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
¨

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

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.

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

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,

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

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

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.

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,

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

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

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.

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

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

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.

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

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
¨