Exploring the aglycon promiscuity of a new glycosyltransferase from Pueraria lobata

Article abstract of DOI:10.1016/j.tetlet.2016.02.088

Enzymatic glycosylation catalyzed by glycosyltransferases has great potential for creating bioactive glycosylated small-molecule compounds. Here, we highlight the aglycon promiscuity of a new glycosyltransferase (PlGT7) from Pueraria lobata. PlGT7 exhibited the capability to glycosylate 26 structurally diverse drug-like scaffolds and simple phenolics with UDP-glucose to form O-, S-, and N-glycosides. The reversibility of PlGT7 coupled with its substrate flexibility was also exploited to generate bioactive glucosides with simple sugar donor. These studies indicate the significant potential of an enzymatic approach to the glycosylation of bioactive natural and unnatural products in drug discovery.

Full text of DOI:10.1016/j.tetlet.2016.02.088

Tetrahedron Letters xxx (2016) xxx–xxx
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Exploring the aglycon promiscuity of a new glycosyltransferase from
Pueraria lobata
b,
Lili Sun a,y, Dawei Chen b,y, Ridao Chen b, Kebo Xie b, Jimei Liu b, Lin Yang a, , Jungui Dai
⇑
⇑
a College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, PR China
b State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, Xian Nong Tan Street 1, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Enzymatic glycosylation catalyzed by glycosyltransferases has great potential for creating bioactive gly-
cosylated small-molecule compounds. Here, we highlight the aglycon promiscuity of a new glycosyl-
transferase (PlGT7) from Pueraria lobata. PlGT7 exhibited the capability to glycosylate 26 structurally
diverse drug-like scaffolds and simple phenolics with UDP-glucose to form O-, S-, and N-glycosides.
The reversibility of PlGT7 coupled with its substrate flexibility was also exploited to generate bioactive
glucosides with simple sugar donor. These studies indicate the significant potential of an enzymatic
approach to the glycosylation of bioactive natural and unnatural products in drug discovery.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 24 January 2016
Revised 20 February 2016
Accepted 23 February 2016
Available online xxxx
Keywords:
Enzyme catalysis
Glycosylation
Plant glycosyltransferase
Aglycon promiscuity
Bioactive glycosides
Sugar moieties often play an essential role in the biological
activity, specificity, and pharmacological properties of many natu-
ral products.1 Attaching the sugar moieties toward unnatural agly-
cons also holds promise for creating new glycosides with novel
biological activities as drug leads.2 Hence, efforts to develop cost-
effective glycosylation strategies have been vigorously pursued in
the chemical and enzymatic synthesis for decades.3 Compared
with chemical glycosylation, enzymatic glycosylation catalyzed
by glycosyltransferases (GTs) has attracted an increasing interest
due to its high efficiency and regio-selectivity under mild condi-
tions.4 A significant process has been made recently in the glycosy-
lation of both natural and unnatural products by GTs from
microbes.5 However, enzymatic glycosylation has been primarily
restricted by enzyme specificity and the availability of suitable
GTs for the targets of interest.6 Studies on plant GTs have revealed
that dramatically varied GTs are involved in plant secondary meta-
bolism and considered to have extensive application prospects.7
However, only a handful of plant GTs have been cloned and func-
tionally verified. Furthermore, most of the reported plant GTs show
relatively narrow substrate spectra and a few plant GTs have been
applied as powerful tools in the glycodiversification of natural
products with structural diversity.8 Thus, mining GTs from plants
with aglycon promiscuity is an important approach to generate
structurally diverse bioactive glycosylated natural/unnatural prod-
ucts. Herein, we report a new glycosyltransferase (PlGT7) from
Pueraria lobata, which can not only glycosylate numerous struc-
turally different aglycons with UDP-a-glucose (UDP-Glc) to gener-
ate O-, S-, and N-glucosides, but also catalyze the reverse reaction
along with its use in the formation of bioactive O-glucosides using
a simple sugar donor.
P. lobata is a traditional Chinese herb with profound pharmaco-
logical effects, including antioxidant, anti-inflammatory, antidia-
betic, estrogenic activities, and anti-cardiovascular diseases.9
Most pharmacological activities of P. lobata are due to the presence
of isoflavone C- and O-glycosides, such as puerarin (daidzein 8-C-
glucoside), daidzein (daidzein 7-O-glucoside), daidzein 40-O-glu-
coside, and genistin (genistein 7-O-glucoside).10 The structurally
diverse glycosylated secondary metabolites imply the existence
of corresponding GTs, which inspired us to search for GTs with
interesting catalytic properties.
To clone permissive GTs from P. lobata, a degenerate PCR primer
for 30-RACE (Table S1 in the Supplementary material) was designed
based on the conserved plant secondary product glycosyltrans-
ferases (PSPG) motif (Fig. S1 in the Supplementary material).11
Combined with 50-RACE, 13 new P. lobata GT genes (PlGTs) were
successfully cloned by RT-PCR amplification using the total RNA
from P. lobata leaves as a template and heterologously expressed
in Escherichia coli as described in the Supplementary material (SM).
To study the glycosylation capability of the PlGTs in vitro, UDP-
Glc donor along with daidzein (1) and genistein (2), which are the
⇑
Corresponding authors. Tel.: +86 10 63165195; fax: +86 10 63017757.
E-mail addresses: 15116995486@163.com (L. Yang), jgdai@imm.ac.cn (J. Dai).
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.tetlet.2016.02.088
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sun, L.; et al. Tetrahedron Lett. (2016), http://dx.doi.org/10.1016/j.tetlet.2016.02.088

