Tài liệu Báo cáo khoa học: Functional expression and mutational analysis of flavonol synthase from Citrus unshiu pptx

Functional expression and mutational analysis of flavonol synthase
from
Citrus unshiu
Frank Wellmann
1,
*, Richard Lukac
ˇ
in
1,
*, Takaya Moriguchi
2
, Lothar Britsch
3
, Emile Schiltz
4
and Ulrich Matern
1
1
Institut fu
¨
r Pharmazeutische Biologie, Philipps-Universita
¨
t Marburg, Germany;
2
National Institute of Fruit Tree Science,
Ibaraki, Japan;
3
Merck kgaA, Scientific Laboratory Products, Darmstadt, Germany;
4
Institut fu
¨

r Organische Chemie
und Biochemie, Universita
¨
t Freiburg, Germany
Flavonols are produced by the desaturation of flavanols
catalyzed by flavonol synthase. The enzyme belongs to the
class of intermolecular dioxygenases which depend on
molecular oxygen and Fe
II
/2-oxoglutarate for activity, and
have been in focus of structural studies recently. Flavonol
synthase cDNAs were cloned from six plant species, but
none of the enzymes had been studied in detail. Therefore, a
cDNA from Citrus unshiu (Satsuma mandarin) designated
as flavonol synthase was expressed in Escherichia coli,and
the purified recombinant enzyme was subjected to kinetic
and mutational chacterizations. The integrity of the recom-
binant synthase was revealed by a molecular ion from
MALDI-TOF mass spectrometry at m/z 37888 ± 40 (as
compared to 37899 Da calculated for the translated poly-
peptide), and by partial N-terminal sequencing. Maximal
flavonol synthase activity was observed in the range of
pH 5–6 with dihydroquercetin as substrate and a tempera-
ture optimum at about 37 °C. K
m
values of 272, 11 and
36 l
M
were determined for dihydroquercetin, Fe
II

and
2-oxoglutarate, respectively, with a sixfold higher affinity
to dihydrokaempferol (K
m
45 l
M
). Flavonol synthase
polypeptides share an overall sequence similarity of 85%
(47% identity), whereas only 30–60% similarity were
apparent with other dioxygenases. Like the other dioxy-
genases of this class, Citrus flavonol synthase cDNA encodes
eight strictly conserved amino-acid residues which include
two histidines (His221, His277) and one acidic amino acid
(Asp223) residue for Fe
II
-coordination, an arginine (Arg287)
proposed to bind 2-oxoglutarate, and four amino acids
(Gly68, His75, Gly261, Pro207) with no obvious function-
ality. Replacements of Gly68 and Gly261 by alanine reduced
the catalytic activity by 95%, while the exchange of these Gly
residues for proline completely abolished the enzyme activ-
ity. Alternatively, the substitution of Pro207 by glycine
hardly affected the activity. The data suggest that Gly68 and
Gly261, at least, are required for proper folding of the
flavonol synthase polypeptide.
Keywords: Citrus unshiu (Rutaceae); flavonoid biosyn-
thesis; flavonol synthase; functional expression; site-directed
mutagenesis.
Flavonoids fulfill vital functions in many plants beyond the
scope of pigmentation and ultraviolet screening, e.g. in

reproduction [1], in the defense against microbial pathogens
and insects or in auxin transport [2], and are accumulated
ubiquitously in flower and green tissues [1]. Their biosyn-
thesis proceeds from 4-coumaroyl- and malonyl-CoAs to
form naringenin chalcone [3] which is cyclized stereospeci-
fically to the flavanone (2S)-naringenin [3]. Naringenin may
be oxidized by flavone synthase (FNS) to yield the flavone
apigenin [4–6] or hydroxylated by flavanone 3b-hydroxylase
(FHT) to form a flavanol (syn. dihydroflavonol) [7–10], i.e.
dihydrokaempferol, which might be reduced subsequently
to a leucoanthocyanidin along the branch leading to
catechins and anthocyanidins [3] (Fig. 1). Alternatively,
flavonol synthase (FLS) catalyzes the oxidation of the
flavanol to a flavonol (Fig. 1). FLS had been reported
initially from irradiated parsley cells as a soluble dioxygen-
ase requiring 2-oxoglutarate and Fe
II
/ascorbate for full
activity [11]. The activity was subsequently detected in
flower tissues of Matthiola incana [12], Petunia hybrida [13]
or Dianthus caryophyllus [14]. The first FLS cDNA was
cloned in 1993 from Petunia hybrida [15] and identified by
functional expression in yeast, while the FLS-antisense
transformation of petunia or tobacco intensified the red
flower pigmentation [15]. Further FLS cDNAs were
isolated later from Arabidopsis thaliana [16], Eustoma
grandiflorum, Solanum tuberosum [17], Malus domestica
and Matthiola incana, and approximately 85% similarity
was determined for the translated polypeptides, mostly in
the C-terminal 40% region based on total length of 335

residues. None of these enzymes has been satisfactorily
expressed and characterized.
Correspondence to U. Matern, Institut fu
¨
r Pharmazeutische Biologie,
Philipps-Universita
¨
t Marburg, Deutschhausstrasse 17A,
35037 Marburg, Germany.
Fax: + 49 6421 282 6678, Tel.: + 49 6421 282 2461,
E-mail:
Abbreviations: ACC, aminocyclopropane-1-carboxylic acid;
DAOCS, deacetoxcephalosporin C synthase; FHT, flavanone
3b-hydroxylase; FLS, flavonol synthase; FNS, flavone synthase;
IPNS, isopenicillin N synthase.
*Note: these authors contributed equally to the work presented.
Note: flavonol synthase NCBI database accession numbers: Citrus
unshiu, AB011796; Eustoma grandiflorum, AAF64168; Malus domes-
tica, AAD26261; Matthiola incana, O04395.
(Received 17 April 2002, revised 4 July 2002, accepted 11 July 2002)
Eur. J. Biochem. 269, 4134–4142 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03108.x
Several intermolecular dioxygenases, particularly those of
microbial or human origin, catalyze reactions of medicinal
and industrial relevance, and their spatial organization and
mode of action are under investigation. The reactions are
diverse, such as the desaturation of aliphatic chains or the
oxidative cyclization and the hydroxylation of substrates
[18–22], and depend on the one-, two- or four-electron
reduction of molecular oxygen. Most of these dioxygenases
rely on the concomitant oxidation of 2-oxoglutarate.

