Arg284-X-Ser286 for OsF3H-2; Arg267-X-Ser268
for OsF3H-3) (Lukačin et al., 2000b) are also present in
OsF3H-1~3.
Quantitative real time RT-PCR was used to determined
levels of OsF3H-1~3 in different tissues. Overall expression of OsF3H-2 in all tissues was higher than that of
OsF3H-1. However, OsF3H-1 was the most highly expressed in seeds. The expression of OsF3H-3 was much
lower than that of the other two OsF3Hs (Fig. 2).
In order to identify the function of OsF3H-1~3, the
ORFs of these genes were subcloned into E. coli expres-
314
Flavanone 3β-Hydroxylases from Rice
A
B
Fig. 2. Relative expression of OsF3H-1~3 in different tissues.
Relative transcript levels were measured by real-time RT-PCR
and standardized to the actin gene.
C
D
Fig. 3. Purification of recombinant OsF3H-1~3. 1, 4, 7, E.coli
lysates before induction (OSF3H-1, -2, and -3 respectively); 2, 5,
8 E. coli lysates after induction (OSF3H-1, -2, and -3 respectively); 3, 6, 9 GST-affinity purified OsF3Hs (OSF3H-1, -2, and
-3 respectively).
sion vector pGEX and the recombinant proteins were induced and purified. All there OsF3Hs were expressed in
soluble form. SDS-PAGE analysis showed that all three
OsF3Hs was expressed as glutathione S-transferase fusion
proteins, and they were purified to homogeneity (Fig. 3).
The results of a BLAST analysis indicated that the
OsF3Hs would use flavanone as substrate and convert it
into 3-hydroflavanone. Accordingly, the purified recombinant OsF3Hs reacted with flavanones, eriodictyol, and
naringenin. However, both eriodictyol and naringenin are
mixtures of two enantiomers. It is known that F3Hs preferentially bind the S-form (Turnbull et al., 2004). We
therefore separated the two enantiomers by HPLC
equipped with a chiral column. The R-form did not yield
any new peak with the enzyme, while the S-form did. The
products of OsF3H-1~3 with S-narignenin formed new
peaks with retention times of 10.56 min, whereas naringenin has a retention time of 15.03 min (Fig. 4D; only the
reaction product is shown). The products of OsF3H-1~3
with S-eriodictyol had similar retention times and UV
spectra to those of taxifolin, 3-hydroxyl eriodictyol (Fig.
4B; only the reaction product is shown). This strongly
suggested that all the OsF3Hs were flavanone 3β-hy-
Fig. 4. Analysis of reaction products by HPLC A. authentic Seriodictyol (S1) and authentic taxifolin (S2). B. reaction product
of OsF3H with S-eriodictyol (P1), and S-eriodictyol itself (S1).
C. authentic S-naringenin (S3). D. reaction product of OsF3H
with S-naringenin (P2), and S-naringenin itself (S3).
droxylases. The structures of the reaction products with
OsF3H-1 were next established by NMR spectroscopy.
First the 1H NMR spectra of the eriodictyol and naringenin reaction products were compared, respectively, with
the published data for taxifolin and dihydrokaempferol,
(Baderschneider et al., 2001; Prescott et al., 2002). The
1
H chemical shifts of the eriodictyol reaction product
were indeed identical to those of taxifolin (Table 1), and
the coupling constant (J2,3 = 11.5 Hz) between H-2 and H3 suggested that its stereochemistry was 2,3-trans. Furthermore, the 1H NMR data for the naringenin reaction
product agreed well with those of dihydrokaempferol. The
stereochemistry of the naringenin reaction product also
turned out to be 2, 3-trans, which was confirmed by its
coupling constant of 11.6 Hz. Thus, it can be concluded
that OsF3H-1 encodes a flavanone 3β-hydroxylase.
Next, we investigated the influence of the hydroxyl and
methoxy groups on the reactivity of OsF3H-1. The presence of methoxy groups in the B-ring of eriodictyol has
various effects on the activity of OsF3H-1. For example,
3′-O-methylated eriodictyol (homoeriodictyol) had 68% of
the activity of eriodictyol itself, while 4′-O-methylated
eriodicyol (hesperitin) had 105% of the activity of eriodictyol. However, the bifunctional flavonol synthase from
Arabidopsis thaliana uses eriodictyol but not hesperetin
Jeong Ho Kim et al.
