Molecular cloning and biochemical characterization of a new coumarin glycosyltransferase CtUGT1 from Cistanche tubulosa

Article abstract of DOI:10.1016/j.fitote.2021.104995

UDP-glycosyltransferases (UGTs) are an important and functionally diverse family of enzymes involved in secondary metabolite biosynthesis. Coumarin is one of the most common skeletons of natural products with candidate pharmacological activities. However, to date, many reported GTs from plants mainly recognized flavonoids as sugar acceptors. Only limited GTs could catalyze the glycosylation of coumarins. In this study, a new UGT was cloned from Cistanche tubulosa, a valuable traditional tonic Chinese herb, which is abundant with diverse glycosides such as phenylethanoid glycosides, lignan glycosides, and iridoid glycosides. Sequence alignment and phylogenetic analysis showed that CtUGT1 is phylogenetically distant from most of the reported flavonoid UGTs and adjacent to phenylpropanoid UGTs. Extensive in vitro enzyme assays found that although CtUGT1 was not involved in the biosynthesis of bioactive glycosides in C. tubulosa, it could catalyze the glucosylation of coumarins umbelliferone 1, esculetine 2, and hymecromone 3 in considerable yield. The glycosylated products were identified by comparison with the reference standards or NMR spectroscopy, and the results indicated that CtUGT1 can regiospecifically catalyze the glucosylation of hydroxyl coumarins at the C7-OH position. The key residues that determined CtUGT1's activity were further discussed based on homology modeling and molecular docking analyses. Combined with site-directed mutagenesis results, it was found that H19 played an irreplaceable role as the crucial catalysis basis. CtUGT1 could be used in the enzymatic preparation of bioactive coumarin glycosides.

Full text of DOI:10.1016/j.fitote.2021.104995

Fitoterapia 153 (2021) 104995
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Molecular cloning and biochemical characterization of a new coumarin
glycosyltransferase CtUGT1 from Cistanche tubulosa
Xiping Xu, Yaru Yan, Wenqian Huang, Ting Mo, Xiaohui Wang, Juan Wang, Jun Li,
Shepo Shi*, Xiao Liu*, Pengfei Tu*
Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic
of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
UDP-glycosyltransferases (UGTs) are an important and functionally diverse family of enzymes involved in sec-
ondary metabolite biosynthesis. Coumarin is one of the most common skeletons of natural products with
candidate pharmacological activities. However, to date, many reported GTs from plants mainly recognized fla-
vonoids as sugar acceptors. Only limited GTs could catalyze the glycosylation of coumarins. In this study, a new
UGT was cloned from Cistanche tubulosa, a valuable traditional tonic Chinese herb, which is abundant with
diverse glycosides such as phenylethanoid glycosides, lignan glycosides, and iridoid glycosides. Sequence
alignment and phylogenetic analysis showed that CtUGT1 is phylogenetically distant from most of the reported
flavonoid UGTs and adjacent to phenylpropanoid UGTs. Extensive in vitro enzyme assays found that although
CtUGT1 was not involved in the biosynthesis of bioactive glycosides in C. tubulosa, it could catalyze the glu-
cosylation of coumarins umbelliferone 1, esculetine 2, and hymecromone 3 in considerable yield. The glyco-
sylated products were identified by comparison with the reference standards or NMR spectroscopy, and the
results indicated that CtUGT1 can regiospecifically catalyze the glucosylation of hydroxyl coumarins at the C7-
OH position. The key residues that determined CtUGT1’s activity were further discussed based on homology
modeling and molecular docking analyses. Combined with site-directed mutagenesis results, it was found that
H19 played an irreplaceable role as the crucial catalysis basis. CtUGT1 could be used in the enzymatic prepa-
ration of bioactive coumarin glycosides.
Plant glycosyltransferases
Coumarin glycosides
Cistanche tubulosa
Homology modeling
Site-directed mutagenesis
1. Introduction
distributed in many plant species. Naturally occurring coumarins are
often existed as glycoconjugates, and these derivatives exhibited a broad
Glycosylation is one of the most common structure modification
types of secondary natural products. Glycosylation could change the
stability and/or solubility of the aglycon, and contribute a lot to the
diversity of natural/unnatural products due to the multiple conjugation
types. In plants, these reactions were usually conducted by uridine
diphosphate (UDP)-dependent glycosyltransferases (UGTs), a kind of
enzymes that catalyze the transfer of monosaccharide moieties from
activated nucleotide sugar to a glycosyl acceptor molecule which can be
a carbohydrate, glycoside, oligosaccharide, or a polysaccharide [1,2].
To date, many genes encoding UGTs have been isolated from several
plants [3]. The glycosylation mainly happened at hydroxyl groups or
carboxyl groups of a wide range of secondary products, such as flavo-
noids [4], phenylpropanoids [5], terpenoids [6], steroids [7], alkaloids
[8], and so on. Coumarins are phenylpropanoids metabolites widely
spectrum of pharmacological activities, including antioxidant [9],
antiviral [10,11], hepatoprotective [12], anti-inflammatory [13], anti-
cancer [14], antimutagenic [15], and cholinesterase (ChE) & mono-
amine oxidase (MAO) inhibitory activities [14,15], etc. However, to
date, in comparison with the most frequently reported and extensively
investigated flavonoids UGTs, only limited UGTs were reported able to
transfer the sugar moiety to coumarins [16–18].
