Molecular Characterization and Phylogenetic Analysis of Two Novel Regio-specific Flavonoid Prenyltransferases from Morus alba and Cudrania tricuspidata

Article abstract of DOI:10.1074/jbc.M114.608265

Prenylated flavonoids are attractive specialized metabolites with a wide range of biological activities and are distributed in several plant families. The prenylation catalyzed by prenyltransferases represents a Friedel-Crafts alkylation of the flavonoid skeleton in the biosynthesis of natural prenylated flavonoids and contributes to the structural diversity and biological activities of these compounds. To date, all identified plant flavonoid prenyltransferases (FPTs) have been identified in Leguminosae. In the present study two new FPTs, Morus alba isoliquiritigenin 3′-dimethylallyltransferase (MaIDT) and Cudrania tricuspidata isoliquiritigenin 3′-dimethylallyltransferase (CtIDT), were identified from moraceous plants M. alba and C. tricuspidata, respectively. MaIDT and CtIDT shared low levels of homology with the leguminous FPTs. MaIDT and CtIDT are predicted to be membrane-bound proteins with predicted transit peptides, seven transmembrane regions, and conserved functional domains that are similar to other homogentisate prenyltransferases. Recombinant MaIDT and CtIDT were able to regioselectively introduce dimethylallyl diphosphate into the A ring of three flavonoids with different skeleton types (chalcones, isoflavones, and flavones). Phylogenetic analysis revealed thatMaIDT and CtIDT are distantly related to their homologs in Leguminosae, which suggests that FPTs in Moraceae and Leguminosae might have evolved independently. MaIDT and CtIDT represent the first two non-Leguminosae FPTs to be identified in plants and could thus lead to the identification of additional evolutionarily varied FPTs in other non-Leguminosae plants and could elucidate the biosyntheses of prenylated flavonoids in various plants. Furthermore, MaIDT and CtIDT might be used for regiospecific prenylation of flavonoids to produce bioactive compounds for potential therapeutic applications due to their high efficiency and catalytic promiscuity.

Full text of DOI:10.1074/jbc.M114.608265

Plant Biology:
Molecular Characterization and
Phylogenetic Analysis of Two Novel
Regio-specific Flavonoid Prenyltransferases
from Morus alba and Cudrania tricuspidata
Ruishan Wang, Ridao Chen, Jianhua Li, Xiao
Liu, Kebo Xie, Dawei Chen, Yunze Yin,
Xiaoyu Tao, Dan Xie, Jianhua Zou, Lin Yang
and Jungui Dai
J. Biol. Chem. published online October 31, 2014
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JBC Papers in Press. Published on October 31, 2014 as Manuscript M114.608265
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.608265
Moraceous flavonoid prenyltransferases
Molecular Characterization and Phylogenetic Analysis of Two Novel Regio-specific Flavonoid
Prenyltransferases from Morus alba and Cudrania tricuspidata*Ⓢ
Ruishan Wang, Ridao Chen, Jianhua Li, Xiao Liu, Kebo Xie, Dawei Chen, Yunze Yin, Xiaoyu Tao,
Dan Xie, Jianhua Zou, Lin Yang, and Jungui Dai 1
1From the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, 1 Xian Nong
Tan Street, Beijing 100050, China
*Running Title: Moraceous flavonoid prenyltransferases
1To whom correspondence should be addressed: State Key Laboratory of Bioactive Substance and
Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College & Chinese
Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, China. Tel.: 86-10-6316-5195;
Fax: 86-10-6301-7757; E-mail: jgdai@imm.ac.cn
Keywords: flavonoid; flavonoid prenyltransferase; gene expression; plant biochemistry; molecular
evolution; phylogenetics; regiospecificity; promiscuity; Morus alba; Cudrania tricuspidata
Background: Plant flavonoid prenyltransferases
(FPTs) transfer prenyl moiety to flavonoid cores
and have previously been identified only in
Leguminosae.
prenylated flavonoids and contributes to the
structural diversity and biological activities of
these compounds. To date, all identified plant
flavonoid prenyltransferases (FPTs) have been
identified in Leguminosae. In the present study,
two new FPTs, MaIDT and CtIDT, were
identified from moraceous plants Morus alba
and Cudrania tricuspidata, respectively. MaIDT
and CtIDT shared low levels of homology with
the leguminous FPTs. MaIDT and CtIDT are
predicted to be membrane-bound proteins with
Results: The newly identified moraceous FPTs,
MaIDT and CtIDT, are distantly related to
leguminous
FPTs
and
feature
catalytic
regioselectivity and promiscuity.
Conclusion: MaIDT and CtIDT evolved
independently from leguminous FPTs.
Significance: These findings are valuable for
identifying additional evolutionarily different
non-Leguminosae FPTs.
predicted
transit
regions
peptides,
and
seven
transmembrane
conserved
functional domains that are similar to other
homogentisate prenyltransferases.
Recombinant MaIDT and CtIDT were able to
regioselectively introduce dimethylallyl
ABSTRACT
Prenylated flavonoids are attractive
specialized metabolites with a wide range of
biological activities and are distributed in
several plant families. The prenylation
diphosphate (DMAPP) into the A ring of three
flavonoids with different skeleton types
catalyzed
by
prenyltransferases
(PTs)
(chalcones,
isoflavones
and
flavones).
represents a Friedel–Crafts alkylation of the
Phylogenetic analysis revealed that MaIDT and
CtIDT are distantly related to their homologs in
flavonoid skeleton in the biosynthesis of natural
1
Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Moraceous flavonoid prenyltransferases
structural diversity and further modification of the
prenylated flavonoids (7). For example,
prenylation is a key step in the biosynthesis of
diverse Diels-Alder-type adducts in mulberry trees
(8, 9). Consequently, intensive biochemical studies
of FPTs have been performed for over two decades
(10, 11). However, the genes coding for these
prenyltransferases (PTs) have not been isolated
from plants until the recent identification of the
first flavonoid-specific PT gene SfN8DT-1 from
Sophora flavescens, which is responsible for the
specific prenylation of the flavanone naringenin at
C-8 (12). Subsequently, further progress was
achieved in the molecular biological and
biochemical investigations of plant FPTs, and
additional relevant genes have been cloned and
functionally characterized (Table 1). Moreover,
due to their substrate promiscuity in vitro, some
soluble types of aromatic PTs from microbes have
been demonstrated to be capable of catalyzing the
prenylation of flavonoids; e.g., 7-DMATS, a
member of DMATS superfamily from Aspergillus
fumigatus (18, 19), and some PTs of the
CloQ/NphB group from the actinomycetes
Streptomyces (20, 21).
Leguminosae, which suggests that FPTs in
Moraceae and Leguminosae might have
evolved independently. MaIDT and CtIDT
represent the first two non-Leguminosae FPTs
to be identified in plants and could thus lead to
the identification of additional evolutionarily
varied FPTs in other non-Leguminosae plants
and could elucidate the biosyntheses of
prenylated flavonoids in various plants.
Furthermore, MaIDT and CtIDT might be used
for regiospecific prenylation of flavonoids to
produce bioactive compounds for potential
therapeutic applications due to their high
efficiency and catalytic promiscuity.
Prenylated flavonoids are an attractive class
of plant-specialized metabolites that are primarily
distributed in Leguminosae (Fabaceae), Moraceae,
Umbelliferae,
Guttiferae,
Euphorbiaceae,
Celastraceae, Compositae, Paulowniaceae and
Zingiberaceae (15). These compounds are hybrid
molecules that contain structurally divergent
flavonoid skeletons and extensively modified
prenyl moieties with different chain lengths, and
these compounds exhibit a wide range of
biological activities; e.g., antioxidative, antitumor,
antibacterial, antiviral, and estrogenic activities (1,
3). Notably, the substitution of the flavonoid ring
system with prenyl group(s) contributes strongly
to these biological activities because the prenyl
group(s) increases the lipophilicity of the
flavonoids and confers to these molecules a strong
affinity to biological membranes that results in
greater bioavailability (6).
However, to date, all of the functionally
characterized plant FPTs have been cloned from
Leguminosae (Table 1). The genes, phylogeny (in
relation to the reported FPTs), and substrate
spectra (specificity or promiscuity) of the
non-Leguminosae FPTs remain enigmatic. These
questions prompted us to search for such enzymes.
Moraceous plants are rich sources of
isoprenoid-substituted phenolic compounds and
their Diels-Alder-type adducts (22, 23). In our
previous work, cell suspension cultures of two
closely related moraceous trees, M. alba (mulberry)
and C. tricuspidata, were employed to prenylate
different types of flavonoids, and the same
bioconversions were observed when microsomes
of the cell cultures were used (24). Further
Due to their diverse chemical structures and
impressive biological activities, prenylated
flavonoids have been studied in many fields
including plant physiology, natural product
chemistry, and synthetic chemistry. The
prenylation reactions catalyzed by flavonoid
prenyltransferases (FPTs) have received particular
attention and significantly contribute to the
2

