UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation

Article abstract of DOI:10.1021/acs.orglett.7b03619

A permissive steroid glycosyltransferase (UGT74AN1) from Asclepias curassavica exhibited robust capabilities for the regiospecific C3 glycosylation of cardiotonic steroids and C₂₁ steroid precursors, and unprecedented promiscuity toward 53 structurally diverse natural and unnatural compounds to form O-, N-, and S-glycosides, along with the catalytic reversibility for a one-pot transglycosylation reaction. These findings highlight UGT74AN1 as the first regiospecific catalyst for cardiotonic steroid C3 glycosylation and exhibit significant potential for glycosylation of diverse bioactive molecules in drug discovery.

Full text of DOI:10.1021/acs.orglett.7b03619

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
UGT74AN1, a Permissive Glycosyltransferase from Asclepias
curassavica for the Regiospecific Steroid 3‑O‑Glycosylation
Chao Wen, Wei Huang, Xue-Lin Zhu, Xiao-San Li, Fan Zhang, and Ren-Wang Jiang*
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Institute of Traditional
Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
S
* Supporting Information
ABSTRACT: A permissive steroid glycosyltransferase
(UGT74AN1) from Asclepias curassavica exhibited robust
capabilities for the regiospecific C3 glycosylation of cardiotonic
steroids and C21 steroid precursors, and unprecedented
promiscuity toward 53 structurally diverse natural and
unnatural compounds to form O-, N-, and S-glycosides,
along with the catalytic reversibility for a one-pot trans-
glycosylation reaction. These findings highlight UGT74AN1 as
the first regiospecific catalyst for cardiotonic steroid C3
glycosylation and exhibit significant potential for glycosylation
of diverse bioactive molecules in drug discovery.
ardiotonic steroid glycosides, an important class of natural
products that have been used to treat congestive heart
glucosides. Especially, UGT74AN1 catalyzes the glycosylation
of C21 steroid precursors. Moreover, this work highlights an
unexpected promiscuity of UGT74AN1 toward a diverse range
of drug-like scaffolds to produce O-, N-, and S-glycosides, along
with the catalytic reversibility for a one-pot transglycosylation
reaction.
C
failure since the 18th century, have shown highly potent
anticancer and anti-inflammatory activity.1 C3 glycosylation of
the steroidal core has been recognized as a practical way to
improve their therapeutic index and enable their wider
application for the treatment of other diseases.2 Chemical
synthesis of steroid 3-O-glycosides is faced with poor regio- and
stereoselectivities, and the protection and deprotection of
functional groups.2a,3 Glycosyltransferases (GTs) are generally
recognized as powerful synthetic tools which can alleviate these
disadvantages.4 Significant progress has been achieved in the
application of promiscuous microbial or plant GTs for natural
product glycodiversification.5 However, the enzymatic C3
glycosylation of the cardiotonic steroids remains restricted by
the availability of regiospecific GTs.6 The plant steroid GTs
which are naturally responsible for the formation of 3-O-
cardiotonic glycosides remain undiscovered.
Cardenolides or bufadienolides attached with different types
of sugar units are present in different plant groups and have been
well characterized for their chemical structures.7 However,
biosynthesis of these compounds has not been studied to date.8 A
putative biosynthetic pathway has been derived from precursor
feeding studies and in vitro crude enzyme assays,9 indicating that
steroid glycosylation may take place at various stages and should
no longer be regarded as terminal biosynthetic steps.9b However,
the powerful steroid GTs remain undetected.10 Therefore, it is of
great significance to discover novel steroid GTs from
cardenolide- or bufadienolide-producing plants for cardiotonic
steroid glycosylation.
A. curassavica (L.) is a well-known cardenolide-containing
species in the milkweed family Asclepiadaceae.11 In our previous
work, a wide variety of bioactive cardenolides and C21 steroidal
glycosides had been isolated from its seeds, leaves, and stems,12
which inspired us to search for specific GTs. We confirmed
uzarigenin (1) was enzymatically glycosylated when incubated
with UDP-glucose (UDPG) and the crude enzymes from A.
curassavica leaves (Figure S1), which implied the corresponding
steroid GT was probably a soluble enzyme that possessed the
conserved PSPG (plant secondary product glycosyltransferases)
motif.13 As cardenolides evolved as a means of the plant defense
system,8a,11 the plant used in this study was treated with 100 μM
methyl jasmonate (MeJA) for 24 h in order to improve the
expression level of the defense-related genes.14 To clone the
specific steroid GTs, total RNA was isolated from the MeJA
treated A. curassavica leaves and a degenerate PCR primer for 3′
RACE was designed based on the PSPG motif (Figure S2).