Plants rich in diverse glycosides could serve as an ideal gene pool of
UGTs with novel catalysis activities. Furthermore, numerous UGTs have
been reported to possess considerable acceptor promiscuity to catalyze
the glycosylation of multiple substrates, some of which were even not
separated in the plants [19]. In this article, a novel glycosyltransferase
CtUGT1 was identified from Cistanche tubulosa, a traditional medicinal
herb that is abundant with diverse glycosides such as phenylethanoid
* Corresponding authors.
E-mail addresses: shishepo@163.com (S. Shi), fcliuxiao@163.com (X. Liu), pengfeitu@bjmu.edu.cn (P. Tu).
https://doi.org/10.1016/j.fitote.2021.104995
Received 7 May 2021; Received in revised form 21 June 2021; Accepted 11 July 2021
Available online 20 July 2021
0367-326X/© 2021 Elsevier B.V. All rights reserved.

X. Xu et al.
Fitoterapia 153 (2021) 104995
glycosides (PhGs), lignan glycosides and iridoid glycosides. Heterolo-
gous expression and function characterization demonstrated that
although recombinant CtUGT1 was not involved in the biosynthesis of
these glycosides in C. tubulosa, it can catalyze the glucosylation of hy-
droxyl coumarins, and the reaction regiospecifically happened on the 7-
OH position of coumarins umbelliferone 1, esculetine 2, and hymecro-
mone 3 to produce three pharmacologically active compounds skimmin
(1a), cichoriin (2a), and 4-methylumbelliferyl glucoside (3a) with
considerable yield, respectively. In addition, benzophenone substrate
4,4′-dihydroxybenzophenone (4), isoflavone substrate genistein (5), and
anthraquinone substrate aloe-emodine (6) could also be accepted by
CtUGT1. The catalytic properties and key residues underlining the
catalysis ability of CtUGT1 were also evaluated based on homology
modeling, AutoDock analysis, and site-directed mutagenesis.
conducted using KOD-Plus-Neo DNA Polymerase (TOYOBO, Osaka,
Japan). The obtained sequences were verified by cloning into the
pEASY-Blunt vector (TransGen Biotech, China). The confirmed ampli-
fication product of CtUGT1 was subsequently digested and sub-cloned
into the expression vector pET-28a(+) (Novagen, USA). The verified
construct was then transformed into E. coli Transetta (DE3) to obtain the
recombinant strain. Overnight culture of recombinant strain was inoc-
ulated into Luria-Bertani (LB) medium containing 50
μ
g⋅mLꢀ 1 kana-
mycin and 40
μ
g⋅mLꢀ 1 chloromycetin at a ratio of 1:100. The cultures
were grown at 37 ◦C, 200 rpm until the OD600 value reached 0.4–0.6.
Isopropyl-β-D-thiogalactopyranoside (IPTG) was subsequently added to
a final concentration of 0.5 mM and the cells were grown for 16 h at
18 ◦C, 180 rpm. Then cell pellets were harvested by centrifugation
(7600 ×g, 10 min, 4 ◦C), and re-suspended in 3 mL/g chilled lysis buffer
(Supplementary Data Note 1) and disrupted by sonication on ice. The
◦
2. Experimental
cell debris was removed by centrifugation at 7600 ×g, 4 C for about
30 min. A pre-equilibrated Histrap column (GE Healthcare, Uppsala,
Sweden) was used for affinity chromatography according to the manu-
facturer’s instructions. The recombinant protein was eluted by 10 col-
umn volumes of elution buffer (Supplementary Data Note 1) containing
250 mM imidazole. Protein purity was analyzed by 10% SDS-PAGE. The
purified protein was concentrated by a 30 kDa ultrafiltration tube
(Sigma-Aldrich, USA) and desalted using a PD–10 column (GE Health-
care, Uppsala, Sweden) with desalting buffer (Supplementary Data Note
1). Protein concentration was determined by the Bradford method using
BSA as a standard.
2.1. Plant material and chemicals
Cistanche tubulosa used in this study was collected from desert areas
in Hetian, Xinjiang autonomous regions. Their botanical identity was
confirmed by Professor Pengfei Tu at Peking University. The tested
coumarins and other substrates in this study were purchased from
Sigma-Aldrich (St. Louis, USA), Chengdu Push Biotechnology Co., Ltd.
(Chengdu, China) and Chengdu Biopurify Phytochemicals Co., Ltd.
(Chengdu, China) unless otherwise stated. Reference standards of
skimmin (1a) and cichoriin (2a) were purchased from Wuhan Chem-
Faces Biochemical Co., Ltd. (Wuhan, China).