Moraceous flavonoid prenyltransferases
chemical investigations confirmed that the
prenylated flavonoids formed the main chemical
compositions of both of the two plant cell
suspension cultures, and that Diels-Alder-type
adducts were present in large amounts in the cell
suspension cultures of M. alba (25, 26). These
observations indicated that FPTs existed in the cell
suspension cultures of each of the two plants.
Therefore, in our ongoing work on plant FPTs (14,
15), cell suspension cultures of M. alba and C.
tricuspidata were selected as materials from which
to isolate non-Leguminosae FPTs.
(v/v) methanol in water over a period of 20 min.
Plant Materials—Cell suspension cultures of
M. alba were maintained in Murashige and
Skoog’s medium with 1.0 mg/L -naphthalene
acetic acid (NAA), 0.5 mg/L 6-benzylaminopurine
(6-BA), and 0.2 mg/L 2, 4-dichlorophenoxy acetic
acid (2, 4-D). Cell suspension cultures of C.
tricuspidata were maintained in 6, 7-V medium
supplemented with 0.5 mg/L NAA, 0.2 mg/L
6-BA, and 0.1 mg/L 2, 4-D as described
previously (24). The cell cultures were
sub-cultured every 15 days.
In
this
report,
we
describe
the
Chemicals—The prenyl donors dimethylallyl
diphosphate (DMAPP), geranyl diphosphate
(GPP), farnesyl diphosphate (FPP), geranylgeranyl
diphosphate (GGPP), and phytyl diphosphate (PPP)
were chemically synthesized as described
previously (27). Chalcones 24 were prepared
according to the literatures (28, 29). The other
characterization of two new FPTs, MaIDT and
CtIDT, from the cell suspension cultures of M.
alba and C. tricuspidata, respectively. As the first
identified non-Leguminosae FPTs, these FPTs
share fairly low identities with their homologs in
Leguminosae and feature catalytic regioselectivity
and promiscuity. These findings not only imply the
diversity of FPTs in the plant kingdom, but may
also illuminate the discoveries of other FPTs in
non-Leguminosae plants.
tested
substrates
were
purchased
from
Sigma-Aldrich (St. Louis, MO, USA) and
BioBioPha (Kunming, Yunnan, China).
Isolation of cDNAs Homologous to AtVTE2-1
from M. alba—Ten-day-old M. alba cell cultures
were treated with 0.1 mM methyl jasmonate (MJ)
for 20 h, and total RNA was subsequently
extracted with an E.Z.N.A.TM Plant RNA Kit
(Omega Bio-tek Inc., Doraville, GA, USA) and
reverse-transcribed (RT) using a SMARTer™
RACE cDNA Amplification Kit (Clontech Inc.,
Mountain View, CA, USA). The RT products were
subjected to rapid amplification of cDNA ends
(RACE) according to the manufacturer’s protocol.
The 3′ ends of MaIDT was obtained using the
EXPERIMENTAL PROCEDURES
General Experimental Procedures—1H and
13C NMR spectra were recorded on Varian NMR
System 600 spectrometers (Varian Inc., Palo Alto,
CA, USA). The HPLC-UV/ESI-MS analyses of
the enzymatic products were performed as
previously described (15) with the exception that
the solvent system consisted of a linear gradient
from 50% to 100% (v/v) methanol in water with
0.1% formic acid over a period of 30 min followed
by an isocratic elution with 100% methanol for 10
min and that the UV detector was set at 350 nm or
265 nm. For the isolation of the enzymatic
products, an approach similar to that previously
described was employed (15) with the exception
that the semi-preparative reverse-phase HPLC was
primer
EST-3′GSP
(5′-TGATGCCGATATTGACAGGATAAATAAG
CC-3′) that was specific for an expressed sequence
tag (EST) sequence of the mulberry root
(GenBank accession no. GT734921), which is
homologous to AtVTE2-1 (GenBank accession no.
performed using a linear gradient from 50% to 100% AY089963). The 5′ ends of MaIDT were
3