315
Table 1. Comparisons of the 1H NMR data for the eriodictyol and naringenin products with those of taxifolin and dihydrokaempferol,
respectively.
Substrate: eriodictyol
Position
Taxifolin*
Product I
2
4.90 (d, J = 11.5)
4.92 (d, J = 11.5)
3
4.51 (d, J = 11.5)
4.50 (d, J = 11.5)
6
5.92 (d, J = 2.0)
5.92 (d, J = 2.0)
Position
Substrate: naringenin
Dihydrokaempferol*
2
Product II
4.98 (d, J = 11)
4.98 (d, J = 11.6)
3
4.54 (d, J = 11)
4.54 (d, J = 11.6)
6
5.93 (d, J = 2)
5.92 (d, J = 2.1)
8
5.88 (d, J = 2.0)
5.88 (d, J = 2.0)
8
5.88 (d, J = 2)
5.88 (d, J = 2.1)
2′
6.94 (d, J = 2.0)
6.96 (d, J = 1.9)
2′/6′
7.35 (m)
7.35 (d, J = 8.7)
5′
6.80 (d, J = 8.0)
6.80 (d, J = 8.1)
3′/5′
6.83 (m)
6.83 (d, J = 8.7)
6′
6.84 (dd, J = 8.0, 2.0)
6.85 (dd, J = 8.1, 1.9)
*Data from Baderschneider and Winterhalter (2001) and Prescott et al. (2002).
Table 2. Kinetic parameters of OsF3Hs for S-eriodictyol.
OsF3H-1
Km
(μM)
Vmax
(μkat/mg)
Kcat (S-1)
Kcat/Km
(S-1 μM-1)
57.8
3.1
2.1 × 10-1
4 × 10-2
OsF3H-2
5.70
0.4
2.4 × 10
OsF3H-3
6.30
0.5
3 × 10-3
-4
4.2 × 10-5
4.7 × 10-4
as substrate (Prescott et al., 2002).
OsF3H-2 and OsF3H-3 also converted naringenin and
eriodictyol into dihydrokaempferol and taxifolin, respectively. They had more activity with eriodictyol than with
naringenin (data not shown).
The Vmax and Km for S-eriodictyol were determined by
Lineweaver-Burk plots (Table 2). On the basis of the kinetic analyses, OsF3H-1 was the most efficient of the
three OsF3Hs followed by OsF3H-3 and OsF3H-2. The
effects of cofactors such as ascorbate, catalase, 2oxoglutaric acid, and Fe2+ on OsF3H activity were also
investigated. The omission of either 2-oxoglutaric acid or
ascorbate resulted in almost complete loss of activity for
all three OsF3Hs. In order to study the effect of the omission of Fe2+ ions, the purified recombinant proteins were
treated with 0.2 M EDTA and kept on ice for 30 min. After this treatment OsF3H-1~3 had completely lost activity
without added Fe2+, indicated that the OsF3Hs are Fe2+and 2-oxoglutarate-dependent oxygenases.
These three OsF3Hs are the first flavanone 3β-hydroxylases from rice to be characterized. Flavanone 3β-
hydroxylases are key enzymes in the pathway leading to
flavonol and anthocyanin. OsF3H-1 and -2 are expressed at
much higher levels than OsF3H-3 in rice cultivars producing high contents of anthocyanins (unpublished results).
This suggests that the products of both genes are active in
the biosynthesis of anthocyanin. Blocking F3Hs in Arabidopsis thaliana resulted in decreased levels of flavonol and
antocyanin (Wiseman et al., 1998). An Arabidopsis F3H
mutant has been used for metabolic engineering of flavonoids in Arabidopsis (Liu et al., 2002). The characterization
of the OsF3Hs reported in the present study may help in
developing OsF3Hs as targets for flavonoid metabolic engineering in rice.
Acknowledgment This work was supported by the Biogreen 21
Program, Rural Development Administration, Republic of Korea,
and partially by grant KRF-2006-005-J0340.
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