Isolation and characterization of a glycosyltransferase with specific catalytic activity towards flavonoids from Tripterygium wilfordii

Article abstract of DOI:10.1080/10286020.2019.1642330

Flavonoids are important secondary metabolites that exist in many medicinal plants. Flavonoid glycosyltransferases can transfer sugar moieties to their parent rings, producing various flavonoid glycosides with significant pharmacological activities. Here, we report the molecular cloning of the O-glycosyltransferase TwUGT2 from Tripterygium wilfordii and its catalytic activity was explored by heterologous expression in E. coli. The results showed that TwUGT2 has specific glycosyltransferase activity towards C-3 and 7 hydroxyl groups of flavonoids, thereby converting quercetin and pinocembrin into isoquercitrin and pinocembrin 7-O-beta-D-glucoside, respectively. The identification of TwUGT2 will provide a useful molecular tool for synthetic biology and contribute to drug discovery. (Figure presented.).

Full text of DOI:10.1080/10286020.2019.1642330

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Isolation and characterization of a
glycosyltransferase with specific catalytic activity
towards flavonoids from Tripterygium wilfordii
Yun Lu, Bao-Wei Ma, Jie Gao, Li-Chan Tu, Tian-Yuan Hu, Jia-Wei Zhou, Yuan
Liu, Yu-He Tu, Zhao-Shou Lin, Lu-Qi Huang & Wei Gao
To cite this article: Yun Lu, Bao-Wei Ma, Jie Gao, Li-Chan Tu, Tian-Yuan Hu, Jia-Wei Zhou, Yuan
Liu, Yu-He Tu, Zhao-Shou Lin, Lu-Qi Huang & Wei Gao (2019): Isolation and characterization of
a glycosyltransferase with specific catalytic activity towards flavonoids from Tripterygiumꢀwilfordii,
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2019.1642330
To link to this article: https://doi.org/10.1080/10286020.2019.1642330
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1642330
Isolation and characterization of a glycosyltransferase
with specific catalytic activity towards flavonoids from
Tripterygium wilfordii
a
a
a
a
a
a
Yun Lu
, Bao-Wei Ma , Jie Gao , Li-Chan Tu , Tian-Yuan Hu , Jia-Wei Zhou ,
a
b
c
d
a,e,f
Yuan Liu , Yu-He Tu , Zhao-Shou Lin , Lu-Qi Huang and Wei Gao
a
b
School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China; Yong’an
c
State Owned Forest Farm, Yong’an 366000, China; Taoyuan State Owned Forest Farm, Datian
3
Medica, China Academy of Chinese Medical Sciences, Beijing 100069, China; School of
Pharmaceutical Sciences, Capital Medical University, Beijing 100069, China; Advanced Innovation
d
65000, China; State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia
e
f
Center for Human Brain Protection, Capital Medical University, Beijing 100069, China
ABSTRACT
ARTICLE HISTORY
Flavonoids are important secondary metabolites that exist in many
medicinal plants. Flavonoid glycosyltransferases can transfer sugar
moieties to their parent rings, producing various flavonoid glyco-
sides with significant pharmacological activities. Here, we report the
molecular cloning of the O-glycosyltransferase TwUGT2 from
Tripterygium wilfordii and its catalytic activity was explored by heter-
ologous expression in E. coli. The results showed that TwUGT2 has
specific glycosyltransferase activity towards C-3 and 7 hydroxyl
groups of flavonoids, thereby converting quercetin and pinocembrin
into isoquercitrin and pinocembrin 7-O-beta-D-glucoside, respect-
ively. The identification of TwUGT2 will provide a useful molecular
tool for synthetic biology and contribute to drug discovery.
Received 14 March 2019
Accepted 7 July 2019
KEYWORDS
Flavonoid glycosyltransfer-
ase; catalytic activity;
quercetin; pinocembrin
CONTACT Wei Gao
weigao@ccmu.edu.cn
School of Traditional Chinese Medicine, Capital Medical University,
Beijing 100069, China
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2019.1642330.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Y. LU ET AL.
1. Introduction
Glycosylation is one of the major biochemical modifications [1], and it is an effective
approach to improve the pharmacological features of natural chemicals by increasing
their water solubility [2]. Glycosyltransferases (GTs) catalyze the formation of a glyco-
sidic bond between a sugar and an acceptor including a wide range of biomolecules