2.5. Enzymatic activity assays and substrate specificity of CtUGT1
2.2. Molecular cloning of CtUGT1 from Cistanche tubulosa
To investigate the glycosylation activity of CtUGT1, enzyme assays
were performed in a reaction mixture (150 μL) composed of 0.4 mM
The total RNA of C. tubulosa was extracted from the fleshy stem using
an OMEGA Plant RNA Kit (GA, USA) and reverse-transcribed to cDNA
with PrimeScript™ RT reagent Kit (TaKaRa, Japan) following the
manufacturer’s instructions. A degenerate primer was designed for 3′
RACE based on the aminoacid sequences in the conserved PSPG (Plant
Secondary Product Glycosyltransferases) -box of plant glycosyl-
transferases. The 3′-end and 5′-end amplifications of CtUGT1 were car-
ried out using Smart RACE cDNA Amplification Kit (Clontech, USA)
according to the manufacturer’s protocol using primers (Table S1). The
full-length cDNA of the CtUGT1 was amplified by PCR using KOD-Plus-
Neo DNA Polymerase (TOYOBO, Japan) with gene-specific primer pairs
(Table S1) under the following conditions: an initial denaturation step at
94 ◦C for 2 min, followed by 35 cycles of denaturation at 98 ◦C for 15 s,
annealing at 55 ◦C for 30 s, and extension at 68 ◦C for 55 s, with a final
extension step at 68 ◦C for 7 min. All the DNA fragments obtained were
cloned into pEASY-Blunt vector (TransGen Biotech, China) and
sequenced.
aglycone, 0.8 mM UDP-glucose (UDPG), and 50
μ
g of purified CtUGT1
protein in the desalting buffer. All reactions were incubated at 30 ◦C for
12 h, and terminated by adding 300
μL of cold methanol. The mixtures
were centrifuged at 15,000 ×g for 30 min to collect the supernatant for
HPLC-UV/ESI-MS analyses. Three parallel assays were routinely carried
out for each reaction. HPLC (Agilent 1260, USA) was equipped with a
diode array detector and
a
CAPCELL PAK C18 column
(250 mm × 4.6 mm, 5
μ
m; Shiseido, Japan) at a flow rate of 1 mL⋅minꢀ 1
,
and the column temperature was maintained at 30 ◦C. The mobile phase
consisted of A (i.e., 0.1% formic acid aqueous solution) and B (i.e.,
acetonitrile). The gradient programs were listed in Table S4. HRESI-MS
data was recorded on an LCMS-IT-TOF system, fitted with an ESI
interface (Shimadzu, Kyoto, Japan) with ultra-high purity He as the
collision gas, and N2 as the nebulizing gas. The optimized ESI source
parameters were as follows: sheath gas flow rate, 1.5 mL⋅minꢀ 1; auxil-
iary gas flow rate, 1.5 mL⋅minꢀ 1; spray voltage, 4.5 KV; capillary tem-
perature, 200 ◦C. The spectra were recorded in the 100–1500 m/z range
for a full scan MS analysis.
2.3. Sequence alignment and phylogenetic analysis
2.6. Effects of reaction time, pH value and temperature on enzyme
activity, and kinetic studies of CtUGT1
Sequence alignment of CtUGT1 with reported glycosyltransferases
(Table S2) was performed using DNAMAN 4.0 software package (Lyn-
non Biosoft, Canada). The motifs and domains were analyzed using a
conserved domain tool (http://www.ncbi.nlm.nih.Gov/Structure/cdd/
wrpsb.cgi). The phylogenetic tree was constructed by bootstrapping
MEGA 7.0.14 software using the neighbor-joining method with 1000
bootstrap replicates [20]. The translated protein sequence of CtUGT1
was aligned with the known plant glycosyltransferases deposited in the
NCBI GenBank database (Table S3) with ClustalW.
Effects of reaction time, pH value, and temperature on the enzyme
activity of CtUGT1 were tested using UDPG as the sugar donor and 3 as
the sugar acceptor in the reaction conditions as described above. For the
determination of optimal pH, the enzyme activity was compared in
100 mM buffer (citric acid‑sodium citrate) with pH values ranged from
3.0 to 6.0, 100 mM buffer (KH2PO4-K2HPO4) with pH values ranged
from 6.0 to 8.0, 100 mM buffer (Tris-HCl) with pH values ranged from
7.0 to 9.0, 100 mM buffer (Na2CO3-NaHCO3) with pH values ranged
from 9.0 to 11.0. To assay for the optimal reaction temperature, the
reactions were incubated at various temperatures ranging from 0 to
65 ◦C. The time courses of the reaction were evaluated at 12 different
time points between 0 and 24 h. All experiments were performed in
triplicate. The reaction mixtures were analyzed by HPLC-MS as
2.4. Heterologous expression and protein purification of CtUGT1 in
Escherichia coli
The coding region of CtUGT1 was amplified with BamH I and Not I as
restriction sites using primers shown in Table S1. PCR reactions were
2

X. Xu et al.
Fitoterapia 153 (2021) 104995
described above.
Protein purification and enzyme reactions were performed and analyzed
as described above.
For kinetic studies of CtUGT1, enzymatic assays were performed in a
final volume of 100
μL containing 100 mM KH2PO4-K2HPO4 buffer
(pH 8.0), 25
centrations (50–3000
for 30 min and then terminated with 100
μ
g of purified CtUGT1, 3 mM of UDPG, and varying con-
M) of 3. The reactions were conducted at 37 ◦C
L ice-cold methanol. All of
3. Results and discussion
μ
μ
3.1. Full length cDNA cloning of the CtUGT1 gene from C. tubulosa
these reactions were performed in triplicate, and enzyme activity was
evaluated via quantification of the corresponding glucosylated de-
rivatives. Kinetic parameters, including the Michaelis–Menten constant
(Km) and Vmax, were calculated by nonlinear regression analysis using
GraphPad Prism 5 software.