Deacetoxycephalosporin C synthase (DAOCS) serves as a
precedent enzyme in comparison to the 2-oxoglutarate-
independent isopenicillin N-synthase (IPNS), as the modu-
lar composition and spatial configuration of IPNS and
DAOCS appear to be rather similar [23] irrespective of only
19% primary structure identity. Both enzymes form a
b-strand core folded into a distorted jelly roll motif [23,24]
known also from viral capsid proteins and other enzymes
[24,25]. During the preparation of this manuscript, an
equivalent configuration was proposed for a putative
anthocyanidin synthase from Arabidiopsis thaliana [26],
although this enzyme has not been functionally proved and
the lability of substrate and product rule out any cocrys-
tallization. The setting of a helices and b-strands causes very
similar CD spectra for this class of enzymes [27–29], which
was confirmed recently also for Petunia FHT [9] sharing
30% sequence similarity with the DAOCS and IPNS
polypeptides.
The IPNS, DAOCS or FHT sequences also range in the
order of 30% similarity to the translated FLS sequences,
but the biochemical characterization of FLSs has remained
very preliminary. In the course of our investigations on the
related dioxygenases FHT [7–10] and flavone synthase I
[4–6] we became interested in the molecular evolution of
diversity concerning the enzymes of flavonoid biosynthesis.
We report the expression of highly active Citrus unshiu
FLS in Escherichia coli and the purification of the labile
enzyme by a modified protocol devised for the isolation of
Petunia hybrida FHT [8]. This enzyme served to deter-
mine for the first time the kinetic parameters of an

FLS. The relevance of three amino-acid residues which
appear to be highly conserved in all plant intermolecular
dioxygenases for FLS activity was examined by site-direc-
ted mutagenesis.
MATERIALS AND METHODS
Expression vector
The FLS cDNA from satsuma mandarin fruits, C. unshiu
[30], was excised with EcoRI and XhoI from the Bluescript
vector (Stratagene) and subcloned in pTZ19R [31]. The
construct was used for the transformation of E. coli RZ1032
(Stratagene), ssDNA was isolated by the addition of phage
M13K07 and used for site-directed mutagenesis by the site-
elimination technique according to Zakour [32]. Hybridiza-
tion of the mismatch primer 5¢-CTCCACCTCCATG
GATTTTATTTTCC-3¢ to the FLS 5¢ coding region
introduced a unique NcoI site at the start of translation,
which was verified by DNA sequencing [33]. The DNA
encoding FLS was subsequently isolated by digestion with
NcoI and PstI and subcloned into the expression vector
pQE6 (FLS-pQE6) as described previously [7,9].
Recombinant FLS
E. coli strain M15 harboring the plasmid pRep4 was
transformed with the FLS-pQE6 constructs containing the
coding sequence of the wild-type or mutant enzymes and
subcultured subsequently to a density of 0.8 in Luria–
Bertani medium (typically 400 mL in 2 L flasks) containing
ampicillin (100 lgÆmL
)1
) and kanamycin (25 lgÆmL
)1

).
Isopropyl thio-b-
D
-galactoside (1 m
M
) was added for the
induction of FLS expression, and the bacteria were
harvested after another 3 h at 37 °C and stored frozen at
) 70 °C as described previously [7–9]. Wild-type FLS was
purified from the crude bacterial extracts by a modified
procedure devised for Petunia FHT [8]. Briefly, the bacterial
pellet (up to 6 g wet mass) was suspended in 20 mL of
50 m
M
potassium phosphate buffer pH 5.5, 10 m
M
EDTA,
5m
M
dithiothreitol and 15 m
M
MgCl
2
, and the suspensions
were sonicated for 2 min and cleared by centrifugation
(30 000 g,10min,5°C). Solid ammonium sulfate was
Fig. 1. Reaction catalyzed by FLS.
(2S)-Naringenin, formed from 4-coumaroyl-
CoA and malonyl-CoAs by the action of
chalcone synthase and chalcone isomerase, is

3b-hydroxylated by flavanone 3b-hydroxylase
(FHT) to furnish the substrate (2R,3R)-
dihydrokaempferol. Alternatively, the B-ring
hydroxylation of naringenin to (2S)-
eriodictyol preceeding the 3b-hydroxylation
yields the substrate (2R,3R)-dihydroquercetin.
Both dihydrokaempferol and dihydroquerce-
tin are accepted by the FLS to produce
kaempferol and quercetin, respectively. The
flavanones naringenin and eriodictyol might
also be oxidized by FNS to the flavones
apigenin or luteolin.
Ó FEBS 2002 Flavonol synthase (Eur. J. Biochem. 269) 4135
added to the clear supernatant, and the protein precipitating
at 40–50% saturation was redissolved in 50 m
M
potassium
phosphate buffer pH 5.5, containing 5 m
M
dithiothreitol
(1 mL) for successive size exclusion and anion exchange
chromatographies on Fractogel EMD BioSEC (S) (Merck,
Darmstadt, Germany) and Fractogel EMD DEAE 650 (S)
(Merck) as described previously [8]. The purification of
wild-type FLS was monitored by enzyme assays and SDS/
PAGE.
Site-directed mutagenesis
Site-directed mutagenesis was accomplished by site-elimin-
ation using the oligonucleotide- directed in vitro mutagenesis
technique [32]. Oligonucleotides were synthesized (G. Igloi,

Institut fu
¨
r Biologie III, Universita
¨
t Freiburg, Germany) as
required for the substitution of glycine by alanine and
proline, respectively, or of proline by glycine (Table 1). Each
individual mutation was verified by DNA sequencing using
the dideoxynucleotide chain-termination method [33] with
the universal and reverse sequencing primers. Following the
confirmation of successful mutation the mutated genes were
ligated into the expression vector pQE6 in the same way as
described for the wild-type cDNA.
Data base retrieval
Data base searches and sequence alignments were carried
out with the
ENTREZ
and
BLAST
software (National Library
of Medicine and National Institute of Health, Bethesda,
MD, USA). The
PROSIS
package (Hitachi, San Bruno, CA,
USA) was used for the analysis of multiple alignments and
consensus sequences with minor adjustment the computer
alignments.
Circular dichroism spectroscopy
Circular dichroism measurements of homogeneous FLS
were performed on a Jasco-720 spectropolarimeter (Tokyo,