Moraceous flavonoid prenyltransferases
re-isolated by RT-PCR using the primer
MaIDT-5′GSP
Heterologous Expression in Yeast—The
expression vectors pESC-HIS-MaIDT and
pESC-HIS-CtIDT were introduced into the yeast
strain YPH499. The microsomal fractions of
YPH499 expressing pESC-HIS-MaIDT and
pESC-HIS-CtIDT were prepared as previously
described (14, 15) and re-suspended in 100 mM
Tris-HCl (pH 9.0). The total protein concentration
was determined by the Bradford method (30).
(5′-AGAGGAGGAGCAGAATAGAAGTGGGCC
A-3′). Its full-length clone was acquired by
RT-PCR using the gene-specific primer pairs
MaIDT-Fw
ATGGAGCTCTCAATCTCTCACTCT-3′)
MaIDT-Rv (5′-GCGGCCGC
(5′-GAATTC
and
TTATATGAAAGGAAATATGACAAACTCCA-3′
). In these sequences, the restriction sites for
subcloning are underlined. The PCR products were
cloned into pEASY-Blunt Simple vector
(TransGen Biotech Co., Ltd., Beijing, China) for
sequencing and then subcloned into pESC-HIS
vector (Stratagene Inc., La Jolla, CA, USA), which
Prenyltransferase
Activity—For
the
quantitative determination of the PT activity, the
basic reaction mixture (100 µL) contained 100
mM Tris-HCl (pH 9.0), 10 mM MgCl2, 200 µM
prenyl acceptor, 400 µM DMAPP and 100 µg
protein of the recombinant yeast microsome. The
reaction mixtures were incubated at 30 °C, and the
reactions were terminated by the addition of 200
µL of methanol. The protein was removed by
centrifugation at 14,000 × g for 20 min. The
resulted
in
the
expression
construct
pESC-HIS-MaIDT.
Isolation of cDNAs Homologous to MaIDT
from C. tricuspidata—Ten-day-old C. tricuspidata
cell cultures were treated with 0.1 mM methyl
jasmonate (MJ) for 20 h, and total RNA was
subsequently extracted with an E.Z.N.A.TM Plant
RNA Kit (Omega Bio-tek Inc., Doraville, GA,
USA) and reverse-transcribed as mentioned above.
The CtIDT fragment was obtained using the
enzymatic
products
were
analyzed
by
HPLC-UV/ESI-MS under the conditions described
above. For the quantitative measurements of the
enzyme activity, three parallel assays were carried
out routinely, and the values are obtained in
duplicate for each assay.
degenerated
primer
pairs
YZ-1
and
Biochemical Properties of the Recombinant
Enzymes—The assays for the determinations of
the kinetic parameters (100 µL) of the prenyl
acceptors contained 400 µM DMAPP, 100 µg
protein of the recombinant yeast microsome, and
prenyl acceptors at final concentrations of 5, 10,
20, 40, 80, 160 and 400 µM. For the
determinations of the kinetic parameters of
DMAPP, isoliquiritigenin (1) at 400 µM and
DMAPP at final concentrations of 5, 10, 20, 40, 80,
160 and 400 µM were used. The incubation time
was 30 min. The apparent Km values were
calculated from Lineweaver-Burk plots using the
(5′-TAAAYAAGCCKTATYTACCTAT-3′)
YZ-2 (5′-CMAAATGKAAKTCCAAGGG-3′).The
complete coding sequence of CtIDT was obtained
with 5′- and 3′-RACE with the internal
gene-specific
(5′-GAGCTCTCACTTAAGCAAGCATGGTT-3′)
and CtIDT-5′GSP
primers
CtIDT-3′GSP
(5′-GTGAAGAATTCCGGCCATCAGAGGAT-3′).
The full-length CtIDT was acquired using the
gene-specific
(5′-GCGGCCGC ATGGCGTTCTCAATCTC-3′)
and CtIDT-Rv (5′-ATCGAT
primer
pairs
CtIDT-Fw
CTATATAAAGGGAAAAATGACAAATTC-3′).
The expression vector pESC-HIS-CtIDT was
constructed as described above.
Hyper32
software
(http://homepage.ntl
world.com/john.easterby/hyper32.html).
To investigate the optimal pH, the enzyme
4