C. tubulosa (L.) (RouCongRong) is a traditional Chinese medicinal
herb producing a wide variety of bioactive natural glycosides. There
should be a considerable number of new glycosyltransferases genes that
existed in its genome. To seek novel glycosyltransferases with inter-
esting catalysis activities, a degenerate primer for 3′-RACE was designed
based on the conserved PSPG motif to clone permissive UGTs from
C. tubulosa. The obtained 3′ end sequence was further analyzed by NCBI
blast and the specific primer for 5′-RACE was designed based on the
obtained sequence. The amplified 5′ cDNA ends and 3′ cDNA ends were
spliced and the full-length of the CtUGT1 sequence, including 1739
nucleotides, was obtained (Fig. S3) and listed in Supplementary data
Note 3. Subsequently, an open reading frame (ORF) of CtUGT1 was
found by NCBI ORF Finder tools and the 1482 bp coding sequence was
successfully cloned by RT-PCR amplification using specific primers in
Table S1. The cDNA sequence of CtUGT1 has been submitted to NCBI
(accession No. MW629113).
2.7. Substrate and sugar donor selectivity of CtUGT1
To explore the substrate preferences of CtUGT1, totally of 27 sub-
strates belonging to different structural types were tested using UDPG as
the donor. Their chemical information was listed in Table S5, and their
structures were shown in Figs. S1 and S2. To investigate the sugar do-
nors selectivity of CtUGT1, assays were conducted using umbelliferone
(1), esculetine (2), hymecromone (3), 4,4′-dihydroxybenzophenone (4),
genistein (5) and aloe-emodine (6) as substrates with UDPG, UDP-N-
acetyl galactosamine, UDP-galactose, GDP-mannose, GDP-fucose, UDP-
galacturonic acid, UDP-rhamnose as sugar donors, respectively. All ex-
periments were performed in triplicate. The reaction mixtures were
analyzed by HPLC-MS as described above.
3.2. Sequence analysis and heterologous expression of CtUGT1
2.8. Preparation of 4-methylumbelliferyl glucoside (compound 3a) by
The CtUGT1 gene was expected to encode a protein of 493 amino
acid residues with an estimated molecular mass of 55.78 kDa. NCBI blast
revealed that the deduced protein sequence of CtUGT1 shares the
highest identity (76.39%) with UGT86A1-like enzymes. The biochem-
ical functions of this kind of enzyme have not been fully characterized. It
is predicted that they may be involved in the plant adaptation to abiotic
stresses. The alignment of the amino acid sequences of CtUGT1 and
other plant glycosyltransferases is shown in Fig. S4.
CtUGT1
The preparative reaction mixture consisted of 0.034 mmol substrate
compound 3, 0.04 mmol UDPG, 5 mg purified CtUGT1 in 50 mL reaction
buffer. The reactions were gently agitated at the optimum conditions
(100 mM KH2PO4-K2HPO4 with pH 8.0; 37 ◦C). After incubation at 37 ◦C
for 24 h, the reaction supernatant was separated by column chroma-
tography with macroporous resin (MCI GEL CHP) after centrifuging at
12,000 ×g for 30 min. The mobile phase was a gradient elution of 100%
water to 100% methanol. Each fraction was analyzed by HPLC-UV.
Fractions contained only targeted product were evaporated to dryness
under reduced pressure and characterized by 1H nuclear magnetic
resonance (NMR) spectroscopy and 13C NMR spectroscopy.
The highly conserved 44 residues of PSPG motif were found at the C-
terminal of CtUGT1. Within the PSPG-box, a peptide sequence of
HCGWNS was located at the 373rd amino acid, which has been detected
in 95% of all glycosyltransferases [31]. The final residue of the PSPG
motif was glutamine, which indicated that this enzyme tends to use
UDPG as its sugar donor [32] (Fig. S4). The phylogenetic tree was
constructed based on the predicted polypeptide sequences of CtUGT1
and different kinds of plant origin glycosyltransferases which were
involved in the biosynthesis of various secondary metabolites. So far,
most of the reported glycosyltransferases from plants recognized flavo-
noids as their substrates, such as the flavone 7-O-glycosyltransferases,
flavonoid 3-O-glucosyltransferases, isoflavone 7-O-glycosyltransferases,
and chalcone glycosyltransferases. Phylogenetic analysis results showed
that CtUGT1 has not grouped into the clade of flavonoid GTs. Instead, it
is located adjacent to UGT74T1 and UGT74S1 (lignan glycosyl-
transferases from Linum usitatissimum) and NtGT2 (a coumarin glyco-
syltransferase from Nicotiana tabacum), which indicated that CtUGT1
may have some novel catalysis activities towards phenylpropanoid
substrates (Fig. 1), especially considered that only limited members
have been functionally identified belonging to these clades.
2.9. Molecular docking and site-directed mutagenesis of CtUGT1
The protein model of CtUGT1 was established by using SWISS-
MODEL [21–25]. The crystal structure of Medicago truncatula glycosyl-
transferase UGT85H2 (PDB ID 2PQ6) was chosen as the template
structure [26]. The established model was assessed by VERIFY-3D
[27,28]. Auto-Dock Vina was used for molecular docking studies [29].