Japan) interfaced to an 486/33 PC and controlled by Jasco
software. The spectropolarimeter was equipped with a
cylindrical quartz cuvette with a pathlength of 0.05 cm. The
temperature of the cell holder was maintained at 5 °Cbya
circulating water thermostat and the instrument was
calibrated with 0.06% ammonium
D
–10-camphor sulfonate.
FLS spectra were recorded in potassium phosphate buffer,
pH 6.8, as described previously for FHT [9], and the protein
concentration was adjusted to 0.371 mgÆmL
)1
in the sample.
The documented spectra show the accumulation of 10 scans
with 50 nmÆmin
)1
. The CD spectra of the FLS sample were
analyzed for the secondary structure content by the self-
consistent method [34] included in the program package
DICHROPROT
V2.4 by Delage and Geourjon [35].
Protein analysis and immunoassay
Partial N-terminal sequencing was carried out by Edman
degradation in a pulsed liquid sequencer (Model 477 A,
Applied Biosystems Inc.) with a Model 120 A PTH-analyzer
for on-line identification, following the supplier’s guidelines.
Mass spectra were recorded on a Bruker Reflex II MALDI-
TOF mass spectrometer in the linear mode. The protein
solution (100 lg per 300 lL20m
M

potassium phosphate
buffer) was diluted with an equal volume of 0.1% trifluo-
roacetic acid/acetonitrile (1 : 1, v/v), and this acidified
solution was then mixed with an equal volume of a saturated
solution of sinapic acid in 0.1% trifluoroacetic acid/aceto-
nitrile (1 : 1, v/v) and applied on the SCOUT MTP
TM
MALDI-TOF target plate target in 0.5 lL portions [8].
SDS/PAGE separation of protein extracts was performed
on 5% stacking and 12.5% separation gels in a Mini-
Protean II cell (Bio-Rad, Mu
¨
nchen) according to Laemmli
[36]. Antibodies were raised in rabbit by repeated injection
of the recombinant homogeneous FLS (1 mg total), and the
antiserum was diluted 10,000-fold for Western blotting [37].
Enzyme assays
FLS activity of filtered (PD10 column, Pharmacia, Frei-
burg) plant or bacterial extracts was routinely assayed at
37 °C and pH 5.0. The assay mixture (total volume 360 lL)
contained 100 l
M
dihydroquercetin as a substrate, 83 l
M
2-oxoglutarate, 42 l
M
ammonium iron(II) sulfate, 2.5 m
M
sodium ascorbate, and 2 mgÆmL
)1

bovine catalase, and the
incubation was carried out in open vials under gentle
shaking. The reaction linearity was assessed by proper
choice of protein amounts (4.5–22 lg) and incubation
periods (0.5–10 min). The reaction was stopped by the
addition of 15 lL saturated aqueous EDTA solution.
The flavonoids were isolated by repeated extraction with
Table 1. Oligonucleotides for site-directed mutagenesis. The Citrus FLS coding sequence flanking the desired site of mutation (top) is aligned with
the complementary oligonucleotide used to create the mutation (bottom). The triplets encoding glycine and proline are bold-printed, and the base
changes are underlined.
Mutant FLS Oligonucleotide Codon change
Gly68Ala
5¢-CGGGAGTGGGGGATTTTCCAG-3¢ GGGfiGCG
3¢-GCCCTCACCCGCTAAAAGGTC-5¢
Gly68Pro
5¢-CGGGAGTGGGGGATTTTCCAG-3¢ GGGfiCCG
3¢-GCCCTCACCGGCTAAAAGGTC-5¢
Gly261Ala
5¢-CATCCACATCGGGGACCAGATC-3¢ GGGfiGCG
3¢-GTAGGTGTAGCGCCTGGTCTAG-5¢
Gly261Pro
5¢-CATCCACATCGGGGACCAGATC-3¢ GGGfiCCG
3¢-GTAGGTGTAGGGCCTGGTCTAG-5¢
Pro207Gly
5¢-GATTAATTATTATCCGCCATGCCC-3¢ CCGfiGGG
3¢-CTAATTAATAATACCCGGTACGGG-5¢
4136 F. Wellmann et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ethylacetate (twice, 75 lL) and reversed-phase HPLC
(Shimadzu, Tokyo, Japan) on a Nucleosil C18-column
(125 · 4 mm, 5 lm; Machery and Nagel, Du

¨
ren, Germany).
The column was equilibrated with solvent A (20% aqueous
methanol), and quercetin or kaempferol and dihydroqu-
ercetin or dihydrokaempferol were eluted in a linear
gradient of solvent A and solvent B (100% methanol) at
0.5 mLÆmin
)1
over 3 min, followed by solvent B for 5 min
[38]. The elution was monitored by the absorption profile at
290 nm (dihydroquercetin, dihydrokaempferol) or 368 nm
(quercetin, kaempferol), and authentic flavonoid samples
were employed as references for calibration. The reaction
catalyzed by FLS follows second order kinetics, and the
apparent Michaelis constants for the wild-type enzyme were
determined with 11 lg of the homogeneous enzyme [7,9].
Protein concentrations were determined according to Lowry
[39] following the precipitation with trichloroacetic acid in
the presence of deoxycholate [40] and using bovine serum
albumin as a standard.
Mass spectrometry
The FLS assay, routinely carried out in Tris/HCl buffer
pH 7.5 prior to the final assessment of pH optima, was
scaled up for preparative purposes to a volume of 40 mL (20
incubations of 2 mL each). The assay contained 6 mg
HPLC-purified dihydroquercetin total, 170 l
M
sodium
ascorbate, 35 l
M

ammonium iron(II) sulfate, 70 l
M
2-oxoglutarate, 2 mgÆmL
)1
bovine catalase and 1.1 mg
(55 lg per 2 mL incubation) of the homogeneous FLS. The
mixture was incubated for two hours at 37 °C on a rotary
shaker (300 r.p.m.), and the flavonoids were extracted
subsequently with ethylacetate (twice 500 lLper2mL
incubation) and isolated by successive cellulose thin-layer
chromatography in 15% aqueous acetic acid (solvent
system I) and trichloromethane/acetic acid/water 10 : 9 : 1
(v/v/v) (solvent system II). The developed cellulose plates
were dried for 2 h in a cold air stream, the substrate
(dihydroquercetin) and product (quercetin) were spotted by
absorbance at 366 nm, and the product was extracted with
methanol. The solution was filtered and concentrated for
EI-MS and MALDI-TOF-MS analyses.
The EI-MS were recorded on a Finnigan MAT 70S mass
spectrometer by direct inlet and an accelerating voltage of
6 kV at injection temperatures of 130 °C, 250 °Cor280 °C.
Product identification was also accomplished on a Bruker
Reflex MALDI-TOF-MS in the positive ion reflectron
mode using an accelerating voltage of 23 kV. The mass
spectra were analyzed over a range of m/z 50–750, and
the [M + H]
+
ions of a-cyano-4-hydroxycinnamic acid
(a-HCCA) and 2,5-dihydroxybenzoic acid (DHB) were
employed for the internal calibration across the mass range.