Combined with 3′ and 5′ RACE PCR, the full length cDNAs of
11 new GT candidates (AcGTs) were finally obtained and
subcloned into pET28a for expression in E. coli. To screen for the
specific steroid GTs capable of glycosylating steroids, the
detecting reactions (50 mM Tris-HCl, pH 7.5; 500 μM UDP-
Glu; 200 μM uzarigenin; 5 mM MgCl2; 100 μL crude AcGTs in
200 μL final volume; 37 °C, 12 h) were performed and analyzed
Herein, we report, for the first time, the identification of a
novel steroid GT (UGT74AN1) from Asclepias curassavica,
which displays catalytic efficiency and regiospecificity toward
cardenolide and bufadienolide aglycons to form 3-O-β-D-
Received: November 21, 2017
© XXXX American Chemical Society
A
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Figure 1. Exploring the substrate promiscuity of UGT74AN1 with a substrate library. (A) Percent conversion of each library number catalyzed by
UGT74AN1. The library numbers are listed based on the structural scaffolds shown in the Supporting Information (SI) Figure S7. The colors in the bar
graphs (Prod. a, Prod. b, Prod. c, Prod. d, and Prod. e) represent different ratios of various glycosylated products in the total product yield of each
compound. The asterisks (*) represent the glycosylated products that are isolated and confirmed by LC-MS and 1H and 13C NMR spectroscopy. D
means trace amount of products that are detected in LC-MS. (B) The structures of steroid compounds in the substrate library and corresponding
glycosylated products. Compounds leading to no conversion are listed in Figure S8.
by HPLC-DAD and LC-MS. Of the 11 recombinant AcGTs,
AcGT5 was the unique GT which exhibited glycosylation activity
toward uzarigenin (1) and showed unanticipated high
conversion rates (>99%) (Figure 1). Three more representative
cardenolide and bufadienolide aglycons (4, 6, and 16) were
tested, and all these substrates were almost entirely transformed
by AcGT5 (Figure 1). After scaled-up preparative reactions, the
structures of the glycosylated products were elucidated by LC-
of UGT74AN1 toward uzarigenin (1), digitoxigenin (4), bufalin
(6), and 3α-hydroxybufalin (16) were found to be 10.3, 8.2, 20.7,
and 7.3 μM, respectively (Figure S6), and the corresponding kcat/
Km values for 1, 4, 6, and 16 were 885.4, 706.1, 439.6, and 442.5
M−1 s−1, respectively, which indicated the high affinity of
UGT74AN1 toward the cardiotonic steroid.
To study the catalytic promiscuity, regio- and stereospecificity
of UGT74AN1 for cardiotonic steroid glycosylation, 22
structurally diverse steroid compounds (1−17, 54−58) were
selected as substrates (Figures S7 and S8) with UDPG as the
donor. Glycoside production was determined by HPLC-DAD
and LC-MS analysis. The results revealed that UGT74AN1
performed unprecedented catalytic promiscuity and proficiency
to glycosylate structurally different cardiotonic steroids (1−17)
(Figure 1). Although the A/B ring of cardenolides from the
Asclepiadaceae family was generally trans-fused,12a UGT74AN1
exhibited unexpected identical activity toward cardenolide and
bufadienolide aglycons with a cis A/B ring juncture (1, 4, 13, 14).
It is important to know that UGT74AN1 was flexible enough to
catalyze cardiotonic steroids with various functional groups at
C5, C11, C12, C16, C19 (2, 3, 5, 7, 8, 10−15) and capable of
catalyzing stereoisomers with opposite configuration of the C3
1
MS, H NMR, and 13C NMR spectroscopic analysis. The large
anomeric proton coupling constants (J > 7.7 Hz) support the
formation of the β-anomers and an inverting mechanism for
AcGT5 (Table S3). The above results unambiguously
established AcGT5 as the first isolated plant GTs capable of
glycosylating cardiotonic steroids. The cDNA sequence of
AcGT5 contained an ORF of 1416 nucleotides (GenBank
accession No. MF942416) encoding a protein of 464 amino
acids. AcGT5 was further named UGT74AN1 by the UGT
Naming Committee15 and showed the highest identity (60%) to
CrUGT9, a predicted GT from Catharanthus roseus.16
A
phylogenetic tree was constructed and revealed UGT74AN1
was clustered into a new clade of UGT74 subfamily (Figure S3).
