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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Institute of Biological Chemistry, Washington State University, PO Box 646340, Pullman, WA 99164-6340, USA
NeweYaar Research Center, Agriculture Research Organization, PO Box 1021, Ramat Yishay 30095, Israel
c
Fondazione Edmund Mach, Centro Ricerca e Innovazione, Department of Food Quality and Nutrition, Via E. Mach, 1 38010 San Michele allAdige (TN), Italy
b
a r t i c l e
i n f o
Article history:
Received 20 February 2014
Received in revised form 10 July 2014
Available online xxxx
Keywords:
Apple
Malus domestica
Rosaceae
p-Coumaroyl-CoA
Feruloyl-CoA
p-Dihydrocoumaroyl-CoA
Dihydrochalcones
Hydroxycinnamoyl-CoA double bond
reductase
a b s t r a c t
The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages.
Many of the health benecial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specic reactions that lead to the biosynthesis of
the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in
the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple
genes with signicant sequence similarity to Arabidopsis alkenal double bond reductases. Herein
described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to
p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3 lM, respectively. The Malus double bond
reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing
catalytic efciencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase
gene revealed that its transcript levels showed signicant variation in tissues of different developmental
stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the
hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the
a,b-unsaturated double bond of p-coumaroyl-CoA, the rst step of dihydrochalcone biosynthesis in apple
tissues, and may be involved in the production of these compounds.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
Polyphenols constitute one of the largest and most diverse classes of specialized (secondary) metabolites in plants. These
metabolites can be divided into non-soluble metabolites, such as
lignins, condensed tannins (proanthocyanidins) and cell-wall
Abbreviations: ACN, acetonitrile; CHS, chalcone synthase; DBR, double bond
reductase; DHC, dihydrochalcone; Enoyl-ACP, enoyl acyl carrier protein; GT,
glycosyltransferase; IPTG, isopropyl-1-thio-b-D-galactopyranoside; m/z [M], mass
spectrometry; MdHCDBR, Malus domestica hydroxycinnamoyl-CoA double bond
reductase; MWCO, molecular mass cut-off; PGT1, phloretin 20 -O-glucosyltransferase; PulR, pulegone reductase; RZS, raspberry ketone/zingerone synthase.
Corresponding author. Tel.: +1 509 335 0550; fax: +1 509 335 7643.
E-mail address: gangd@wsu.edu (D.R. Gang).
1
Contributed equally to this work.
http://dx.doi.org/10.1016/j.phytochem.2014.07.027
0031-9422/ 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027
OH
SCoA
H2N
SCoA
NADPH
NADP+
MdHCDBR
L-Phenylalanine (1)
OH
OH
p-Coumaroyl-CoA (2)
p-Dihydrocoumaroyl-CoA (3)
3 x Malonyl-CoA 3 x Malonyl-CoA
CHS
CHS
CoA-SH
CoA-SH
OH
HO
OH
HO 4
3
OH
6 OH
2
OH
B
1
OH
4
3
Phloretin (5)
CHI
PGT1
OH
HO
OH
OH
Naringenin (6)
[Flavonoids]
OH
HO
O
Glc
Phloridzin (7)
[Dihydrochalcones]
Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027
kDa
130
100
70
55
MdHCDBR
35
25
10
87
98
79
97
72
96
71
93
Fig. 2. Neighbor-joining tree of deduced amino acid sequences of plant double bond reductase (DBR), enoyl-ACP reductase and dehydrogenase proteins. The MdHCDBR
protein identied in this study is highlighted by bold text. The bootstrap method was performed with 1000 replicates.
Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027
5.30
p-dihydrocoumarate (9)
-O
p-coumarate (8)
-O
5.01
relative abundance
165.0555
100
0
145 150 155
100
OH
OH
relative abundance
166.0589
167.0609
160
165
170 175
180
m/z
222.2678
relative abundance
100
5.65
276.2678
100
0
220
4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0
6.2
relative abundance
-O
280
nm
300
ferulate (10)
5.84
196.0685
197.0701
0
180
OCH3
OCH3
185
190
195
200
205
210
215
m/z
100
222.2678
OH
relative abundance
OH
320
195.0652
100
dihydroferulate (9)
-O
260
6.4
5.50
240
relative abundance
0
4.2
279.2678
0
4.2
4.4
4.6
4.8
5.0
5.2 5.4
Time (min)
5.6
5.8
6.0
6.2
6.4
220
240
260
nm
280
300
320
Fig. 4. Enzymatic activity of the recombinant MdHCDBR with p-coumaroyl-CoA (2) and feruloyl-CoA as substrate, after products (and left over reactants) were hydrolyzed by
NaOH as outlined in the text. Total ion chromatograms for reaction mixtures with p-coumaroyl-CoA (2) (A) and feruloyl-CoA (D) as substrates. Mass spectra of product peaks
matched the expected m/z values for p-dihydrocoumarate (9) (B) and dihydroferulate (E). Inline UV analysis of products showed spectra as expected for p-dihydrocoumarate
(9) (C) and dihydroferulate (F). S: substrate; P: product. Relative abundance is shown in arbitrary units.
Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027
0
0
600
80
60
40
20
0
0
-1
pkatmg protein
60
50
40
30
20
-1
pkatmg protein
0.4
f
f
f
g
0.2
0
1.0
PGT1
0.8
f
0.6
c
0.4
f,e
c
f,g
0.2
d
g
10
0
0
d
c
C
Bu los
ds ed
80 bu
% d
O o s
pe pe
n n
bu
ds
pkatmg-1 protein
B 100
es
in
Sk sta
in ge
Sk sta 1
in ge
Sk sta 2
in ge
Sk sta 3
in ge
st 4
ag
e
Fl
5
es
h
Fl s
es ta
g
Fl h s e 1
es ta
ge
h
Fl s
es ta 2
g
Fl h s e 3
es ta
h ge
st 4
ag
e
5
10
0.8
0.6
DBR
e
av
20
Le
30
1.0
Sk
40
pkatmg-1 protein
500
2500
60
50
40
30
20
10
0
0
10
20
30
Temperature (C)
40
50
Table 1
Kinetic parameters of MdHCDBR, obtained using the His-tagged and afnity
chromatography puried protein.
Substrate
Km (lM)
p-Coumaroyl-CoA
Feruloyl-CoA
NADPH
96.6 5.9
92.9 1.5
101.3 2.8
47.7 2.4
102.9 2.6
56.4 0.3
1.9 0.1
4.2 0.1
2.3 0.0
dihydrocoumaroyl-CoA (3) synthesis. Moreover, none of these species are known to accumulate DHCs. Thus, it is unknown how
widespread this catalytic function may be in plants or whether it
is always catalyzed by an enzyme belonging to the specic
dehydrogenase subfamily that contains the MdHCDBR gene.
2.3. Tissue specic expression of MdHCDBR
The level of expression of the MdHCDBR gene was examined by
qRT-PCR of leaf, ower and fruit tissues of M. domestica Golden
Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027
Egoi
DCt;ref
Eref
where the E is the PCR efciency, goi the gene of interest and ref a
reference gene (Pfaf, 2001). Gene expression was analyzed using
analysis of variance (One-way ANOVA) between different ripening
stages, followed by Tukeys test into homogeneous subsets. Data
are expressed as mean standard deviation. The signicance level
was set at 5%. Statistical analysis was performed using Statistica
software version 9.1 (StatSoft Inc., 2010). STATISTICA (data analysis
software system), version 9.1. http://www.statsoft.com). All analyses were performed using three technical replicates.
4.6. Enzyme activity measurements
Initial reactions were carried out in 50 mM TrisHCl pH 7.5 and
contained 1020 lg His-tag puried (high purity level) recombinant protein, 2 mM NADPH, and 500 lM p-coumaroyl-CoA (2).
Stock buffers for determination of pH optimum were prepared by
mixing 0.5 M citric acid and 1 M dibasic sodium phosphate solutions. Final pH values ranged between 4.0 and 7.6 with an interval
Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027
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Please cite this article in press as: Ibdah, M., et al. Identication and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase
involved in dihydrochalcone formation in Malus domestica Borkh. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.027