RESULTS
Heterologous expression of
Citrus
FLS
The unequivocal assignment of genes requires the functional
characterization of the corresponding polypeptides, which
has been occasionally neglected in the process of recent gene
bank accessions. FLS cDNAs were accessed from several
plant species (Petunia hybrida, Arabidopsis thaliana,
Solanum tuberosum, Eustoma grandiflorum, Malus domestica
and Matthiola incana), but functionality was only verified in
case of Petunia and Arabidopsis [15,16]. In the course of our
research on Rutaceae [41–43], we became interested in a
clone from C. unshiu recently designated as FLS [30].
Accordingly, this cDNA containing an ORF of 1005 bp
was ligated into the pQE6 vector (FLS-pQE6 construct),
expressed in E. coli, and the product was purified under
conditions that had been successfully employed for the
isolation of another labile 2-oxoglutarate-dependent diox-
ygenase from Petunia [8]. The cDNA from Citrus encoded
a 335-residue polypeptide, which was extracted from the
induced, recombinant bacteria with potassium phosphate
buffer at pH 5.5, and the crude extract was fractionated by
successive size exclusion and ion exchange chromatogra-
phies at pH 7.5. This rapid protocol was very efficient and
yielded elution profiles for the recombinant Citrus enzyme
resembling those of the Petunia FHT [8]. The apparently
homogeneous FLS eluted from the anion exchanger was
used for spectroscopy, the generation of antibodies and
activity assays.

Polypeptide analysis
The homogeneous Citrus enzyme revealed only one band of
about 38 kDa on SDS/PAGE separation (Fig. 2), which
correlated to the molecular mass of 37 899 Da calculated
for the translated polypeptide. Furthermore, partial N-ter-
minal sequencing of the polypeptide yielded a sequence,
Met-Glu-Val-Glu-Arg-Val-Gln-Ala-Ile-Ala-Ser-Leu-Ser-His,
identical to the N-terminal 14 amino acids translated from
the FLS-pQE6 construct. Moreover, the pure polypep-
tide was subjected to MALDI-TOF-MS which revealed a
molecular ion at 37888 ± 40 fully matching the mass
Fig. 2. SDS/PAGE separation of recombinant Citrus FLS. Crude
extracts in phosphate buffer at pH 5.5 of E. coli expressing the FLS
(lane 1) were subjected to 40–50% ammonium sulfate fractionation
followed by size-exclusion (lane 2) and anion-exchange (lane 3) chro-
matographies. The proteins were separated in 5% stacking and 12.5%
separation gels and stained with Coomassie Brilliant Blue R250.
Commercial molecular mass markers (lane M) served for calibration.
Ó FEBS 2002 Flavonol synthase (Eur. J. Biochem. 269) 4137
calculated for the translation product. These data corro-
borated the integrity of the recombinantly expressed
polypeptide, an essential prerequisite for further structural
investigations. A polyclonal antiserum to the pure recom-
binant polypeptide was raised in rabbit, which recognized
one protein band of 38 kDa in Western blots of crude
enzyme extracts. The homogeneous Citrus FLS was
subjected to CD spectroscopy in order to substantiate its
structural relationship with mechanistically related enzymes.
The CD profile revealed a characteristic double minimum at
222 nm and 208–210 nm and a maximum at 191–193 nm,

which indicated the presence of extended a helical regions
interrupted by b sheet strands. Very similar profiles had
been recorded for Streptomyces IPNS [28,29] and Petunia
FHT [9].
Catalytic activity
The enzymatic activity of the recombinant protein was
examined in FLS incubations employing dihydroquercetin
or dihydrokaempferol as a substrate. Both these flavanols
were accepted, and the reaction products were identified as
the flavonols quercetin and kaempferol, respectively, by
their mobility on RP-HPLC and cellulose-TLC in compar-
ison to authentic reference samples. Thoroughly purified
dihydroquercetin was additionally employed for preparative
incubations, and the product was identified as quercetin by
EI-MS and MALDI-TOF-MS showing the molecular ion
at m/z 302. The conversion of flavanols to flavonols
depended on the presence of ferrous iron and 2-oxogluta-
rate, thus establishing that the clone from Citrus unshiu
encoded the 2-oxoglutarate-dependent dioxygenase FLS.
Dihydroquercetin is commercially available and seems to
be the predominant substrate for Citrus unshiu flavonol
biosynthesis [30]. Therefore, this substrate was employed in
order to define the optimal assay conditions. At aerobic and
saturating conditions for ferrous iron and 2-oxoglutarate,
the rate of conversion depended exclusively on the substrate
concentration. Maximal activity was observed at pH 5.0
(Fig. 3) and over a temperature range from 35 to 40 °C.
Under these conditions, the apparent K
m
values were

determined at 272, 11 and 36 l
M
for dihydroquercetin, Fe
II
and 2-oxoglutarate, respectively. Reexamination of the
conversion rate of dihydrokaempferol under same condi-
tions, however, revealed an apparent K
m
at 45 l
M
and, thus,
dihydrokaempferol as the preferred Citrus FLS substrate
in vitro. Nevertheless, rutin (quercetin 3-O-rutinoside) was
identified as the major flavonol in satsuma mandarin plants
[30].
Sequence analysis and mutagenesis
The alignment of the polypeptide sequences of 2-oxogluta-
rate-dependent dioxygenases and related enzymes retrieved
from data banks (59 sequences total) revealed only 8 strictly
conserved amino-acid residues which cluster in three regions
of high overall similarity (Fig. 4). Three of these residues
(His221, His277 and Asp223; the numbering refers to the
Citrus FLS sequence) are essential for the coordination of
ferrous iron as had been demonstrated with Petunia FHT by
kinetic and mutational studies [7] as well as with Aspergillus
IPNS by X-ray diffraction of the Fe
II
-IPNS complex [24,25].
A further residue (Arg287) is involved in 2-oxoglutarate
binding as had been proved with Petunia FHT [7,9] and by