Moraceous flavonoid prenyltransferases
reactions were performed in reaction buffers with
pH values in the range of 6.0 to 8.0 (sodium
phosphate buffer), 8.0 to 10.0 (Tris-HCl buffer)
and 10.0 to 11.0 (Caps-NaOH buffer) at 30 °C. To
assay the optimal reaction temperature, the
reaction mixtures were incubated at eight different
temperatures that ranged from 4 to 50 °C in 100
mM Tris-HCl buffer (pH 9.0). To test the
requirement of PT activity for divalent cations,
MgCl2, BaCl2, CaCl2, FeCl2, CoCl2, CuCl2, NiCl2,
and MnCl2 were individually used with DMAPP
and isoliquiritigenin (1) in 100 mM Tris-HCl
buffer (pH 9.0) at 30 °C.
165.3 (C-2'), 114.5 (C-3'), 162.7 (C-4'), 108.1
(C-5'), 130.4 (C-6'), 22.4 (C-1''), 123.4 (C-2''),
131.6 (C-3''), 18.0 (C-4''), 26.0 (C-5'')
(Supplemental Fig. S2). (31, 32)
3'-Dimethylallyl-2',4'-dihydroxychalcone (2a):
1
ESI-MS, m/z 309.1 [M+H]+; H NMR (600 MHz,
acetone-d6): δ 7.96 (1H, d, J = 15.6 Hz, H-β),7.87
(1H, d, J = 15.6 Hz, H-α), 7.85 (overlapped, 2H;
H-2 and H-6), 7.47 (overlapped, 3H; H-3, H-4 and
H-5), 6.55 (1H, d, J = 8.4 Hz, H-5'), 8.02 (1H, d, J
= 8.4 Hz, H-6'), 3.38 (2H, d, J = 7.2 Hz, H-1''),
5.28 (1H, t, J = 7.2 Hz, H-2''), 1.78 (1H, s, H-4''),
1.65 (1H, s, H-5'') (Supplemental Fig. S3); 13C
NMR (150 MHz, acetone-d6): δ 144.6 (C-β), 121.9
(C-α), 193.0 (CO), 131.7 (C-1), 129.7 (C-2), 129.9
(C-3), 131.5 (C-4), 129.9 (C-5), 129.7 (C-6), 114.4
(C-1'), 165.4 (C-2'), 116.2 (C-3'), 163.2 (C-4'),
108.3 (C-5'), 130.7 (C-6'), 22.4 (C-1''), 123.3
(C-2''), 131.6 (C-3''), 18.0 (C-4''), 26.0 (C-5'')
(Supplemental Fig. S4). (33)
Preparative Synthesis of the Enzymatic
Products for Structural Elucidation—The assays
for the isolation of the enzymatic products (1015
mL) contained 100 mM Tris-HCl (pH 9.0), 10 mM
MgCl2, 1 mM DMAPP, 500 µM prenyl acceptors,
and 30 mg protein of the recombinant yeast
microsome. The reaction mixtures were incubated
at 30 °C for 16 h and subsequently extracted with
ethyl acetate (20 mL  5). After evaporation of the
solvent, the residues were dissolved in methanol
and purified by reverse-phase semi-preparative
HPLC under the conditions described above. The
isolated products were subjected to MS and 1H and
13C NMR spectroscopic analyses, which yielded
the following results:
3'-Dimethylallyl-2,4,2',4'-tetrahydroxychalco
ne (3a): ESI-MS, m/z 340.9 [M+H]+;1H NMR (600
MHz, acetone-d6): δ 8.22 (1H, d, J = 15.6 Hz,
H-β),7.79 (1H, d, J = 15.6 Hz, H-α), 6.50 (1H, d, J
= 2.4Hz, H-3), 6.46 (1H, dd, J = 8.4, 2.4Hz, H-5),
7.69 (1H, d, J = 8.4Hz, H-6), 6.52 (1H, d, J =
8.4Hz, H-5'), 7.89 (1H, d, J = 8.4Hz, H-6'), 3.37
(2H, d, J = 7.2Hz, H-1''), 5.28 (1H, t, J = 7.2Hz,
H-2''), 1.78 (1H, s, H-4''), 1.64 (1H, s, H-5'')
(Supplemental Fig. S5); 13C NMR (150 MHz,
acetone-d6): δ 140.8 (C-β), 117.6 (C-α), 193.5
(CO), 114.8 (C-1), 159.9 (C-2), 103.7 (C-3), 162.3
(C-4), 109.2 (C-5), 131.8 (C-6), 114.6 (C-1'),
162.5 (C-2'), 115.3 (C-3'), 165.2 (C-4'), 107.9
(C-5'), 130.0 (C-6'), 22.4 (C-1''), 123.4 (C-2''),
131.5 (C-3''), 18.0 (C-4''), 25.9 (C-5'')
(Supplemental Fig. S6). (34, 35)
3'-Dimethylallylisoliquiritigenin
(1a):
1
ESI-MS, m/z 325.3 [M+H]+; H NMR (600 MHz,
acetone-d6): δ 7.83 (1H, d, J = 15.6 Hz, H-β), 7.76
(1H, d, J = 15.6 Hz, H-α), 7.74 (2H, d, J = 8.4 Hz,
H-2), 6.93 (1H, d, J = 8.4 Hz, H-3), 6.93 (1H, d, J
= 8.4 Hz, H-5), 7.74 (1H, d, J = 8.4 Hz, H-6), 6.53
(1H, d, J = 8.4 Hz, H-5'), 7.98 (1H, d, J = 8.4 Hz,
H-6'), 3.37 (2H, d, J = 7.2 Hz, H-1''), 5.28 (1H, t, J
= 7.2 Hz, H-2''), 1.78 (1H, s, H-4''), 1.64 (1H, s,
H-5'') (Supplemental Fig. S1); 13C NMR (150
MHz, acetone-d6): δ 145.0 (C-β), 118.6 (C-α),
193.1 (CO), 127.8 (C-1), 131.9 (C-2), 116.9 (C-3),
3'-Dimethylallylbutein (4a): ESI-MS, m/z
341.0 [M+H]+;1H NMR (600 MHz, acetone-d6): δ
7.76 (1H, d, J = 15.6 Hz, H-β),7.69 (1H, d, J =
161.0 (C-4), 116.9 (C-5), 131.8 (C-6), 116.2 (C-1'), 15.6 Hz, H-α), 7.34 (1H, d, J = 2.4 Hz, H-2), 6.91
5

Moraceous flavonoid prenyltransferases
(1H, d, J = 8.4 Hz, H-5), 7.23 (1H, dd, J = 8.4, 2.4
Hz, H-6), 6.53 (1H, d, J = 8.4 Hz, H-5'), 7.97 (1H,
scale bar corresponds to 0.1 estimated amino acid
changes per site. The exon-intron structure
d, J = 8.4 Hz, H-6'), 3.37 (2H, d, J = 7.2 Hz, H-1''), diagram was generated using the online Gene
5.27 (1H, t, J = 7.2 Hz, H-2''), 1.77 (1H, s, H-4''),
1.64 (1H, s, H-5'') (Supplemental Fig. S7); 13C
NMR (150 MHz, acetone-d6): δ 145.4 (C-β), 118.6
(C-α), 193.1 (CO), 128.4 (C-1), 116.0 (C-2), 146.5
(C-3), 149.2 (C-4), 116.5 (C-5), 123.4 (C-6), 114.5
(C-1'), 162.7 (C-2'), 116.2 (C-3'), 165.2 (C-4'),
108.1 (C-5'), 130.3 (C-6'), 22.3 (C-1''), 123.4
(C-2''), 131.5 (C-3''), 18.0 (C-4''), 25.9 (C-5'')
(Supplemental Fig. S8). (36)
Structure
Display
Server
(GSDS:
http://gsds.cbi.pku.edu.cn) and the exon position
and gene length method.
Nucleotide
Sequence
Accession
Number—The nucleotide sequences of MaIDT
and CtIDT have been deposited in the GenBank
database under the accession numbers KM262659
and KM262660, respectively.
6-Dimethylallylgenistein (7a): ESI-MS, m/z
338.9 [M+H]+; 1H NMR (600 MHz, acetone-d6): δ
8.14 (1H, s, H-2), 6.50 (1H, s, H-8), 7.45 (1H, d, J
= 8.4 Hz, H-2'), 6.90 (1H, d, J = 8.4 Hz, H-3'),
6.90 (1H, d, J = 8.4 Hz, H-5'), 7.45 (1H, d, J = 8.4
RESULTS
Cloning of MaIDT from the Cultured Cells of
M. alba—According to previous studies (1217),
plant FPTs from leguminous plants are closely
related to each other and cluster together in the
Hz, H-6'), 3.36 (2H, d, J = 7.2 Hz, H-1''), 5.27 (1H, phylogenetic tree. Therefore, we initially
t, J = 7.2 Hz, H-2''), 1.78 (1H, s, H-4''), 1.64 (1H, s, hypothesized that the FPTs of M. alba would
H-5'') 13.32 (1H, s, 5-OH) (Supplemental Fig. S9).
(37)
cluster together with the leguminous FPTs and
used degenerated primer pairs based on these
FPTs to clone the candidate gene(s). Similar
cloning strategies proved to be successful in the
isolation of SfFPT and GuA6DT from S. flavescens
and G. uralensis in our previous work (14, 15).
However, following RT-PCR and RACE, we failed
to achieve active PTs. A similar cloning strategy
also failed to lead to the identification of active
FPT from Epimedium acuminatum (41). Hence,
we switched to the Morus EST library for the
cloning of these candidate genes. Utilizing the
internal sequence of an EST clone that is
homologous to the homogentisate PT AtVTE2-1,
we succeeded in amplifying the full length cDNA
of MaIDT, and PT activity was observed following
the heterologous expression of MaIDT in yeast.
The encoded polypeptide of MaIDT (402
amino acids) had seven putative transmembrane
α-helices as predicted by the TMHMM 2.0
program. The polypeptide possessed putative
transit peptide sequences for targeting plastids as
6-Dimethylallyl-2'-hydroxygenistein
(8a):
1
ESI-MS, m/z 354.8 [M+H]+; H NMR (600 MHz,
acetone-d6): δ 8.12 (1H, s, H-2), 6.52 (1H, s, H-8),
6.45 (1H, d, J = 2.4 Hz, H-3'), 6.42 (1H, dd, J =
8.4, 2.4 Hz, H-5'), 7.10 (1H, d, J = 8.4 Hz, H-6'),
3.36 (2H, d, J = 7.2 Hz, H-1''), 5.27 (1H, t, J = 7.2
Hz, H-2''), 1.77 (1H, s, H-4''), 1.64 (1H, s, H-5'')
13.01 (1H, s, 5-OH) (Supplemental Fig. S10). (38)
Computer-assisted Sequence Analysis—The
sequence identities were obtained via alignment of
the amino acid sequences performed with the
program
“BLAST
2
SEQUENCES”
(www.ncbi.nlm.nih.gov). The PT proteins of the
plants were aligned using ClustalW (39). From the
alignments, a consensus phylogenetic tree was
generated by the neighbor-joining method using
MEGA6 (40). Bootstrap values are indicated in
percentages (only those greater than 70% are
presented) on the nodes. The bootstrap values
were obtained from 1,000 bootstrap replicates. The
6