such as proteins, lipids and small molecules [3]. GTs involved in secondary metabol-
ism often display obvious promiscuity with structurally altered donors and/or accept-
ors [4]. Glycosylation is a powerful approach for the synthesis or modification of
biologically active compounds, which alters the physicochemical characteristics, par-
ticularly of largely hydrophobic compounds, such as flavonoids [5]. It has been
reported that the water-solubility and stability of bavachin was enhanced by glycosy-
lation via the UDP-glycosyltransferase YjiC [6].
Secondary metabolites like flavonoids, terpenoids, and alkaloids exhibit a wide
range of pharmacological effects including immunosuppressive [7], anti-inflammatory
[
8], antimicrobial [9], antioxidant activities, and so on [10]. Additionally, glycosyla-
tion of biologically relevant compounds is of considerable importance for the discov-
ery of new drugs or the production of valuable glycosides. To date, a significant
number of glycosyltransferases have been isolated from different species and they
were classified according to their functions. For example, three new glucosyltransfer-
ases from Carthamus tinctorius exhibited robust catalytic promiscuity towards benzy-
lisoquinoline alkaloids, which were used as enzymatic tools for the glycosylation of
bioactive products [11]. And a diterpene glycosyltransferase ApUGT was reported to
have a broad substrate scope, which could generate O-, S-, and N-glycosides [12].
Similarly, a multifunctional flavonoid glycosyltransferase from Ornithogalum cauda-
tum was observed to exhibit both glycosyltransferase and glycosidase activities [13].
However, there are few reports on flavonoid glycosyltransferases from Tripterygium
wilfordii, which is a traditional Chinese medicine and belongs to the Celastraceae
family [14].
In this work, we screened glycosyltransferases of Tripterygium wilfordii through
transcriptome analysis and a sequence of glycosyltransferase was successfully cloned,
namely TwUGT2. We explored the catalytic capabilities of glycosyltransferase
TwUGT2 with different kinds of chemical components as substrates. As a result,
TwUGT2 showed the specific catalytic activity towards two flavonoids including quer-
cetin and pinocembrin, which indicated that TwUGT2 might be a useful molecular
tool for production of various flavonoid glycosides.
2. Results and discussion
2.1. Sequence analysis
The cDNA with the open reading frame of TwUGT2 (GenBank accession number:
MK035745) was 1428 bp, coding for 475 amino acid (aa) residues, with a relative
molecular weight (MW) of 52.681 kDa and isoelectric point (pI) of 6.25. The second-
ary structure of the protein was mainly composed of alpha helices (42.74%) and ran-
dom coils (30.53%), along with extended strands (16.42%) and beta turns (10.32%).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 1. Alignment of amino acid sequences of highly conserved regions in TwUGT2 and other
plant UGTs. The proteins and GenBank accession numbers are: UGTPg21 (AKA44598), UGT89B2
(Q6VAA5), UGT73B2 (Q94C57), UGT73B5 (Q9ZQG4.1), AcUGT (PSR89095). The PSPG motif is shown
in the blue box.
The amino acid sequence of TwUGT2 protein shared 64% and 61% identity with the
UDP-glycosyltransferase from Panax ginseng (UGTPg21, AKA44598.1) and Stevia
rebaudiana (UGT89B2, Q6VAA5.1), respectively. It is reported that secondary struc-
ture prediction can provide important information about the functions of a given
protein [15]. 3D modeling of TwUGT2 was conducted using the structure 5nlm.1.A
as template, an indoxyl UDP-glucosyltransferase with which it shares 33.18%
sequence identity [16]. The modeled 3D structure is shown in supplementary material
Figure S1. Multiple sequence alignment revealed that the plant secondary product gly-
cosyltransferase motif (PSPG) is conserved among the glycosyltransferase sequences
(Figure 1). The PSPG motif has been proposed as the sugar donor binding site [17]
and plays an important role in the catalytic function of UGTs [18].
2
.2. Phylogenetic analysis
To predict the function of glycosyltransferase TwUGT2, a phylogenetic tree (Figure 2)
was constructed based on the full-length protein sequences of 21 functionally