X-ray diffraction of the Streptomyces clavuligerus DAOCS
complexed with Fe
II
and oxoglutarate [23]. However, no
particular function was assigned to the additional four
conserved residues (Gly68, His75, Pro207, Gly261). This is
compatible with the observation that the mutation of His78
in P. hybrida FHT, corresponding to His75 in FLS, only
had a marginal effect on the enzyme activity [7].
On the assumption that Gly68, Pro207 or Gly261 might
be required for structural integrity of the active FLS, point
mutations were initiated aiming at the substitution of
glycine residues by alanine or proline and the exchange of
proline by glycine. Glycine residues are frequently found at
the C-terminal ends of a helices providing the conforma-
tional flexibility required at these sites of the polypeptide
backbone. Accordingly, at least the substitution of glycine
residues by proline was expected to interfere with the folding
process of the FLS polypeptide. Conversely, the Pro207fi
Gly mutation should increase the degree of structural
freedom.
Five mutants (Gly68fiAla, Gly68fiPro, Pro207fiGly,
Gly261fiAla, Gly261fiPro) were generated, cloned into
the expression vector pQE6 and expressed in E. coli strain
M15prep4 as described for the wild-type FLS cDNA.
Examination of crude cell-free extracts or of the solubi-
lized pellet of E. coli expressing wild-type and the mutant
Gly68fiAla, Pro207fiGly or Gly261fiAla FLSs by
Fig. 3. Relative activity of recombinant Ci t rus
FLS depending on the pH of the assay. The

enzyme assays were performed in 200 m
M
buffers composed of glycine/HCl pH 2.0–3.5,
sodium acetate pH 4.5–5.5, potassium phos-
phate pH 5.0–7.5, Bis-Tris/HCl pH 6.5–7.0,
Tris/HCl pH 7.0–8.5 or sodium glycinate
pH 8.5–10.0.
4138 F. Wellmann et al. (Eur. J. Biochem. 269) Ó FEBS 2002
SDS/PAGE and Western blotting revealed no differences
in the mobilities of the wild-type and mutant FLS
polypeptides (Fig. 5). Replacement of either glycine resi-
due by alanine reduced the enzyme activity below 10% of
control, while the substitution in Pro207fiGly did not
affect the FLS activity to a significant extent (Table 2).
Extraction of the mutants Gly68fiPro and Gly261fiPro,
however, failed to yield immunoreactive FLS polypeptide
in the soluble supernatant. Considerable amounts of the
immunopositive polypeptide were recovered from the
solubilized bacterial pellets, which showed no change in
relative mobility on SDS/PAGE separation (Fig. 5).
Nevertheless, this fraction had completely lost the FLS
activity, presumably as the result of major structural
changes.
DISCUSSION
Plants of the Rutaceae family are a rich source of flavonol
glycosides such as the abundant rutin (quercetin rutinoside)
which had been described initially from Ruta species.
Flavonols originate from flavanones, i.e. (2S)-naringenin,
by the consecutive action of FHT and FLS (Fig. 1), and
both of these enzymes use molecular oxygen for catalysis

and are referred to as 2-oxoglutarate-dependent dioxygen-
ases [18–21]. These types of dioxygenases are encoded by
genes of moderate to high sequence identity (from 19% to
75%), which might catalyse very diverse reactions irrespect-
ive of their relative degree of sequence conservation.
Nevertheless, many of these enzymes expressed so far adopt
a highly homologous jelly roll topology [44]. Therefore,
Fig. 4. Alignment of the FLS polypeptide from C. unshiu (FLS-Cit) with the FLS consensus sequence derived from the FLS polypeptide sequences of
P. hybrida, E. gr andiflorum, M. d omestica, S. tube rosum and A. tha liana. The consensus sequence is composed of the identical (marked by asterisks)
or the most abundant amino acids with conservative exchanges (marked by dots). Residues of equivalent hydropathy (STPAG or NDEQ) were
replaced by an x, and gaps were introduced for maximal alignment. Three regions of high similarity were inferred from 59 data base accessions
(National Library of Medicine, NIH or EMBL library) of 2-oxoglutarate-dependent and related enzymes, which include five FLSs as above, 18
FHTs (Petunia hybrida, Zea mays, Hordeum vulgare, Malus sp., Matthiola incana, Vitis vinifera, Citrus sinensis, Daucus carota, Dianthus caryo-
phyllus, Callistephus chinensis, Chrysanthemum morifolium, Anthirrhinum majus, Bromhaedia finlaysonia, Arabidopsis thaliana, Persea americana,
Ipomea purpurea, Ipomea nil and Medicago sativa), three anthocyanidin synthases (Zea mays, Anthirrhinum majus and Oryza sativa), five gibberellin
C20 oxidases (Arabidopsis thaliana, Cucurbita maxima, Pisum sativum, Spinacia oleracea and Marah macrocarpa), hyoscyamine 6b-hydroxylase
from Hyoscyamus niger, the iron-deficiency-specific proteins 2 and 3 from Hordeum vulgare; desacetoxyvindoline-4-hydroxylase from Catharanthus
roseus, 11 aminocyclopropane-1-carboxylic-acid oxidases (Actinidia deliciosa, Arabidopsis thaliana, Brassica juncea, Dianthus caryophyllus, Lyco-
persicum esculentum I and II, Malus domestica, Nicotiana tabacum, Persea americana, Petunia hybrida and Pisum sativum), 11 isopenicillin
N-synthases (Acremonium chrysogenum, Flavobacterium sp., Lysobacter lactamgenus, Nocardia lactamdurans, Streptomyces cattleya, Streptomyces
clavuligerus, Streptomyces jumonjinensis, Streptomyces lipmanii, Aspergillus nidulans, Cephalosporium acremonium and Penicillium chrysogenum),
deacetoxycephalosporin C synthase and deacetylcephalosporin C synthase from Streptomyces clavuligerus. These regions are underlined, and four
amino acids shown to ligate the ACV substrate in IPNS [25] and Fe
II
in IPNS [24] and FHT [7] as well as 2-oxoglutarate in FHT [7] are highlighted
in red and green. The additional four conserved amino acids (Gly68, His75, P207, Gly261) of unknown function [7] are shaded.
Ó FEBS 2002 Flavonol synthase (Eur. J. Biochem. 269) 4139
sequence alignments per se support only a preliminary
functional assignment. In the course of our studies on Ruta
graveolens secondary metabolism [41–43], the report of a