Moraceous flavonoid prenyltransferases
predicted
by
Chlorop
1.1
linear dependence of the product formation on the
amount of microsome protein was found up to 200
µg per 100 µL assay and on the reaction time was
up to 30 min at 30 °C.
(http://www.cbs.dtu.dk/services/Chlorop/), TargetP
1.1 (http://www.cbs.dtu.dk/services/TargetP/), and
iPSORT
(http://ipsort.hgc.jp/).
A
multiple
alignment of the encoded polypeptide and the
reported leguminous FPTs is shown in Fig. 1. The
polypeptide possesses a conserved aspartate-rich
motif of PTs (N(Q/D)XXDXXXD) in loop 2 and
another characteristic sequence that is conserved
in the flavonoid/homogentisate PTs in loop 6
(KD(I/L)XDX(E/D)GD). However, several highly
conserved sequences, such as SD(L/I)S, SWP(L/S)
H(M/I)QT, IC(I/V)SLL(E/Q) and W(S/T)KI were
observed in the alignment of the leguminous FPTs,
and were not apparent in this polypeptide; the
functions of these domains are not yet known.
Moreover, the polypeptide shared identities of
only 24%30% at the amino acid level with the
reported leguminous FPTs, although the above
data fulfilled the three criteria for selecting FPT
candidates that have been adopted in a previous
report (12).
To clarify the prenylation properties, the
enzymatic product (1a) was subsequently prepared
1
and subjected to NMR analysis. In the H NMR
spectrum, the presence of the prenyl moiety was
suggested by the appropriate signals for two
methyls (δH 1.64 and δH 1.78, s, 3H each), a
methylene (δH 3.37, d, J = 7.2 Hz, 2H) and a
methine group (δH 5.28, t, J = 7.2 Hz, 1H). The
location of the prenyl moiety was indicated by the
disappearance of the H-3' signal (1H at δH 6.35)
and the change in the H-5' peak shape, which
became an ortho-coupled doublet (δH 6.53, d, J =
8.4 Hz) (Supplemental Fig. S1). This observation
is supported by a downfield chemical shift of C-3'
from δC 103.7 in 1 to δC 114.5 in 1a (Supplemental
Fig. S2). The NMR data of 1a were in good
agreement
with
those
(31,
of
3'-dimethylallylisoliquiritigenin
32).
Enzymatic
Activity
of
MaIDT and
Therefore, the enzymatic product of MaIDT was
Identification of the Enzymatic Products—In
previous studies, mulberry trees and related plants
have been found to be rich sources of prenylated
flavonoids (22, 23), and prenylation activity has
been confirmed in microsomal fractions of
mulberry tree cell cultures (42, 43). Accordingly,
various flavonoids, including chalcones, flavones,
flavanones and isoflavones have been employed as
acceptors to determine enzymatic activities. Of
these, isoliquiritigenin (1) is accepted to have the
highest efficiency. HPLC-UV/ESI-MS analysis
revealed that a product at tR 24.4 min with a
68-amu higher shift in its molecular peak was
detected in the incubation mixture with the
microsomes from the yeast cells transformed with
MaIDT but not with an empty vector (Fig. 2).
Product formation was strictly dependent on the
presence of MaIDT, DMAPP, Mg2+, and 1. A
unambiguously
identified
(1a),
as
3'-dimethylallylisoliquiritigenin
which
indicates that this enzyme regiospecifically
transferred a dimethylallyl moiety to 1 at the C-3'
position; thus, this recombinant enzyme was
designated as MaIDT (M. alba isoliquiritigenin
3'-dimethylallyltransferase).
Cloning and identification of CtIDT from
Cultured Cells of C. tricuspidata—To increase
understanding of the diversity of the FPTs in
moraceous plants, a cell suspension culture of C.
tricuspidata was chosen for further study. Due to
the absence of genetic information about C.
tricuspidata, homologous PCR was used to clone
candidate gene(s) as previously described (14, 15).
Similarly, degenerated primer pairs based on the
conserved regions of the leguminous FPTs failed
to produce an active FPT. Next, we designed
7