The crystal structure of UGT74F2 [30], a glycosyltransferase from
Arabidopsis thaliana, in complex with ligand UDP and salicylic acid (PDB
ID 5U6M) was used as the control structure for sugar donor pocket and
sugar acceptor pocket analysis, respectively. The Auto-Dock-Tools 1.5.6
from the MGLTools (Molecular Graphics Laboratory tools) was used to
prepare the sugar donor UDPG and substrate 1, 2 for molecular docking.
The side chains of residues around the active-site cavity were set flex-
ible, and the rotatable bonds of ligands were left free to rotate. The top-
ranked pose as judged by the Vina docking model with the highest (kcat/
mol) affinity was subjected to visual analysis using PyMOL 2.0 software.
For mutagenesis investigations, the sequence optimized gene of
CtUGT1 (Supplementary data Note 2) was synthesized and tested as the
wild type. Site-directed mutagenesis of the CtUGT1 was conducted using
the Fast Mutagenesis System (TransGen Biotech). The mutants were
amplified from the template of the optimized gene which has been
constructed into the pET-28a vector using the specific primers shown in
Table S1. The constructed mutants were verified by PCR and sequencing
before being transformed into E. coli (DE3) for heterologous expression.
To further reveal its catalysis activity, the CtUGT1 sequence was
expressed under the control of T7 lac promoter in E. coli Transetta (DE3)
cells as His6-tagged fusion protein. The His-tagged recombinant CtUGT1
was efficiently purified to homogeneity by Ni–NTA affinity chroma-
tography. The molecular weight of the purified recombinant CtUGT1
was approximately 55 kDa (Fig. S5), which is consistent with the pre-
dicted molecular mass.
3.3. In vitro acceptor substrate specificity of CtUGT1
To test the glycosylation activity of CtUGT1, in vitro enzymatic assays
were performed. UDPG was used as the sugar donor. Sugar acceptors
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X. Xu et al.
Fitoterapia 153 (2021) 104995
Fig. 1. Phylogenetic analysis of CtUGT1 with other known glycosyltransferases. The accession numbers of the sequences used in this study are shown in Table S3.
4

X. Xu et al.
Fitoterapia 153 (2021) 104995
were selected based on different concerns. Firstly, the CtUGT1 gene was
cloned from C. tubulosa whose main chemical components are PhGs. To
verify whether CtUGT1 is involved in the biosynthesis of PhGs, the
common aglycones of PhGs in C. tubulosa such as tyrosol (NP1, Fig. S2)
and salidroside (NP2, Fig. S2) were firstly selected as substrates. Un-
fortunately, HPLC and HRESI-MS analyses didn’t find any glycosylated
products, which indicated that CtUGT1 may not be involved in the
biosynthesis of PhGs.
natural products, including flavonoids, phenolic compounds, couma-
rins, stilbene, anthraquinones, benzophenone, naphthalene, and iridoid
(Figs. S1-S2), using UDPG as the sugar donor. It was found that the
benzophenone substrate 4,4′-dihydroxybenzophenone (4), isoflavone
substrate genistein (5), and anthraquinone substrate aloe-emodine (6)
could also be catalyzed by CtUGT1 to form the corresponding glucosy-
lated product 4a, 5a, 6a with higher polarity (Fig. 3). HRESI-MS analysis
showed that the ion peaks of the products were all 162 amu greater than
that of the substrates, suggesting that CtUGT1 could catalyze the glu-
cosylation of these compounds to form their corresponding mono-
glucoside product. The conversion rates of 4, 5 and 6 were 4.12%,
14.14% and 23.33%, respectively (Table 1).
Secondly, based on the sequence alignment and phylogenetic anal-
ysis results (Fig. 1), CtUGT1 was phylogenetically related to lignanoid
and coumarin glycosyltransferases, which suggested that CtUGT1 is
probably a phenylpropanoid glycosyltransferase. To verify this predic-
tion, three coumarin substrates 1, 2, and 3 together with one lignanoid
substrate magnolol NP17 were tested as sugar acceptors, respectively.
Heat-inactivated enzymes (100 ◦C, 10 min) were used as a negative
control. HPLC-HRESI-MS analysis of reactions using NP17 as substrate
did not find any product. By contrast, CtUGT1 showed obvious gluco-
sylation abilities against the tested coumarin substrates (1,2,3). Using
substrate 1 as an example, as shown in Fig. 2A, a new peak (1a) with a
decreased retention time (11.46 min) compared with that of substrate 1
(19.75 min) was produced by CtUGT1 at a yield of 37.78%. Its UV ab-
sorption spectrum is corresponding to 1. HRESI-MS spectrum showed
that peak 1a exhibited a [M + H]+ at m/z 325.1029, and a [M + Na]+ at
m/z 347.0710 (calcd. 347.0737 for C15H16O8Na) with the predicted
formula of C15H16O8, which was 162 amu greater than that of 1 and
consistent with the theoretical formula of a glucosylated product
(Fig. 2A). Similarly, the glucosylated product of substrate 2 (14.21 min,
Fig. 2B) and 3 (21.92 min, Fig. 2C) was also found at the retention time
of 10.85 min and 13.80 min, with the conversion rate of 22.03% and
68.67%, respectively. The molecular mass of the products matched with
the calculated mass of glucosylated products exactly, which confirmed
that CtUGT1 could catalyze the glucosylation of coumarin compounds
1–3 (Table 1).