cDNA from C. unshiu [30] assigned to FLS appeared
relevant and led us to express and characterize this
enzyme for comparison with dioxygenases from other
sources [18–21].
Plant 2-oxoglutarate-dependent dioxygenases, unfortu-
nately, are commonly rather labile enzymes which might be
digested partially after heterologous expression in E. coli [8],
and this hampers their functional characterization. Based
on our previous experience [7–10], the full size Citrus FLS
was expressed and rapidly purified to a homogeneous
polypeptide of 38 kDa (Fig. 2) corresponding to 335
amino-acid residues. The identity of the recombinant
enzyme was verified by FLS assays employing dihydroqu-
ercetin or dihydrokaempferol as a substrate (Table 2), and
antibodies raised to the pure polypeptide detected exclu-
sively the FLS polypeptide in crude extracts of recombinant
E. coli (Fig. 5). FLS cDNAs had been reported before from
P. hybrida [13], A. thaliana [16], E. grandiflorum, S. tubero-
sum [17], M. domestica and M. incana, and the translated
polypeptides share about 85% sequence similarity with the
Citrus FLS with 158 identical residues (47%). The differ-
ences in the FLS sequences were mostly confined to the
N-terminal portion (approx. 38% identity), while 62%
identity was observed for the C-terminus (amino-acid
residues 200–335). Surprisingly, the kinetic data revealed a
much higher affinity of the recombinant enzyme to dihydro-
kaempferol as compared to dihydroquercetin, although
the satsuma mandarin accumulates mainly the quercetin
3-O-rutinoside (rutin) [30]. This discrepancy might suggest
the expression of more than one FLS in C. unshiu.

Polypeptide alignments of FLSs with plant 2-oxogluta-
rate-dependent dioxygenases of different functionality
(FHT, anthocyanidin synthase, gibberellin C20-oxidase,
hyoscyamine 6b-hydroxylase, barley Ids2, Ids3 and Cath-
aranthus desacetoxyvindoline 4-hydroxylase) as well as with
related 2-oxoglutarate-independent enzymes (ACC oxidase
and microbial DAOCS and IPNS) revealed eight strictly
conserved amino acids in three regions (Fig. 4). These
enzymes probably evolved from a common ancestral gene,
and the essence of most of the conserved amino acids has
been further substantiated by site-directed mutagenesis of
FHT [7,9] and by documentation of ligand binding in
crystalline DAOCS and IPNS complexes [23–25]. The
coordination of Fe
II
is commonly mediated by two histidine
and one aspartate residues (correspondingly His221, His277
Fig. 5. Western blotting of Citr us wild-type and mutant FLSs in crude
extracts of recombinant E. co li. The extracts were filtered through
PD10 columns, the proteins (15 lg per lane) were separated subse-
quently by SDS/PAGE on 5% stacking and 12.5% separation gels and
transferred to polyvinylidene difluoride membranes for immuno-
staining [7]. Soluble extracts of the bacteria expressing the wild-type
FLS (lane 1) or the mutant enzyme Pro207fiGly (lane 3),
Gly261fiAla (lane 5), Gly68fiAla (lane 7), Gly68fiPro (lane 9) and
Gly261fiPro (lane 11), respectively, as well as the solubilized pellet
fractions of these wild-type (lane 2) and mutant bacteria (lanes 4, 6, 8,
10 and 12) were subjected to Western blotting with reference to mo-
lecular mass markers (indicated in the left margin). The Western blots
were developed with goat anti-rabbit IgG conjugated to alkaline

phosphatase and 5-bromo-4-chloro-3-indolyl phosphate as described
elsewhere [7,9]. The relative FLS contents of the supernatant and pellet
of wild-type and Pro207fiGly, Gly261fiAla, Gly68Ala mutant ex-
tracts (lanes 1–8) as well as the Gly68fiPro mutant membrane extract
(lane 10) were comparable, while the amount in the solubilized pellet of
the Gly261fiPro mutant (lane 12) was negligible and the band could
be hardly recognized in the soluble Gly68fiPro and Gly261fiPro
mutant extracts (lanes 9 and 11).
Table 2. Specific activities of the wild-type and the Gly68fiAla, Gly68fiPro, Gly261fiAla, Gly261fiPro and Pro207fiGly mutant flavonol
synthases. Soluble extracts of E. coli expressing wild-type or mutant FLS were filtered through PD10 columns, and the specific activities were
examined under standard assay conditions (360 lL total) using dihydroquercetin or dihydrokaempferol as a substrate. The wild-type activity
reached 0.5 mkatÆkg
)1
on average with either substrate, and the level of expression was equivalent for the wild-type and mutant FLSs except for the
Gly68fiPro and Gly261fiPro mutants as determined by Western blotting (Fig. 5).
FLS
Relative specific FLS activities
with Dihydroquercetin (%) Dihydrokaempferol (%)
Amount of protein in the
standard assay (lg)
Wild-type 100 100 288
Gly68Ala 3.8 6 396
Gly68Pro 0
a
0
a
360
Gly261Ala 7.5 10 252
Gly261Pro 0
a

0
a
288
Pro207Gly 83.7 70 360
a
The solubilized bacterial pellet of these mutants also lacked FLS activity up to 1 mgÆmL
)1
protein.
4140 F. Wellmann et al. (Eur. J. Biochem. 269) Ó FEBS 2002
and Asp223 in Citrus FLS), whereas only in clavaminate
synthase the aspartate is replaced by a glutamate residue
[44], and an arginine residue (Arg287 in Citrus FLS) can be
ascribed to 2-oxoglutarate-binding [7,23]. This arginine is
also conserved in IPNS and ACC oxidase with slightly
different functionalities, binding the substrate carboxylate,
d-(
L
-a-aminoadipoyl)-
L
-cysteinyl-
D
-valine, in IPNS [25] and
presumably ascorbate in ACC oxidase. This left two glycine
and one proline residues unaccounted for (Gly68, Pro207
and Gly261 in Citrus FLS), but these amino acids are
presumed to be required for proper folding of the enzyme
polypeptide. The data obtained by site-directed mutagenesis
supported this assumption, as the substitution of either
glycine residue (Gly68fiAla or Gly261fiAla) reduced the
enzyme activity to only about 5% and the Gly68fiPro or