4
Y. LU ET AL.
Figure 2. Phylogenetic analysis of glucosyltransferase TwUGT2 and other GTs of known functions.
The tree was constructed using MEGA 6.0 software with an evaluation method of Bootstrap for
1000 times.
characterized GTs from other plants (supplementary material Table S2), including fla-
vonoid glucosyltransferases, coumarin glucosyltransferases, terpenoid glycosyltransfer-
ases, lignan glucosyltransferases as well as sterol glucosyltransferases. These GTs were
grouped into five main branches and TwUGT2 clustered together with flavonoid glu-
cosyltransferases, such as flavonoid 7-O-glucosyltransferase (BAA83484) and flavon-
oid 3-O-glucosyltransferase (Q2V6J9, ARW73634), which encouraged us to speculate
that TwUGT2 might have specific glycosyltransferase activity towards flavonoids.
2
.3. In vitro enzyme assays of TwUGT2
To investigate the catalytic activity of the glycosyltransferase, recombinant TwUGT2
protein was obtained by heterologous expression in E. coli. After purification via resin
binding followed by SDS-PAGE analysis (supplementary material Figure S2), in vitro
enzyme assays of the recombinant TwUGT2 protein were performed. The substrate
library (Figure 3) encompassed 8 flavonoids (1–8), 2 coumarins (9–10), 1 lignan (11),
1
phenolic compound (12), and 4 terpenoids (13–16). Subsequently, the products of
catalysis were analyzed by UPLC-Q-TOF-MS.
Surprisingly, TwUGT2 showed specific glycosyltransferase activity towards two fla-
vonoids (1–2) while no products were detected with any of the other substrates.
Especially, TwUGT2 showed the highest catalytic activity towards quercetin (1) and
the product (1a) could be identified as isoquercitrin (quercetin-3-O- glucoside) in

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 3. Screening the glycosyltransferase activity of TwUGT2 against a substrate library.
Compounds 1–16 represent the substrates: (1) Quercetin; (2) Pinocembrin; (3) Genistein; (4)
Liquiritigenin; (5) Kaempferol; (6) Phloretin; (7) Daidzein; (8) Luteolin; (9) 4-Methylumbelliferone;
(10) Nodakenitin; (11) Arctigenin; (12) Rhapontigenin; (13) Triptolide; (14) Celastrol; (15)
Triptophenolide; and (16) Neotriptophenolide. The corresponding products generated by TwUGT2
catalysis are marked with 1a and 2a, respectively.
Figure 4(A) by comparing the retention times and mass spectra with an authentic ref-
erence standard (supplementary material Figure S3(A)). The flavonoid glycoside iso-
quercitrin is commonly present in medicinal herbs, where it exerts significant
pharmacological activities such as anti-tumour effects [19], and it may be potentially
used for preventing disorders of lipid metabolic and non-alcoholic fatty liver disease
[
20]. Isoquercitrin (quercetin-3-O-glucoside) showed higher levels of antioxidant
00
activity than quercitrin (quercetin-3-O-rhamnoside) because of the 6 -OH (x-OH)
group [21]. Although isoquercitrin is widely distributed, its content in plants is
extremely low [22]. Therefore, it is difficult to obtain isoquercitrin by direct separ-
ation and extraction, and TwUGT2 is a potential enzymatic tool for its synthesis.
In addition, TwUGT2 was able to attach sugars to the C-7 hydroxyl groups of
pinocembrin (2). The corresponding product (2a) was identified as pinocembrin 7-O-
beta-D-glucoside (Figure 4(B)) by comparing the retention times and mass spectra
with an authentic reference standard (supplementary material Figure S3(B)). It has
been reported that pinocembrin-7-O-b-D-glucoside (PCBG) has significant hepato-
protective effects as well as antimicrobial and antioxidant activities [23].
Therefore, in vitro enzyme assays showed that TwUGT2 was able to attach sugars
to C-3 and 7 hydroxyl groups of quercetin and pinocembrin, respectively. These
results were generally in agreement with the bioinformatical analysis. To our best