To study the biochemical properties and dynamic parameters,
the recombinant His6-UGT74AN1 was expressed in a large-scale
culture and purified to near-homogeneity by His-tag affinity
chromatography (Figure S4). By using uzarigenin (1) as an
acceptor and UDPG as a donor, the purified UGT74AN1 was
found to perform the maximum conversion rates at 45 °C, pH
8.0, and was divalent cation-independent (Figure S5). Km values
1
hydroxy group (15−17). Based on H and 13C NMR analysis,
UGT74AN1 was verified to be a permissive but regiospecific
steroid 3-O-GT and established to be the first identified GT to
glycosylate 3α-hydroxysteroids. It had been proven that the
regiospecific C3 glycosylation of cardiotonic steroid could
enhance water solubility with increased bioactivity.2,6b In
B
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comparison, the promiscuous microbial GT YjiC1 exhibited
limited regiospecificity to form varied regioisomeric species with
reduced bioactivity,6b and OleD showed low tolerance to the
functional groups at the C19 position,6a which was also
recognized to be vital for anticancer activity.17
scaffolds to form mono- or multiple products (Figures S21−
S41). It is noteworthy that chloramphenicol (47) and
erythromycin (48) led to monoglucosylated products, which
implied UGT74AN1 as the first reported plant GT capable of
catalyzing antibiotics. Besides, UGT74AN1 exhibited robust N-
and S-glycosylation activity toward representative compounds
containing N-based (49−51) and S-based (52, 53) nucleophiles.
Overall, UGT74AN1 had higher efficiency and more regiose-
lectivity toward steroid compounds containing a 6/6/6/5 ring
system in comparison with other scaffolds. In this study, a total of
53 drug-like acceptors were glycosylated by UGT74AN1 and the
Based on the above results, we further accessed the catalytic
ability of UGT74AN1 toward other steroid compounds (18−23,
59−62) and observed that C21 steroids (18, 19), steroid
sapogenins (20, 21), and other steroid compounds (22, 23) were
also glycosylated by UGT74AN1 (Figure 1, Figures S15−S20).
Of particular note, UGT74AN1 effectly transformed pregnolone
(18) and isopregnanolone (19) into pregnolone 3-O-glucoside
(18a) and isopregnanolone 3-O-glucoside (19a) (Figures S76−
S81), respectively, which demonstrated UGT74AN1 to be the
first identified GT to glycosylate C21 steroid precursors. Given
the robust catalytic activity toward cardenolide aglycons and C21
steroid precursors, UGT74AN1 was tentatively hypothesized as
a steroid GT which probably plays a role in the 3-O-glycosylation
of cardenolides in vivo (Scheme 1).9d,11 Unlike previously
1
structures of 15 glucosylated products were elucidated by H
NMR and 13C NMR analysis (Figures S45−S85).
To establish a green, cost-effective chemoenzymatic plat-
form,5d,19 we exploited the catalytic reversibility of UGT74AN1
for one-pot transglycosylation reactions. As shown in Figure 2,
Scheme 1. Proposed Biosynthetic Pathway of Cardenolides in
a
A. curassavica
Figure 2. Exploiting the catalytic reversibility of UGT74AN1 to
generate digoxigenin 3-O-β-D-glucoside (5a) from a simple donor (70).
The HPLC analysis is shown in the SI, Figures S42−S44.
the model reactions were carried out in the presence of only a
catalytic amount of UDP and led to the formation of expected
products 1a, 5a, and 15a with an average high yield of 90%
(Figures S42−S44). Given the established substrate promiscuity
of UGT74AN1, the extension of the one-pot reaction suggests
UGT74AN1 can be applied to glycodiversification of bioactive
molecules without adding UDPG.