Gly261fiPro substitution completely abolished the activity.
It is conceivable that these mutations greatly affected the
tertiary structure of the FLS, because upon expression in
E. coli the polypeptides accumulated in inclusion bodies.
The CD spectroscopy of the wild-type FLS revealed an
overall composition of helices and b sheets very similar to
that recorded for FHT [9] or IPNS [28,29]. Unfortunately,
considerable losses of activity occurred on purification of
the enzyme mutants Gly68fiAla or Gly261fiAla, and the
yields were too low for reliable CD spectroscopy. Further
comparison of the Gly68fiPro and Gly261fiPro mutants
was not reasonable, because these FLSs had to be partially
renatured from the membraneous bacterial pellet. Albeit not
absolutely required for activity, the data assign a role to
Gly68 and Gly261 in the FLS functionality.
ACKNOWLEDGEMENTS
The work was supported by the Deutsche Forschungsgemeins-
chaft and Fonds der Chemischen Industrie. We are grateful to
Drs R. Zimmermann and H. Mu
¨
ller (Merck KGaA, Darmstadt) for
EI-MS and MALDI-TOF-MS measurements, to Dr U. Pieper (Institut
fu
¨
r Biochemie, Universita
¨
t Giessen) for CD spectroscopy, and to
Prof E. Wellmann (Institut fu
¨
r Biologie II, Universita

¨
t Freiburg) for
HPLC assays. We would also like to thank Michaela Mu
¨
ller and Olga
Lezrich for their excellent technical assistance.
REFERENCES
1. Deboo, G.B., Albertsen, M.C. & Taylor, L.T. (1995) Flavanone
3-hydroxylase transcripts and flavonol accumulation are tempo-
rally coordinate in maize anthers. Plant J. 7, 703–713.
2. Koes, R.E., Quattrocchio, F. & Mol, J.N.M. (1994) The flavonoid
biosynthetic pathway in plants: function and evolution. Bioessays
16, 123–132.
3. Heller, W. & Forkmann, G. (1994) Biosynthesis of the flavonoids.
In The Flavonoids, Advances in Research Since 1986 (Harborne,
J.B., ed.), pp. 499–536. Chapman & Hall, London.
4. Lukac
ˇ
in,R.,Matern,U.,Junghanns,K.T.,Heskamp,M L.,
Britsch, L., Forkmann, G. & Martens, S. (2001) Purification and
antigenicity of flavone synthase I from irradiated parsley cells.
Arch. Biochem. Biophys. 393, 177–183.
5. Martens, S., Forkmann, G., Matern, U. & Lukac
ˇ
in, R. (2001)
Cloning of parsley flavone synthase I. Phytochemistry 58, 43–46.
6. Martens, S. & Forkmann, G. (1999) Cloning and expression of
flavone synthase II from Gerbera hybrids. Plant J. 20, 611–618.
7. Lukac
ˇ

in, R. & Britsch, L. (1997) Identification of strictly con-
served histidine and arginine residues as part of the active site in
Petunia hybrida flavanone 3b-hydroxylase. Eur. J. Biochem. 249,
748–757.
8. Lukac
ˇ
in, R., Gro
¨
ning,I.,Schiltz,E.,Britsch,L.&Matern,U.
(2000) Purification of recombinant flavanone 3b-hydroxylase from
Petunia hybrida and assignment of the primary site of proteolytic
degradation. Arch. Biochem. Biophys. 375, 364–370.
9. Lukac
ˇ
in, R., Gro
¨
ning,I.,Pieper,U.&Matern,U.(2000)Site-
directed mutagenesis of the active site serine 290 in flavanone
3b-hydroxylase from Petunia hybrida. Eur. J. Biochem. 267, 853–
860.
10. Lukac
ˇ
in, R., Urbanke, C., Gro
¨
ning, I. & Matern, U. (2000) The
monomeric polypeptide comprises the functional flavanone
3b-hydroxylase from Petunia hybrida. FEBS-Letters 467, 353–358.
11. Britsch, L., Heller, W. & Grisebach, H. (1981) Conversion of
flavanone to flavone, dihydroflavonol and flavonol with an
enzyme system from cell cultures of parsley. Z. Naturforsch.

36c, 742–750.
12. Spribille, R. & Forkmann, G. (1984) Conversion of dihydro-
flavonols to flavonols with enzyme extracts from flower buds of
Matthiola incana. Z. Naturforsch. 39c, 714–719.
13.Forkmann,G.,deVlaming,P.,Spribille,R.,Wiering,H.&
Schramm, A.W. (1986) Genetic and biochemical studies on the
conversion of dihydroflavonols to flavonols in flowers of Petunia
hybrida. Z. Naturforsch. 41c, 179–186.
14. Forkmann, G. (1991) Flavonoids as flower pigments: the forma-
tion of the natural spectrum and ist extension by genetic
engineering. Plant Breed 106, 1–26.
15. Holton,T.A.,Brugliera,F.&Tanaka,Y.(1993)Cloningand
expression of flavonol synthase from Petunia hybrida. Plant. J. 4,
1003–1010.
16. Wisman, E., Hartmann, U., Sagasser, M., Baumann, E., Palme,
K.,Hahlbrock,K.,Saedler,H.&Weisshaar,B.(1998)Knock-out
mutants from an En-1 mutagenized Arabidopsis thaliana popula-
tion generate phenylpropanoid biosynthesis phenotypes. Proc.
Natl Acad. Sci. USA 95, 12432–12437.
17. Van Eldik, G.J., Reijnen, W.H., Ruiter, R.K., Van Herpen, M.M.,
Scrauwen, J.A. & Wullems, G.J. (1997) Regulation of flavonol
biosynthesis during anther and pistil development, and during
pollen tube growth in Solanum tuberosum. Plant J. 11, 105–113.
18. Prescott, A.G. (1993) A dilemma of dioxygenases (or where
biochemistry and molecular biochemistry fail to meet). J. Exp.
Bot. 44, 849–861.
19. DeCarolis, E. & DeLuca, V. (1994) 2-oxoglutarate-dependent
dioxygenase and related enzymes: biochemical characterization.
Phytochem. 36, 1093–1107.
20. Prescott, A.G. & John, P. (1996) Dioxygenases: Molecular