The sugar donor selectivity of CtUGT1 was also explored. In addition
to UDPG, six other donors, including UDP-N-acetyl galactosamine, UDP-
galactose, GDP-mannose, GDP-fucose, UDP-galacturonic acid and UDP-
rhamnose, were tested using substrates 1–6 as acceptors. The results
showed that CtUGT1 exclusively selected UDPG as the sugar donor. No
product was observed when other activated sugars were tested (data not
shown).
3.4. Biochemical properties and kinetic parameters of CtUGT1
Biochemical properties of CtUGT1 were investigated using 3 as the
sugar acceptor and UDPG as the sugar donor as indicated in Fig. 4.
Catalysis activity CtUGT1 was tested in a temperature range of 4–60 ◦C,
the optimal activity was detected at 37 ◦C (Fig. 4A). Analysis of the
enzyme activity between pH 3.0 and 9.0 showed that the optimal pH
value was observed at pH 8.0 (100 mM buffer KH2PO4-K2HPO4, Fig. 4C).
The yield of the glucosylation product 3a increased linearly within 1 h
and the growth rate was leveling off after 5 h (Fig. 4B). The apparent Km
value of CtUGT1 for 3 was 218.7 ± 7.176
μM, and Vmax was
0.02255 ± 0.0001796 nmol⋅minꢀ 1
⋅
μ
gꢀ 1
.
Subsequently, considering that most of the recently reported en-
zymes exhibited catalysis promiscuity in vitro, to further explore the
substrate spectrum of CtUGT1, expansive enzyme assays were con-
ducted towards diverse compounds belonging to different kinds of
3.5. Enzymatic preparation and structural identification of glucosylated
products
Based on the enzyme assays results, CtUGT1 tends to be a specific
Fig. 2. HPLC-UV/HRESI-MS spectra of in vitro glycosylation reactions catalyzed by CtUGT1 using UDPG as sugar donor, compound 1 (A), 2 (B), 3 (C) as
sugar acceptors.
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X. Xu et al.
Fitoterapia 153 (2021) 104995
Table 1
Characterization of enzymatic glycosylated products by HPLC and HRESI-MS analyses.
Substrate (No.)
Substrate RT (min)
Product RT (min)
Product Yield (%)
Measured m/z
Predicted formula
Theratical m/z
Error (ppm)
1
2
3
4
5
6
19.75
14.21
21.92
15.27
27.53
15.01
11.46
10.85
13.80
10.82
19.85
10.72
37.78
22.03
68.67
4.12
347.0710 [M + Na]+
339.0711 [M ꢀ H]ꢀ
339.1044 [M + H]+
375.1074 [M ꢀ H]ꢀ
477.1017 [M + HCOO]ꢀ
467.0738 [M + Cl]ꢀ
C15H16O8
C15H16O9
374.0737
339.0722
339.1074
375.1085
477.1038
467.0750
ꢀ 7.78
ꢀ 3.24
ꢀ 8.85
ꢀ 2.93
ꢀ 4.40
ꢀ 2.57
C16 18
H
O8
C19 20
C21 20
C21 20
H
O8
14.14
23.33
H
O10
O10
H
Note: the amount of the product was calculated from the product peak integral, assuming that the extinction coefficient of the glycosylated product was the same as
that of the substrate by HPLC.
Fig. 3. HPLC-UV/HRESI-MS spectra of in vitro glycosylation reactions catalyzed by CtUGT1 using UDPG as sugar donor, compound 4 (A), 5 (B), 6 (C) as
sugar acceptor.
glucosyltransferase that could catalyze the glucosylation of different
kinds of natural products. HPLC-HR-MS information of the products was
summarized in Table 1. Among them, coumarins substrates exhibited a
considerable conversion rate. The enzyme activity against coumarins
was analyzed further. It should be noted that all three tested coumarin
substrates accepted by CtUGT1 have a hydroxyl group at C-7 position,
while esculetin (2) has an extra hydroxyl group at C-6 position. The
HPLC profile of the glucosylated product of esculetin is shown in Fig. 2.
Only a single product was observed, suggesting that the glucosylation
reaction regiospecifically happened at C-6 or C-7 position. To confirm
the glycosylation site, product 2a was identified by comparison with the
authentic glucosides by HPLC-UV/HRESI-MS analysis. When adding the
authentic glucoside cichoriin (glucosylated on 7-OH position of 2) in the
reaction system, the peak overlapped with 2a, co-HPLC profile further
confirmed that the glycosylated product of 2 catalyzed by CtUGT1 is
cichoriin [33], which proved that the glucosyl moiety was specifically
transferred on C7-OH position. Product 1a was also identified by com-
parison with the reference standard. UV-HPLC spectra together with co-
HPLC analysis (Fig. 2) revealed that the chemical structure of 1a was
attributed as skimmin [34], which is the glucosylated product of 1 on
C7-OH position.
glucosylation of hydroxyl coumarin at 7-OH position.