In summary, we reported for the first time, the discovery of a
novel steroid GT from A. curassavica, UGT74AN1, which
displayed catalytic efficiency and regiospecificity toward
cardenolide and bufadienolide aglycons, and even catalyzed
C21 steroid precursors to form 3-O-β-D-glucosides. Unprece-
dentedly, UGT74AN1 was capable of catalyzing structurally
different cardenolide and bufadienolide aglycons with cis or trans
A/B ring fusion. Furthermore, UGT74AN1 showed great
tolerance to various functional groups at the steroid core and
served as the first reported GT catalyzing the glycosylation of 3α-
hydroxy group of cardiotonic steroids. This study highlights
UGT74AN1 to be the first reported promiscuous steroid GT to
catalyze numerous drug-like natural and unnatural products to
form O-, N-, and S-glycosides, along with catalytic reversibility for
the one-pot transglycosylation reaction. UGT74AN1 accepted
antibiotics as a substrate, which had not yet been reported in
plant GTs. Cumulatively, this study demonstrated, for the first
time, that a novel steroid GT, which probably played a role in the
glycosylation of cardenolides in vivo, could serve as a powerful
enzymatic tool for the regiospecific glycosylation of drug-like
scaffolds. The enzyme reported here hints at more exciting and
a
SCCE: cholesterol side chain cleaving enzyme.
reported sterol GTs which are mostly insoluble membrane
bound enzymes with a narrow substrate range,18 UGT74AN1 is a
soluble enzyme with a broad substrate range, which makes it an
unusual kind of steroid GT. UGT74AN1 could glycosylate C21
steroid precursors in vitro, which indicates that the substrate
promiscuity of steroid GT could be one of the reasons why
steroid glycosylation takes place at various stages.
Inspired by the unprecedented catalytic efficiency for steroid
compounds, we further investigated the capability of
UGT74AN1 toward structurally diverse scaffolds by using 37
representative natural and unnatural compounds (24−53, 63−
69) as substrates (Figures S7 and S8). The results were
summarized in Figure 1, which revealed the excellent capability
of UGT74AN1 to catalyze 30 of the 37 structurally diverse
C
DOI: 10.1021/acs.orglett.7b03619
Org. Lett. XXXX, XXX, XXX−XXX

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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b03619.
Experimental procedures, HPLC-HRESIMS character-
ization data, NMR, and HSQC spectra of glucosylated
products (PDF)
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: trwjiang@jnu.edu.cn.
ORCID
Muller-Uri, F.; Brauchler, C.; Heubl, G.; Kreis, W. Plant Syst. Evol. 2008,
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271, 65. (e) Lindemann, P. Steroids 2015, 103, 145.
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(b) Theurer, C.; Treumann, H. J.; Faust, T.; May, U.; Kreis, W. Plant
Cell, Tissue Organ Cult. 1994, 38, 327.
Chao Wen: 0000-0002-4987-4492
Ren-Wang Jiang: 0000-0002-2163-1683
Notes
(11) Agrawal, A. A.; Petschenka, G.; Bingham, R. A.; Weber, M. G.;
Rasmann, S. New Phytol. 2012, 194, 28.
The authors declare no competing financial interest.
(12) (a) Zhang, R. R.; Tian, H. Y.; Tan, Y. F.; Chung, T. Y.; Sun, X. H.;
Xia, X.; Ye, W. C.; Middleton, D. A.; Fedosova, N.; Esmann, M.; Tzen, J.
T.; Jiang, R. W. Org. Biomol. Chem. 2014, 12, 8919. (b) Yuan, W. Q.;
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(14) Munkert, J.; Ernst, M.; Muller-Uri, F.; Kreis, W. Phytochemistry
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ACKNOWLEDGMENTS
■
This work was financially supported by grants from the National
Natural Science Foundation of China (81573315), Guangdong
Natural Science Fund (2015A030313313), and Guangzhou
Industry-University Collaborative Innovation Major Projects
(201508030016).
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Bairoch, A.; Belanger, A.; Fournel-Gigleux, S.; Green, M.; Hum, D. W.;
́
DEDICATION
■
Iyanagi, T.; Lancet, D.; Louisot, P.; Magdalou, J.; Chowdhury, J. R.;
Ritter, J. K.; Schachter, H.; Tephly, T. R.; Tipton, K. F.; Nebert, D. W.
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Oksman-Caldentey, K. M.; Martinoia, E.; Bouwmeester, H.; Goossens,
A.; Memelink, J.; Werck-Reichhart, D. Nat. Commun. 2014, 5, 3606.
Dedicated to Prof. Qi-Tai Zheng on the occasion of his 80th
birthday.
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