2
L. Sun et al. / Tetrahedron Letters xxx (2016) xxx–xxx
main aglycon components of P. lobata, were each incubated with
the crude recombinant enzymes, and analyzed by HPLC–UV/MS
(high performance liquid chromatography–UV absorption/mass
spectrometry), respectively. Of the 13 recombinant PlGTs, only
PlGT7 exhibited glycosylation activity toward daidzein (1) and
genistein (2) with high conversion rates (Fig. 1). The glycosylated
products 1a and 1b were prepared via the preparative-scale reac-
tions, and their structures including the attached positions of the
glucosyl moiety and anomeric stereochemistry, were illuminated
by MS, 1H and 13C NMR spectroscopic data analyses and compared
with the reported data.12 The large coupling constants (J = 6.4,
7.1 Hz) of the anomeric protons (Figs. S24–S27 in SM) indicated
the formation of the b-anomers and the inversion mechanism of
PlGT7. The cDNA sequence of PlGT7 (1425 bp, GenBank accession
No. KU306330) contained an ORF encoding 474 amino acids, and
the PlGT7 showed the highest identity (86%) to a predicted UDP-
glycosyltransferase from Glycine max. Given the glycosylation
activity to daidzein (1) and genistein (2), of which glycosylated
products naturally occur in P. lobata, and the high sequence iden-
tities to other reported isoflavonoid glycosyltransferases (Fig. S2
A
B
R2
O
O
R3
OH
OH
HO
O
O
R1
O
O
R2
HO
O
O
R1
R3
HO
R2
1
R1 = H; R2 = R3 = OH
R1
OH
OH
R
1a
1b
2
R1 = H; R2 = OGlc; R3 = OH
5 R1 = R3 = OH; R2 = H
OH
O
R
R
1 = H; R2 = OH; R3 = OGlc
1 = R2 = R3 = OH
5a R1 = OH; R2 = H; R3 = OGlc
8
R1 = OH; R2 = H
3
4
R = OH
R = H
8a
R
1 = OGlc; R2 = H
6
R1 = R2 = H; R3 = OH
7
R
OH
R2
R3
O
R5
HO
O
O
OH
HO
HO
N
O
O
N
R1
R4
O
H
9
10
O
O
R1 = R4 = H; R2 = R3 = R5 = OH
O
O
HO
12
O
R1 = R2= R4 = R5 = OH; R3 = H
HO
11 R1 = R2 = R3 = OH; R4 = R5 = H
11a R1 = OGlc; R2 = R3 = OH; R4 = R5 = H
11b R2 = OGlc; R1 = R3 = OH; R4 = R5 = H
H
O
14
OH
13
15 R=H
16 R=Ethyl
HO
OH
N+
R2O
OH
R2O
OH
O
R2O
OH
O
RO
H
OH
O
H
H
OR1
O
OR1
OR1
O
RO
22 R1 = R2 = H
22a R1 = Glc; R2 = H
22b R1 = H; R2 = Glc
21
20 R1 = R2 = H
20a R1 = Glc; R2 = H
20b R1 = H; R2 = Glc
R1 = R2 = H
17 R = CH3
18 R = H
19 R = H
21a
R1 = Glc; R2 = H
19a R = Glc
21b R1 = H; R2 = Glc
R2O
OH
NHR
R2O
OH
O
SR
OH
O
OR1
O
HO
HO
Cl
Cl
OR1
24 R1 = R2 = H
24a R1 = R2 = Glc
24b R1 = H; R2 = Glc
O
Cl
Cl
23
R1 = R2 = H
OH
23a R1 = R2 = Glc
23b R1 = Glc; R2 = H
23c R1 = H; R2 = Glc
26 R = H
25 R = H
Glc = -D-Glucosyl
β
26a R = Glc
25a R = Glc
Figure 1. Exploring the aglycon promiscuity of PlGT7. (A) Conversions of glycosylated products catalyzed by PlGT7. Members are listed with the number which
corresponding to the structures listed in part B. The columns with different color represent the percent conversion of different products for each glycosylation reaction. ‘‘⁄”
represents the glycosylated products that were prepared and confirmed by MS, and NMR spectroscopic data analyses. (B) The structures of the library members and
corresponding glycosylated products.
Please cite this article in press as: Sun, L.; et al. Tetrahedron Lett. (2016), http://dx.doi.org/10.1016/j.tetlet.2016.02.088