3.6. Homology modeling, molecular docking and site-directed
mutagenesis of CtUGT1 protein
Extensive enzyme reactions revealed that CtUGT1 could catalyze the
glucosylation of three coumarins into their corresponding glucosylated
product, and the glucosylated position specifically happened at the 7-
OH position. To identify the key residues that determined the catalysis
activities of CtUGT1, homology modeling, molecular docking and site-
directed mutagenesis investigations of CtUGT1 were performed. Ho-
mologous molecular modeling for CtUGT1 was established using SWISS-
MODEL [21–25]. The crystal structure of Medicago truncatula glycosyl-
transferase UGT85H2 (PDB ID 2PQ6), determined at a resolution of
2.1 Å, showed the highest total score with maximum sequence homol-
ogy and a low E-value, was chosen as the template structure [26]. Ra-
tionality assessment of the established model was shown in the
Ramachandran plot (Fig. S6), 88.5% of the residues in the established
model are in the most favored region. The homology model of CtUGT1
consists of two similar Rossmann-type folds motifs at the N- and C-ter-
minal, respectively (Fig. 5A). The N-terminal domain has seven-stranded
Product 3a was prepared from a scaled-up reaction and structurally
characterized by HRESI-MS, 1H NMR and 13C NMR spectroscopy
(Figs. S13, S14 and Note 4). In the 1H NMR signal, compared with
substrate 3, a phenolic hydroxyl signal at the C-7 position disappeared,
and the carbon signal of C-7 shifted δ 1.0 ppm to the high field, while C-6
and C-8 signals shifted to the low field in 13C NMR. These findings
confirmed that the glucopyranosyl residue was attached to the phenolic
hydroxyl group at the C-7 position of 3 [35]. Besides, 1H NMR spectrum
parallel β sheets around by ten
α
helices. The C-terminal domain con-
helices (Fig. 5A, S7).
tains six-stranded β sheets flanked by nine
α
To further clarify the active pocket of CtUGT1, molecular docking
was performed. Since UGT85H2 only has the apo form structure,
another homologue UGT74F2 with both UDP and salicylic acid as two
ligands was selected as the control template. The UDPG was prepared as
the sugar donor. Two coumarin substrates 1 and 2 were prepared as the
sugar acceptors respectively. The binding sites of glycosyl donor and
acceptor were calculated and docking separately as shown in Fig. 5. The
results indicated that both of the two binding pockets are located inside
the deep cleft in the interaction area of the tightly packed N-terminus
showed anomeric proton signals at δ 5.00 (1H, d, J = 7.0 Hz); the sugar
H
component was indicated to be β-D-glucopyranose. This result demon-
strated CtUGT1 is a coumarin O-glucosyltransferase that catalyzed the
6

X. Xu et al.
Fitoterapia 153 (2021) 104995
Fig. 4. Effects of temperature (A), reaction time (B), and reaction buffer pH values (C) on the activities of CtUGT1. (D) Determination of kinetic parameters (Km and
Vmax values) for purified CtUGT1. Hymecromone (3) was used as the glycosyl acceptor and UDPG was used as glycosyl donor.
domain and C-terminus domain of CtUGT1 and spatially close to each
other (Fig. 5A). The C-terminus of CtUGT1 is mainly involved in contact
with the glycosyl donor as described in other plants UGTs, and most of
the surrounding residues are hydrophilic and highly conserved. Such as
the typical PSPG-box, residue W377 in this motif could form hydrogen
bonds with the glucose group of UDPG (Fig. 5B); N378 and S379 in this
motif could form hydrogen bonds with the diphosphate group of UDPG
(Fig. 5B). In addition, according to the docking results, residues of W356
and Q359 may interact with the uridine group through hydrogen bond
interaction to stabilize the donor in the active pocket. To verify the
function of these predicted key residues, a total of 16 sites were selected
and subjected to alanine scanning mutagenesis. The catalysis activities
of these mutants were tested using all six positive substrates mentioned
above. HPLC analyses showed that, when residues of G18, H19, S295,
F296, W356, C357, Q359, H374, G376, W377, N378, S379, E382, Y397
and D398 was mutated into alanine, the glycosylation activity of the
protein was totally lost towards all tested substrates. These results sug-
gested that the above 16 residues are the key residues that determined
the binding of the UDPG donor and the glycosylation activity of CtUGT1.
To identify the crucial residues in the sugar acceptor binding pocket,
molecular docking analyses were performed on 1 and 2, respectively.
These two substrates have the same coumarin core. 2 have two hydroxyl
groups at both C-6 and C-7 position while 1 only has one hydroxyl group
at the C-7 position. The overlapped docking results of these two com-
pounds were shown in Fig. 5C. They exhibited almost the same confir-
mation in the glycosyl acceptor binding pocket (1 in cyan color and 2 in
green color). Two hydrogen bonds were observed between the NE2 atom
of the imidazole ring of H19 and the OH group of coumarins at position
C-6 (~3.1 Å) and C-7(~3.0 Å) respectively (Fig. 5D). It was calculated
that H19 is much closer to the 7-OH group which will make the hydroxyl
group at the C-7 position easily to be deprotonated by H19 to form the
nucleophilic oxyanion that could attack the C1′ carbon of UDPG to
initiate the glycosylation reaction. This may explain why the glycosyl-
ation position was preferred to happen at the 7-OH position of couma-
rins substrates. Besides, it can be seen that H19 is located at the cleft of
the two active pockets, and interacts with both the substrate and UDPG
(Fig. 5B, C, D). Mutation of His19 to alanine will lead to the complete
loss of enzyme activity, which confirmed the irreplaceable role of H19
that plays a role as the crucial catalysis basis, meanwhile, stabilize the
UDPG donor. Alanine scanning mutagenesis of some other sites in the
acceptor binding pocket also resulted in no detectable enzyme activity,
including Y16, L213, F296, or significantly decrease of the enzyme ac-
tivity (> 44%) such as V14, V132, E153, T212, and I209 (Fig. S8). It is
predicted that these hydrophobic residues surrounded the aromatic ring
could form van der Waals interactions with the substrate in the binding
pocket.