Moraceous flavonoid prenyltransferases
degenerated primer pairs based on the conserved
amino acid regions of MaIDT and its closely
related genes that are obtained via a BLASTN
search of Genbank, and eventually cloned a
full-length cDNA possessing FPT activity (CtIDT).
The polypeptide encoded by CtIDT (398 amino
acids) fulfilled the three criteria for selecting FPTs
(12). This polypeptide shared sequence identities
of 66% with MaIDT and only 24%30% with the
reported leguminous FPTs. Enzymatic assays
regioselectively introduced into the A ring of the
skeletons at the same position (C-3' of the
chalcones and C-6 of the isoflavones and
flavones).
The relative reactivities of MaIDT and CtIDT
toward different flavonoids also provided insight
into the “substrate structure-enzyme selectivity
relationships”.
MaIDT
preferred
2',4'-dihydroxychalcones to isoflavonoids and
flavone. The existence of hydroxyls at C-2', 4'
(C-5, 7) of the A ring was crucial for prenylation
because the lack of any one of these hydroxyl
groups led to no enzymatic product generation (5,
6, and 9). Regarding the B ring, the presence of a
hydroxyl group at C-4 position increased the
prenylation efficiency, as the conversion rate of 1
by MaIDT was higher than that of 2 (Fig. 3 A and
B). In contrast, the hydroxyl group at the C-2/3
containing
various
flavonoids
and
HPLC-UV/ESI-MS analyses revealed that CtIDT
exhibited a function similar to that of MaIDT (Fig.
3 A and B); thus, this recombinant enzyme was
designated
as
CtIDT
(C.
tricuspidata
isoliquiritigenin 3'-dimethylallyltransferase).
Substrate Specificities of MaIDT and
CtIDT—To investigate the substrate specificities
of the moraceous FPTs, we tested the activities of
MaIDT and CtIDT via HPLC-UV/ESI-MS
analysis utilizing 18 potential substrates that
included various types of flavonoids and
structurally simple phenols (Fig. 3 A and B). In
addition to isoliquiritigenin (1), MaIDT and CtIDT
were also able to catalyze the prenylation of three
other chalcones (2',4'-dihydroxychalcone (2),
2,4,2',4'-tetrahydroxychalcone (3), and butein (4))
(C-2'/3') position of
prenylation efficiency
B
ring reduced the
as revealed by
comparisons of the relative yields of 3 and 4 to 1
and of 8 and 10 to 7 (Fig. 3 A and B).
The prenylation catalyzed by FPTs represents
a Friedel–Crafts alkylation of flavonoid skeleton.
A
carbocation is generated from prenyl
diphosphate substrate (DMAPP) and carries out
an electrophilic attack at flavonoid ring, leading
to the regio-specific C-C bond formation. Hence,
the electron density around attacked carbon at
flavonoid ring might play an important role in the
course of prenylation. For example, the
insufficient electron density around C-6 of
daidzein (9) without C-5 hydroxyl group
compared with genistein (7) might lead to the
non-occurrence of prenylation.
and
two
isoflavonoids
(genistein
(7),
2'-hydroxygenistein (8)), and the flavone type
substrate apigenin (11) was only accepted by
MaIDT. The enzymatic products were prepared
from larger scale assays and subjected to MS and
NMR spectroscopic analyses with the exception of
apigenin (11) (Supplemental Figs. S3S10), the
prenylated product of which was identified as
6-dimethylallylapigenin (11a) in a comparison
Regarding CtIDT, its substrate specificity was
slightly different from that of MaIDT: firstly,
CtIDT accepted isoflavones with higher
efficiency than did MaIDT, and this finding is
partially in accordance with the fact that 7a has
been identified in large amounts in C.
with
an
authentic
standard
using
a
HPLC-UV/ESI-MS analysis (data not shown). The
results clearly showed that although the three
types of flavonoids could be accepted by MaIDT
and CtIDT, one dimethylallyl moiety was
8

Moraceous flavonoid prenyltransferases
tricuspidata cell suspension cultures (26);
secondly, the hydroxyl group at C-2 of chalcones
decreased the activity of CtIDT as revealed by
comparisons of the relative yields of 3 to 1, while
the corresponding hydroxyl group at the C-2'
position of the isoflavone increased the activity
as revealed by the comparison of the relative
yields of 8 to 7.
high Km values of CtIDT for flavonoids, this
enzyme might act on other flavonoids (or other
aromatic compounds) in C. tricuspidata.
Phylogenetic and Gene Structure Analysis of
the Flavonoid Dimethylallyltransferases and
Related
Enzymes—A
neighbor-joining
phylogenetic tree was constructed to analyze the
evolutionary relationships of MaIDT and CtIDT
with the other plant aromatic PTs (Fig. 5A). As the
first identified FPTs from non-Leguminosae plant
species, MaIDT and CtIDT will have particularly
significant effects on our understanding of the
molecular evolution of plant FPTs. The two
moraceous FPTs MaIDT and CtIDT appeared to
cluster together to form a new branch with a
Other prenyl donors (including GPP, FPP,
GGPP, and PPP) were also tested in the enzymatic
assay. The results revealed that GPP could also act
as prenyl donor when 1 is used as the acceptor
(Fig. 3C).
Biochemical Properties of the Recombinant
MaIDT and CtIDT—Previous studies have
reported that the plant FPT activity is dependent
on divalent cations and strongly affected by pH
and temperature (10). Further investigations using
1 as an acceptor and DMAPP as a prenyl donor in
this study revealed that the highest activities
occurred at approximately 30 °C and that the
activity decreased rapidly at temperatures above
40 °C (Fig. 4A). The analysis of the enzyme
activity within the pH range of 6.0 to 11.0 revealed
that the optimal pH value was between 8.0 and 9.0
(Fig. 4B). MaIDT activity was observed to
common
origin
in
the
homogentisate
phytyltransferases for plastoquinone formation
(AtVTE2-2 and GmVTE2-2). This branch is
clearly divergent from that of the leguminous FPTs,
which were recruited from the vitamin E
biosynthetic pathway. Considering the similar
substrate spectra and sequence divergence of the
leguminous and moraceous FPTs, it can be
concluded that they have evolved independently
via recruitment from the vitamin
E
and
plastoquinone biosynthetic pathways, respectively.
The sequence distance might reflect the
taxonomical distance between Moraceae from
Leguminosae plants (44). This situation would
decrease in the order of Mg2+> Ba2+ > Ca2+
>
Mn2+ > Fe2+ >Ni2+, and Co2+ and Cu2+ did not lead
to the production of 1a; CtIDT activity was found
to decrease in the order of Mg2+> Mn2+ > Ca2+
>
represent
a
somewhat subtler example of
Fe2+ > Ba2+, while Ni2+, Co2+ and Cu2+ did not lead
to the production of 1a (Fig. 4C). No product was
detected in the buffer following the addition of
EDTA.
convergent evolution. Morever, this type of
convergent evolution has been termed “repeated
evolution”, which indicates situations in which “a
similar function arose not from a completely
unrelated sequence but from a homologous,
although not orthologous gene” (45, 46).
Consequently, the search for new FPTs should not
focus solely on the degrees of sequence identity
The apparent Kms of MaIDT and CtIDT for
DMAPP as a prenyl donor was calculated as 28.3
and 173.6 µM, respectively. The apparent Kms of
MaIDT and CtIDT for 1 were calculated as 23.9
and 109.6 µM, respectively. The apparent Kms of
MaIDT and CtIDT for 7 were calculated as 36 and
255.9 µM, respectively. Considering the relatively
with
known
enzymes.
Furthermore,
in
evolutionarily distant plants, it is possible that
as-yet unidentified FPTs might share low
9