Structure and role in plant metabolism. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 47, 245–271.
21. Prescott, A.G. & Lloyd, M.D. (2000) The iron (II) and 2-oxoacid-
dependent dioxygenases and their role in metabolism. Nat. Prod.
Report 17, 367–383.
22. Britsch, L., Dedio, J., Saedler, H. & Forkmann, G. (1993) Mole-
cular characterization of flavanone 3 b-hydroxylases. Consensus
sequence, comparison with related enzymes and the role of con-
served histidine residues. Eur. J. Biochem. 217, 745–754.
23. Valega
˚
rd, K., Van Schelting, T., Lloyd, M.D., Hara, T.,
Ramaswamy,S.,Perrakis,A.,Thompson,A.,Lee,H J.,Baldwin,
J.E., Schofield, C.J., Hajdu, J. & Andersson, I. (1998) Structure of
a cephalosporin synthase. Nature 394, 805–809.
24. Roach, P.L., Clifton, I.J., Fu
¨
lo
¨
p,V.,Harlos,K.,Barton,G.J.,
Hajdu, J., Andersson, I., Schofield, C.J. & Baldwin, J.E. (1995)
Crystal structure of isopenicillin N synthase is the first from a new
structural family of enzymes. Nature 375, 700–704.
25. Roach, P.L., Clifton, I.J., Hensgens, C.M.H., Shibata, N.,
Schofield, C.J., Hajdu, J. & Baldwin, J.E. (1997) Structure of
isopenicillin N synthase complexed with substrate and the
mechanism of penicillin formation. Nature 387, 827–830.
26. Turnbull, J.J., Prescott, A.G., Schofield, C.J. & Wilmouth, R.C.
(2001) Purification, crystallization and preliminary X-ray
Ó FEBS 2002 Flavonol synthase (Eur. J. Biochem. 269) 4141

diffraction of anthocyanidin synthase from Arabidopsis thaliana.
Acta Crystallogr. D 57, 425–427.
27. Zhang, Z., Barlow, J.N., Baldwin, J.E. & Schofield, C.J. (1997)
Metal-catalyzed oxidation and mutagenesis studies on the iron
(II) binding site of 1-aminocyclopropane-1-carboxylate oxidase.
Biochemistry 36, 15999–16007.
28. Borovok, I., Landman, O., Kreisberg-Zakarin, R., Aharonowitz,
Y. & Cohen, G. (1996) Ferrous active site of isopenicillin N syn-
thase: genetic and sequence analysis of the endogenous ligands.
Biochemistry 35, 1981–1987.
29.Durairaj,M.,Leskiw,B.K.&Jensen,S.E.(1996)Geneticand
biochemical analysis of the cysteinyl residues of isopenicillin N
synthase from Streptomyces clavuligerus. Can. J. Microbiol. 42,
870–875.
30. Moriguchi,T.,Kita,M.,Ogawa,K.,Tomono,Y.,Endo,T.&
Omura, M. (2002) Flavonol synthase gene expression during citrus
fruit development. Physiol. Plant. 114, 251–258.
31. Zagursky, R.J. & Berman, M.L. (1984) Cloning vectors that yield
high levels of single-stranded DNA for rapid DNA-sequencing.
Gene 27, 183–191.
32. Kunkel, T.A., Roberts, J.D. & Zakour, R.A. (1987) Rapid and
efficient site-specific mutagenesis without phenotypic selection.
Methods Enzymol. 154, 367–382.
33. Sanger, F., Nicklen, S. & Coulsen, A.R. (1977) DNA sequencing
with chain terminating inhibitors. Proc. Natl Acad. Sci. USA 74,
5463–5467.
34. Sreerama, N. & Woody, R.W. (1993) A self-consistent method for
the analysis of protein secondary structure from circular dichro-
ism. Anal. Biochem. 209, 32–44.
35. Deleage, G. & Geourjon, C. (1993) An interactive graphic pro-

gram for calculating the secondary structure content of proteins
from circular dichroism spectrum. Comput. Appl. Biosci. 9,197–
199.
36. Laemmli, U.K. (1970) Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature 227,
680–685.
37. Towbin, H., Staehelin, T. & Gordon, J. (1979) Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc. Natl Acad. Sci.
USA 76, 4350–4354.
38. Lukac
ˇ
in, R. (1997) Molekulare und Strukturelle Charakterisuring
der Flavanon 3b-Hydroxylase aus Petunia hybrida, einer
2-Oxoglutarrat-abhongigen Dioxygenase der Flavanoidbio-
synthase. Doctoral Thesis, Universita
¨
t Freiburg im Breisgau,
Germany.
39. Sandermann, H. & Strominger, L. (1972) Purification and
properties of C
55
-isoprenoid alcohol phosphokinase from
Staphylococcus aureus. J. Biol. Chem. 247, 5123–5131.
40. Bensadoun, A. & Weinstein, B. (1976) Assay of proteins in the
presence of interfering material. Anal. Biochem. 70, 241–250.
41. Lukac
ˇ
in, R., Springob, K., Urbanke, K., Ernwein, C., Schro
¨

der,
G., Schro
¨
der, J. & Matern, U. (1999) Native acridone synthases I
and II from Ruta graveolens L. form homodimers. FEBS Letters
448, 135–140.
42. Springob, K., Lukac
ˇ
in, R., Ernwein, C., Gro
¨
ning,I.&Matern,U.
(2000) Specificities of functionally expressed chalcone and acri-
done synthases from Ruta graveolens. Eur. J. Biochem. 267, 6552–
6559.
43. Lukac
ˇ
in, R., Schreiner, S. & Matern, U. (2001) Transformation of
acridone synthase to chalcone synthase. FEBS Letters 508,413–
417.
44. Schofield, C.J. & Zhang, Z. (1999) Structural and mechanistic
studies on 2-oxoglutarate-dependent oxygenases and related
enzymes. Curr. Op. Struct. Biol. 9, 722–731.
4142 F. Wellmann et al. (Eur. J. Biochem. 269) Ó FEBS 2002