4. Discussion
Glycosylation is one of the most important modification steps in
many secondary metabolites’ biosynthesis pathways. Glycosylation
could increase the bioavailability of natural products by improving their
water solubility and reducing their toxicity. Coumarin is considered the
simplest form within a huge class of naturally-occurring phenolic sub-
stances constructed from an α-pyrone ring fused with a benzene ring.
Both natural and synthetic coumarin-based compounds have attracted
much attention from medicinal chemists and drug design scientists as a
consequence of their diverse pharmacological and biological properties
[36]. Further glycosylated modification of coumarins could help to
enrich the structural diversity of these compounds and also improve
their solubility and bioavailability. In plants, such glycosylation process
is catalyzed by UDP-glycosyltransferase. Although multiple UGTs have
been identified from different plants, to date, most of the reported UGTs
were involved in the glycosylated modification of flavonoids. Only
7

X. Xu et al.
Fitoterapia 153 (2021) 104995
Fig. 5. Molecular modeling and Auto-Dock analyses of the critical residues of CtUGT1. (A) Overall modeling structure of CtUGT1. (B) Model of the CtUGT1 sugar
donor binding pocket. UDPG is shown in sticks. (C) Model of the CtUGT1 substrate binding pocket shows key residues stabilizing the sugar acceptor. Umbelliferone
(1) is shown in cyan color and esculetine (2) is shown in green color. (D) Interactions between H19 residue and UDPG donor, and hydroxyl groups at C6-OH and C7-
OH positions of coumarins. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
limited UGTs were reported to be able to accept coumarins as substrates.
In this study, a new glycosyltransferase gene CtUGT1 was cloned from
the traditional Chinese herb C. tubulosa. Sequence alignment and
phylogenetic analysis results showed that CtUGT1 is phylogenetically
distant from most of the reported flavonoid UGTs, and much close to
phenylpropanoid UGTs. Extensive enzyme assays revealed that CtUGT1
could not catalyze the glycosylation of phenylethanol compounds to
form PhGs, which is the main chemical component of C. tubulosa.
Instead, it could recognize coumarins as its substrates, and conduct the
glycosylation reaction. Structure elucidation of the products showed
that the glycosylation site catalyzed by CtUGT1 regiospecifically
happened at the C7-OH position of coumarins. Using CtUGT1, three
coumarin glycosides including skimmin (1a), cichoriin (2a) and 4-meth-
ylumbelliferyl glucoside (3a) were successfully enzymatic synthesized
and structurally identified. All these three compounds were reported as
pharmaceutically active molecules. For example, 1a has been consid-
ered to possess various pharmacological activities, including renopro-
tective activity, anti-inflammatory, anti-cancer, and antiamoebic
properties [37]. 2a showed high antibacterial activity against some
Gram–positive bacteria as Bacillus cereus and Staphyllococcus aureus
[38]. 3a can help the plants to improve their response abilities to
chemicals and toxins [39].
6, which indicated that CtUGT1 has certain substrate tolerance.
Nevertheless, CtUGT1 could not catalyze the biosynthesis of glycosides
components in C. tubulosa such as phenylethanoid glycosides (PhGs),
lignanoid glycosides and iridoid glycosides. Besides, according to the
previous investigations, no coumarin glycosides were isolated from
C. tubulosa. So, the in vivo function of CtUGT1 still needs to be further
explored. The glucosylation activity of CtUGT1 towards coumarin sub-
strates make this enzyme a useful tool for the enzymatic glycosylation
modification of coumarins, and also indicated that that plant secondary
metabolic enzymes could perform inestimable catalysis activities that
are far beyond their native functions.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgment
This work was financially supported by Beijing Natural Science
Foundation (Grant No. 7192112); Young Elite Scientists Sponsorship
Program by CAST (Grant No. CACMꢀ 2018ꢀ QNRC1ꢀ 02); National
Natural Science Foundation of China (Grant No. 81402809); China
Scholarship Council State Scholarship Fund (Grant No. 201906555010)
and Science Foundation for Elite Young Scholars of Beijing University of
Chinese Medicine (Grant No. 2018-JYB-XJQ006).
In addition to coumarins, extensive enzyme assays found that
CtUGT1 also showed observable glucosylation activity towards benzo-
phenone substrate 4, isoflavone substrate 5 and anthraquinone substrate
8

X. Xu et al.
Fitoterapia 153 (2021) 104995
glycosyltransferase from Trollius chinensis, Angew. Chem. Int. Ed. Eng. 58 (2019)
11513–11520, https://doi.org/10.1002/anie.201905505.
Appendix A. Supplementary data
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org/10.1016/j.fitote.2021.104995.
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