Moraceous flavonoid prenyltransferases
homology with the known FPTs.
biological/pharmacological activities have been
extensively studied (9, 22, 23). Of these
compounds, optically active Diels-Alder-type
adducts, which are characteristic mulberry
constituents with promising biological activities,
are attractive compounds from a biosynthetic point
of view, and prenylation is a key biosynthetic step
in their formation (8, 9).
Based on a BLASTP search of the Morus
Genome Database (MorusDB) (47, 48), 1 hit with
MorusDB accession no. Morus014065 was found
and shared an identities of 97% with MaIDT, and
its genome structure was compared with those of
the leguminous FPT GmG4DT and homogentisate
PTs involved in vitamin E and plastoquinone
biosyntheses,which are available in GenBank. The
exon–intron structure of MaIDT was quite
different from those of the leguminous FPT
GmG4DT and the related homogentisate PTs
involved in vitamin E biosynthesis, while the
exon–intron organizations of MaIDT and the
homogentisate PTs for plastoquinone biosynthesis
were very similar (Fig. 5B). These observations
confirm the results of the phylogenetic analysis,
which suggested that the leguminous and
moraceous FPTs were derived from homogentisate
PTs involved in vitamin E and plastoquinone
biosynthesis, respectively, and have evolved
independently.
Evolution of FPTs—Plant aromatic PTs are
divided into two distinct subgroups: one is the
4-hydroxybenzoate PTs that are involved in the
biosyntheses of ubiquinone (benzoquinone) and
shikonin (naphthoquinone) derivatives; the second
subgroup is the homogentisate prenyltransferase
(HGPT) family, which is involved in vitamin E
and plastoquinone biosyntheses. Thus far, all the
identified plant FPTs are belong to HGPT family
(1115).
Based on the newly identified moraceous
FPTs, the phylogenetic tree of the HGPT gene
family was rebuilt. A clear phylogenetic separation
of the HGPT genes based on primary metabolism
(i.e., those involved in tocopherol, tocotrienol and
plastoquinone biosyntheses) versus specialized
metabolism can be observed. The formers
phylogenetic group is clustered in a pattern of
functional clades because the molecular evolution
of this group has been severely constrained by
natural selection. However, the latter group
exhibits a remarkable flexibility of the HGPT
family to evolve enzymes with new substrate
specificities. Moreover, several functionally
divergent enzymes, such as FPTs, phloroglucinol
PTs and coumarin PTs, have emerged from the
primary metabolic HGPTs (5053). Interestingly,
repeated evolution has been observed to exist in
the FPTs. The moraceous FPTs MaIDT and CtIDT
are grouped in a branch that is clearly different
from the branch in which the leguminous FPTs
cluster, which implies that FPTs evolved
independently in the two plant lineages. Repeated
DISCUSSION
Prenylation as a Typical Modification of
Aromatic Compounds in Moraceous Plants—A large
variety of biological activities have been reported
for prenylated aromatic compounds, and these
natural products are attractive resources in the
food and pharmaceutical industries. Aromatic PTs
catalyze transfer reactions of prenyl moieties from
prenyl diphosphates to diverse aromatic acceptors.
Theses PTs are involved in both general and
specialized metabolism, play important roles in
living organisms, and strongly contribute to the
structural diversity and biological activities of
aromatic natural products (7, 49).
In moraceous plants, many prenylated
aromatic compounds, such as Diels-Alder-type
adducts, flavonoids, 2-arylbenzofurans, xanthones,
and stilbenes, have been identified, and their
10

Moraceous flavonoid prenyltransferases
evolution might represent the majority of
convergent evolution cases in plant specialized
metabolism, and many such cases have been
identified (46); e.g., in each plant lineage, the
respective linalool synthase is more similar to
other terpene synthases (TPSs) that are responsible
for the synthesis of other monoterpenes within that
lineage than it is to the linalool synthases from
different lineages (54, 55). Furthermore, the
phylogeny of FPTs seems to exhibit a pattern of
species-specific expansion in Leguminosae and
Moraceae. Apparently, the identified moraceous
FPTs and leguminous FPTs arose individually by
repeated gene duplications and divergence that
occurred after the split of the two plant families.
However, our understanding of the plant FPTs is
currently based on just over 10 genes so far; thus,
more different FPTs need to be identified to clarify
the molecular evolution of plant FPTs.
isolated from moraceous plants, a large family of
FPTs can be imagined. Moreover, prenylated
flavonoids are active constituents of many other
non-Leguminosae medicinal plants. As the first
successful
identification
of
FPTs
from
non-Leguminosae plants, this study might inspire
the identification and characterization of
additional and evolutionarily varied FPTs that are
involved in the biosyntheses of pharmacologically
active prenylated flavonoids in plants other than
those of the Leguminosae and Moraceae families.
In summary, we have characterized the first
two non-Leguminosae plant FPTs MaIDT and
CtIDT from M. alba and C. tricuspidata,
respectively. These FPTs feature catalytic
regioselectivity and promiscuity. Multiple
alignments, phylogenetic analyses and gene
structure analyses revealed that these novel FPTs
evolved independently from their counterparts in
Leguminosae. These findings provide useful
guidance for identifying additional and
evolutionarily varied FPTs in non-Leguminosae
plants. Furthermore, based on their substrate
promiscuities and high catalytic efficiencies,
MaIDT and CtIDT might be useful as an
environmentally friendly and efficient biological
catalysts of the regiospecific prenylation of
flavonoids to produce bioactive compounds for
potential therapeutic applications.
In some studies, the above mentioned
species-specific patterns of repeated gene
duplications and sequence divergence are referred
to as “blooms” (56). In practice, the approach of
selecting candidate genes from species-specific
blooms has proven successful for the discovery of
new genes (56, 57). As FPTs have now been
successfully identified in moraceous plants, this
approach is likely worthwhile for attempts to clone
other FPTs from moraceous plants. Given the great
variety of prenylated flavonoids that have been
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Moraceous flavonoid prenyltransferases
FOOTNOTES:
*The study was financially supported by the National Natural Science Foundation of China (Grant
No. 81273405), the Fundamental Research Funds for the Central Universities (Grant No. 2012N06),
the Program for Doctorate Education (Grant No. 20111106110029), and the National Science &
Technology Major Project ′Key New Drug Creation and Manufacturing′, China (No.
2012X09301002-001-005).
Ⓢ
This article contains Supplemental Figures S1S10.
1To whom correspondence should be addressed: State Key Laboratory of Bioactive Substance and
Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College &
Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, China. Tel.:
86-10-6316-5195; Fax: 86-10-6301-7757; E-mail: jgdai@imm.ac.cn
The abbreviations used in this manuscript are as follows: MaIDT, Morus alba isoliquiritigenin 3'-
dimethylallyltransferase;
CtIDT,
Cudrania
tricuspidata
isoliquiritigenin
3'-
dimethylallyltransferase; PT, prenyltransferase; HGPT, homogentisate prenyltransferase; FPT,
flavonoid prenyltransferase; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP,
farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; PPP, phytyl diphosphate; ESI,
electrospray mass ionization; EST, expressed sequence tag; RT, reverse-transcribe; RACE, rapid
amplification of cDNA ends; and BLAST, basic local alignment search tool.
16