6
Y. LU ET AL.
Figure 4. In vitro enzyme assays for determining activity of glucosyltransferase TwUGT2 with substrates:
A (quercetin) and B (pinocembrin). (a) Chemical structures of sugar acceptors and corresponding glycosy-
lated products; (b) UPLC analysis of substrate and glycosylated products of recombinant TwUGT2, using
2
UDP-glucose as the sugar donor. (c) MS of O-glycosylated products. (d) MS of O-glycosylated products.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
knowledge, this is the first study of flavonoid glucosyltransferase from Tripterygium
wilfordii, and the results provide a new genetic resource for the production of differ-
ent types of O-glycosylated flavonoids using synthetic biology.
3
. Experimental
.1. Plant materials
Suspensions cells of T. wilfordii were cultured in Murashige and Skoog (MS) medium
pH 5.8–6.0) containing sucrose (30 g/L), 2,4-dichlorophenoxyacetic acid (2,4-D,
3
(
0
.5 mg/L), kinetin (KT, 0.1 mg/L), and indole-3-butytric acid (IBA, 0.5 mg/L), shaking
ꢀ
at 120 rpm (Eppendorf, 5810R, Germany) at 25 C in the dark. The suspension cells
(2 g) were subcultured in the MS medium (25 ml) every 20 days [24].
3
.2. RNA isolation and cDNA cloning of TwUGT2
The total RNA was isolated using the EastepVR Super Total RNA Extraction Kit
Promega Biological Technology Co. Ltd., Beijing, China) according to the man-
(
ufacturer’s instructions. Total RNA was used as the template to be reversely tran-
scribed into cDNA using the FastKing RT Kit (Tiangen Biotech, Beijing, China)
following the manufacturer’s protocol. Specific primers (supplementary material Table
S1) were designed using Primer Premier 5.0 software according to the selected
sequence. The cDNA served as a template for amplifying the open reading frame
using 2 ꢁ KAPA HiFi HotStart ReadyMix (KAPA Biosystems, USA) with a procedure
ꢀ
ꢀ
comprising an initial 3-min step; followed by 35 cycles of 98 C for 20 s, 53 C for
1
5
ꢀ
ꢀ
ꢀ
5 s, and 72 C for 2 min; a final elongation at 72 C for 7 min and 4 C hold. Then,
ll of the PCR product was used for a second PCR amplification using the same
PCR conditions. The PCR products were purified and ligated into the pEASY-Blunt
Zero cloning vector (TransGen Biotech, Beijing, China) and transformed into E. coli
Trans1-T1 competent cells (TransGen Biotech, Beijing, China). Positive bacterial colo-
nies were selected on ampicillin plates and confirmed by sequencing.
3
.3. Bioinformatics analysis of TwUGT2 gene sequence
The open reading frame was identified using ORF Finder (https://www.ncbi.nlm.nih.
gov/orffinder/). The nucleotide and amino acid sequences were searched using
BLAST on NCBI (http://www.ncbi.NLM.NIH.gov). The theoretical molecular weight
(
(
Mw) and isoelectric point (pI) were determined using the Compute pI/Mw tool
http://Web.ExPASy.org/compute_pi/). Secondary structures were predicted using
PRABIGERLAND (https://npsa-prabi.ibcp.fr/) and the 3-dimensional structural model
was constructed using Swiss-Model (http://swissmodel.expasy.org/). Multiple sequence
alignments were carried out by DNAMAN software. Phylogenetic analysis was per-
formed using MEGA 6.0 software with the neighbor-joining method [25].