L. Sun et al. / Tetrahedron Letters xxx (2016) xxx–xxx
3
in SM), the PlGT7 can be tentatively validated as an isoflavonoid
glycosyltransferase.
Deglycosylation
OH
OH
O
HO
HO
O
O
Recombinant His6-PlGT7 was purified by His-tag affinity chro-
matography and analyzed by SDS–PAGE (Fig. S3 in SM) for the fur-
ther study of its biochemical characteristics. PlGT7 displayed the
maximum activity at pH 9.0 and 40 °C, and was divalent-cation-
independent (Fig. S4 in SM). PlGT7 exhibited the calculated Km val-
HO
HO
Aglycon
UDP
O
OH
NO2
OH
27
Aglycon
PlGT7
HO
OH
ues of 383.3
lM and 310.5 lM for daidzein (1) and genistein (2),
UDP-Glc
NO2
respectively (Fig. S5 in SM).
27a
Aglycon
To investigate the aglycon promiscuity and the synthetic utility
of PlGT7 in vitro, a representative acceptor library with structurally
diverse drug-like scaffolds was established. In this study, 26 differ-
ent members (their structures were shown in Fig. 1), including iso-
flavonoids (1, 2), flavonoids (3–6), chalcones (7), xanthones (8),
benzophenones (9–11), lignans (12, 13), coumarins (14), alkaloids
(15–18), steroid (19), acetophenone (20), valerophenone (21),
deoxybenzoin (22), naphthalene (23), benzoate (24), and simple
aromatics with nucleophilic groups of –NH2 (25) and –SH (26),
were used for enzymatic assays.
Glycosylation
Figure 2. One-pot reactions catalyzed by PlGT7. The bioactive glycosides were
generated from the simple sugar donor (27) with a catalytic amount of UDP. The
aglycons could be magnolol (12) or aesculetin (14). The HPLC–MS chromatograms
(Figs. S23–S24) are shown in the Supplementary material.
Table 1
Neuroprotective effects of phenylpropyl-2,4,6-trihydroxybenzoate (24) and its glu-
coside (24b) on the glutamate-induced toxicity in SK-N-SH cells
An initial hint about the enzyme’s broad capability for glycosy-
lation was provided by the HPLC–UV/MS analysis (Figs. 1 and S6–
S21 in SM), which revealed that PlGT7 could glycosylate all library
members, including 19 different types of structures. Moreover,
PlGT7 exhibited a high substrate conversion rate (>60%) with 16
out of 26 substrates and 15 polyhydroxy phenolic acceptors (1–7,
9–11, 20–24) have at least two glycosylated products, respectively.
Of particular note is that the enzyme exhibited the capability to
glycosylate natural ‘‘drug-like” scaffolds including magnolol (12),
epipodophyllotoxin (13), 10-hydroxycamptothecine (15), 7-ethyl-
10-hydroxycamptothecin (16), jatrorrhizine hydrochloride (17),
demethyleneberberine (18), estriol (19), and even chemical syn-
thesis compounds (21–24). Although there exist engineered GT
from microbe with glycosylation activities toward alkaloids and
steroids,5a to the best of our knowledge, this is the first report on
plant GT (PlGT7) capable of catalyzing alkaloids and steroid.
Interestingly, PlGT7 also exhibited N- and S-glycosylation
Compound
Concentration (
l
M)
Cell viability (%)
Increased cell
viability (%)
Control
0
100
56.64 4.34
0
0
2.7 ꢀ 104
L
-Glutamic acid
Resveratrola
10
10
10
71.29 4.70
73.60 4.41
68.83 5.38
26.03
30.16*
21.54
24b
24
a
Resveratrol was used as a positive control.
P <0.05.
*
glycosides through one-pot reactions, two bioactive aglycons (12,
14) exemplified with PlGT7, simple aromatic glycoside (27) and
UDP, respectively (Figs. S23–S24 in SM). In the coupled reactions
(Fig. 2), UDP-Glc was generated by PlGT7-catalyzed deglycosyla-
tion to 27 with a catalytic amount of UDP, and the sugar moiety
was intermediately transferred to the targeted aglycon acceptors
through glycosylation catalyzed by PlGT7, in which the corre-
sponding glycosides were obtained in high yields. These results
indicated that the coupled reactions could be served as cost-effec-
tive and applicable approach to generate bioactive glycosides with-
out adding expensive UDP-Glc.
As mentioned above, there was no C-glycosylated flavone and
isoflavone product detected and isolated in the 13 new GT-cat-
alyzed reactions, especially for puerarin, a major bioactive C-glyco-