Moraceous flavonoid prenyltransferases
FIGURE LEGENDS
FIGURE 1. Multiple alignment of plant flavonoid prenyltransferases. MaIDT (M. alba;
KM262659); CtIDT (C. tricuspidata; KM262660); SfFPT (S. flavescens; KC513505); SfN8DT-1 (S.
flavescens; AB325579); SfG6DT (S. flavescens; BAK52291); SfiLDT (S. flavescens; AB604223);
LaPT1 (L. albus; JN228254); GmG4DT (G. max; AB434690); GuA6DT (G. uralensis; KJ123716).
The conserved aspartate-rich motifs N(Q/D)XXDXXXD and KD(I/L)XDX(E/D)GD are
highlighted with solid underlines. The sequences conserved only in the leguminous FPTs are
highlighted with black boxes. The transit peptides have been highlighted in blue backgroud.
FIGURE 2. Functional characterization of recombinant MaIDT. A. Reaction catalyzed by the
recombinant MaIDT. B. Incubation containing isoliquiritigenin (1), DMAPP and Mg2+ with
microsomal fractions of yeast strain YPH499 harboring either the expression vector
pESC-HIS-MaIDT or the empty vector pESC-HIS. C. MS spectra of the 1a and 1.
FIGURE 3. Substrate specificities of the recombinant MaIDT and CtIDT. A. Relative enzymatic
activities with various flavonoids and simple phenols acting as the prenyl acceptors and DMAPP
acting as the prenyl donor. B. The reactions catalyzed by the recombinant MaIDT and CtIDT and
the chemical structures of the flavonoids used for the substrate specificity analysis. C. Relative
enzymatic activities with DMAPP, GPP, FPP, GGPP, and PPP as the prenyl donors and 1 as the
prenyl acceptor.
FIGURE 4. Biochemical properties of MaIDT and CtIDT. A. Effects of temperature on the enzyme
activities of MaIDT and CtIDT. B. pH dependencies of MaIDT and CtIDT. C. Effects of various
divalent metal ions on MaIDT and CtIDT activities.
FIGURE 5. Phylogenetic and genome structure analyses of MaIDT and CtIDT. A. The
phylogenetic relationship between the MaIDT and CtIDT proteins and the related plant
prenyltransferases. The protein sequences were aligned using ClustalW. The neighbor-joining
phylogenetic tree was drawn using MEGA6. The bootstrap value was 1,000, and the branch lengths
represent the relative genetic distances. The abbreviations of the protein sequences and their
accession numbers are as follows: MaIDT (M. alba; KM262659); CtIDT (C. tricuspidata;
KM262660); SfFPT (S. flavescens; KC513505); SfN8DT-1 (S. flavescens; AB325579); SfN8DT-2
(S. flavescens; AB370330); SfN8DT-3 (S. flavescens; AB604222); SfG6DT (S. flavescens;
BAK52291); SfiLDT (S. flavescens; AB604223); LaPT1 (L. albus; JN228254); GmG4DT (G. max;
AB434690); GuA6DT (G. uralensis; KJ123716); ZmVTE2-1 (Zea mays; DQ231055); TaVTE2-1
(Triticum aestivum; DQ231056); ApVTE2-1 (Allium porrum; DQ231057); CpVTE2-1 (Cuphea
pulcherrima; DQ231058); AtVTE2-1 (Arabidopsis thaliana; AY089963); GmVTE2-1 (G. max;
DQ231059); PcPT (Petroselinum crispum; AB825956); Cl-PT1 (Citrus limon; AB813876)
OsHGGT (Oryza sativa; AY222862); HvHGGT (Hordeum vulgare; AY222860); TaHGGT (T.
17

Moraceous flavonoid prenyltransferases
aestivum; DQ231056); AtVTE2-2 (A. thaliana; DQ231060); GmVTE2-2 (G. max; DQ231061);
HlPT-1 (Humulus lupulus; AB543053); CsPT (Cannabis sativa; see ref. 51); OsPPT1 (O. sativa;
AB263291); AtPPT1 (A. thaliana; AY089963); LePGT1 (Lithospermum erythrorhizon; AB055078);
LePGT2 (L. erythrorhizon; AB055079). B. Comparisons of the exon–intron structures of MaIDT
and the related prenyltransferases. The gene sequence of MaIDT can be accquired from MorusDB
with the gene ID Morus014065; the Genbank accession numbers of the other gene sequences used
have been provided above.
18

Moraceous flavonoid prenyltransferases
TABLES
Table 1 Identified flavonoid prenyltransferases in plants
Enzyme
Flavonoid substrate
Flavanone
Species of origin
Reference
[12, 13]
SfN8DT-1
SfN8DT-2
SfN8DT-3
S. flavescens
Flavanone, Flavanonol,
Flavone,
Dihydrochalcone
SfFPT
S. flavescens
S. flavescens
[14]
SfiLDT
Chalcone
Flavone
[13]
[15]
Glycyrrhiza
uralensis
GuA6DT
SfG6DT
LaPT1
Isoflavone
Isoflavone
Pterocarpan
S. flavescens
Lupinus albus
Glycine max
[13]
[16]
[17]
GmG4DT
19

Moraceous flavonoid prenyltransferases
Figure 1
20

Moraceous flavonoid prenyltransferases
Figure 2
21

Moraceous flavonoid prenyltransferases
Figure 3
22

Moraceous flavonoid prenyltransferases
Figure 4
23

Moraceous flavonoid prenyltransferases
Figure 5
24