8
Y. LU ET AL.
3.4. Heterologous expression and purification of the TwUGT2 protein
The open reading frame of TwUGT2 was amplified under the guidelines of
2
ꢁ KAPA HiFi HotStart ReadyMix (KAPA Biosystems, USA) with the pEASY-Blunt-
TwUGT2 vector as a template. The PCR product was gel-purified, digested with
BamH I and PstI HF and ligated directly into the expression vector pMAL-c2X with
a maltose-binding protein (MBP) tag to obtain the recombinant plasmid pMAL-c2X-
TwUGT2. Then, the recombinant plasmid was transformed into E. coli BL21 (DE3)
chemically competent cells (TransGen Biotech, Beijing, China) and the empty vector
pMAL-c2X was used as a control.
After verification by sequencing, single colonies of the transgenic E. coli strain
ꢃ1
were cultured in 300 ml LB medium containing 100 mgꢂL of ampicillin. The bac-
ꢀ
teria grown at 37 C with shaking (220 rpm) until the OD600 value reached 0.6–0.8,
at which point the expression of the recombinant protein was induced by the add-
ition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and the cells were incu-
ꢀ
bated at 16 C for 16 h. Then, the cells were harvested by centrifugation (12000 ꢁ g,
ꢀ
4
min, 4 C) and washed with Tris-HCl buffer (100 mM, pH ¼ 7.5) containing 1 mM
DTT. The cell pellets were resuspended in 25 ml of resuspension buffer (50 mM Tris-
HCl, 0.1 mM Na EDTA 2H O 150 mM NaCl, 1 mM DTT, 5% glycerol, 1 mM PMSF,
ꢄ
2
2
pH ¼ 7.5). The resuspended cells were added with chicken egg white lysozyme
ꢃ1
(
0.5 mgꢂml ) and incubated for 20 min on ice. After adding Triton X-100 (0.1%) and
ꢃ1
NaCl (0.5 molꢂL ) to the mixture, the total protein was extracted by ultrasonic dis-
ruption (lysed for 5 s, paused for 5 s) for 30 min in an ice bath.
ꢀ
After centrifugation at 12000 ꢁ g and 4 C for 20 min, the supernatant was mixed
with 1.0 ml of amylose resin (New England Biolabs, Beijing, China) and shaken
(
(
180 rpm) on ice for 1–2 h. Then, the resin was washed with 15 ml of washing buffer
ꢄ
50 mM Tris-HCl, 0.1 mM Na EDTA 2H O, 500 mM NaCl, 1 mM DTT, 5% glycerol,
2
2
pH ¼ 7.5) and 15 ml of resuspension buffer for 3 times, respectively. Then, it was
ꢄ
washed with 2 ml of elution buffer A (50 mM Tris-HCl, 0.1 mM Na EDTA 2H O,
2
2
1
50 mM NaCl, 1 mM DTT, 1 mM maltose, 5% glycerol, pH ¼ 7.5) in order to remove
nonspecifically bound impurities. Subsequently, 5 ml of elution buffer B (50 mM Tris-
HCl, 0.1 mM Na EDTA 2H O, 150 mM NaCl, 1 mM DTT, 10 mM maltose, 5% gly-
ꢄ
2
2
cerol, pH ¼ 7.5) was added to the resin twice for elution of the recombinant protein.
Finally, the recombinant protein was concentrated using an Amicon Ultra-50K filter
ꢀ
(
Millipore) and mixed with 20% (v/v) glycerol, then stored at ꢃ80 C. The purity of
the recombinant TwUGT2 protein was checked via an electrophoresis on 8% SDS-
PAGE. The protein concentration was measured using a modified Bradford Protein
Assay Kit (Sangon Biotech, Shanghai, China).
3
.5. Enzyme assay
Enzyme assays were performed in 100 ll of the reaction mixture, containing 100 mM
Tris-HCl buffer (pH 7.5), 0.5 mM sugar donor (uridine diphosphate glucose, UDPG),
50 lM substrate and 50 mg purified recombinant TwUGT2 protein. The reaction mix-
ture with the addition of the purified empty vector protein was set as a control. The
ꢀ
reactions were incubated at 35 C for 24 h and terminated by the addition of 200 ll of

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
UPLC-grade methanol. After centrifuging at 10,000 g for 2 min, the supernatant was
ꢀ
filtered through a 0.22-mm nylon syringe filter and temporarily stored at 4 C.
3
.6. UPLC-Q-TOF-MS analysis of the catalytic products
The conditions of ultraperformance liquid chromatography analyses were described
in supplementary material Table S3. The UPLC separation was performed using a
Waters Acquity UPLCTM I-Class system (Waters Corp., Milford, MA, USA) with an
ACQUITY UPLCVR HSS T3 column (2.1 mm 100 mm, 1.8 lm, USA) and samples were
scanned with the absorption spectrum set from 190 to 450 nm. The TOF MS experi-
ments were performed using a Xevo G2-S QTOF MS system (Waters Corp., Milford,
MA, USA). Both positive and negative ion modes were employed to ionize the chem-
ical compounds. The detection conditions were as follows: capillary voltage at 1.0 kV,
ꢀ
ꢀ
cone voltage 40 V, source temperature 120 C, desolvation temperature 450 C, cone
gas flow 50 L/h, desolvation gas flow 800 L/h. TOF-MS was set from 50 to 1200 m/z.
The scan time was 0.2 s. Sample data were collected and processed using
MassLynx software.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the Support Project of High-level Teachers in Beijing
Municipal Universities in the Period of 13th Five-year Plan (CIT&TCD20170324), the
National Natural Science Foundation of China (81773830), the Beijing Natural Science
Foundation Program and Scientific Research Key Program of Beijing Municipal Commission
of Education (KZ201710025022), the National Program for Special Support of Eminent
Professionals, and the Key project at central government level: The ability establishment of
sustainable use for valuable Chinese Medicine Resources (2060302-1806-03) to LH.
ORCID
Yun Lu
http://orcid.org/0000-0003-0664-2664
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