sylated isoflavone in P. lobata when daidzein (1) as the postulated
aglycon substrate. It suggests that more PlGTs should be further
cloned and functionally identified. Moreover, the 8-C-glycosylation
in the biosynthesis of puerarin might occur at the stage of 2-
hydroxylisoflavanone,10c the biosynthesis of puerarin should be
further investigated by combining the new PlGTs with isoflavone
synthase (IFS).
In summary, the aglycon promiscuity of a new glycosyltrans-
ferase from P. lobata, PlGT7, was highlighted. PlGT7 exhibited
robust glycosylation activity toward numerous natural and unnat-
ural drug-like compounds and simple phenolics, and the ability to
generate structurally diverse O-, N-, and S-glucosides. The
reversibility of PlGT7 coupled with its catalytic promiscuity was
also exploited as a powerful biocatalyst for the enzymatic synthe-
sis of bioactive glycosides. The present studies not only demon-
strate the application prospect in enriching the structural
diversity of natural/unnatural products and increasing the possibil-
ity to generate new bioactive glycosylated derivatives, but also
facilitate further enzyme engineering to develop novel and general
biocatalysts in the combinatorial biosynthesis of biologically active
small-molecule natural products for drug discovery.
activities
toward
3,4-dichloroaniline
(25)
and
3,4-
dichlorobenzene thiophenol (26) in vitro (Figs. 2 and S20–S21 in
SM), the corresponding products (25a, 26a) were b-N-glucoside
and b-S-glucoside compared with authentic compounds. Based
on the above results, PlGT7 exhibited unusual substrate
promiscuity, which rendered PlGT7 a promising enzyme for the
creation of structurally diverse bioactive glycosides.
To further confirm the catalytic characteristics of PlGT7 in vitro,
18 glycosylated products from 10 aglycon acceptors (1, 5, 8, 11,
19–24) were obtained via preparative-scale reactions, 9 glucosides
(8a, 21b, 22a, 22b, 23a–23c, 24a, 24b) of which were novel com-
pounds. Their structures were identified with MS, 1H and 13C
NMR spectroscopic data analysis (Figs. S25–S68 in SM). All the
anomers were in the b-configuration with the large coupling con-
stants (J > 5.7 Hz) of the anomeric protons. It is important to note
that most glycosylated products exhibited improved water-solu-
bility or biological activities compared to the parent molecules.
For instance, the water-solubility of daidzein 40- and 7-O-glucoside
(1a, 1b) was about 30-fold higher than that of daidzein.13 Espe-
cially, the in vitro biological assay revealed that the glycoside form
(24b) had a more potent neuroprotective effect than the corre-
sponding aglycon (phenylpropyl-2,4,6-trihydroxybenzoate, 24,
Table 1) and the positive control (resveratrol) toward the gluta-
mate-induced toxicity in SK-N-SH cells,14 indicating its potential
as a drug lead.
According to the protocols for reverse assays of GT-catalyzed
reactions,15 we found that PlGT7 could deglycosylate 4-nitro-
phenyl-b-D-glucopyranoside (27) to 27a in the presence of UDP
(Fig. S22 in SM), confirming the reaction reversibility of PlGT7. To
further exploit the reversibility of PlGT7 in generating bioactive
Please cite this article in press as: Sun, L.; et al. Tetrahedron Lett. (2016), http://dx.doi.org/10.1016/j.tetlet.2016.02.088

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This work was financially supported by the National Natural
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Supplementary data
Supplementary data (experimental operations, including PlGT7
cloning, expression, activity analysis, products purification and
one-pot reactions, as well as HPLC-UV/MS, HRESIMS, and NMR
characterization data and spectra of products) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.tetlet.2016.02.088.
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Please cite this article in press as: Sun, L.; et al. Tetrahedron Lett. (2016), http://dx.doi.org/10.1016/j.tetlet.2016.02.088