Identification and Characterization of Two New 1- O-Acyl-glucose-ester Forming Glucosyltransferases from Erigeron breviscapus

Article abstract of DOI:10.1021/acs.jafc.8b07215

Two versatile UDP-glucosyltransferases, UGT75L25 and UGT75X1, were isolated from Erigeron breviscapus. The enzymes display high sequence identity to flavonoid 7-O-glucosyltransferase from Malus species and cluster to the phylogenetic group L of plant glucosyltransferases, also involved in the formation of hydroxycinnamoyl glucose esters, which are used as bifunctional donors in the glucosylation or acylation of anthocyanins. The enzymes, functionally expressed in Escherichia coli, exhibit broad substrate specificity toward 21 structurally diverse types of phenolic acids, including (hydroxy)cinnamates, vanillic acid, 3-hydroxycoumarin, and 7-hydroxyflavonoids. The catalytic characteristics of UGT75L25 and UGT75X1 were exploited to generate the corresponding acyl-glucose-esters or glucosides with high efficiency. These findings demonstrate the significant potential of acyl-glucose-esters in the further enzymatic synthesis of bioactive anthocyanins.

Full text of DOI:10.1021/acs.jafc.8b07215

Article
pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Identification and Characterization of Two New 1‑O‑Acyl-glucose-
ester Forming Glucosyltransferases from Erigeron breviscapus
Yan Yang,†,‡ Minzhi Liu,‡ Wenxuan Zhang,‡ Yunsong Cao,‡ Changkun Li,# and Wei Wang*,†,‡
†State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences & Peking Union Medical College, Beijing 100050, China
‡
́
Key Laboratory of Biosynthesis of Natural Products of National Health Commission of the Peoples Republic of China, Institute of
Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
#Beijing Branch, Shimadzu (China) Co., Ltd., Beijing 100020, China
S
* Supporting Information
ABSTRACT: Two versatile UDP-glucosyltransferases, UGT75L25 and UGT75X1, were isolated from Erigeron breviscapus.
The enzymes display high sequence identity to flavonoid 7-O-glucosyltransferase from Malus species and cluster to the
phylogenetic group L of plant glucosyltransferases, also involved in the formation of hydroxycinnamoyl glucose esters, which are
used as bifunctional donors in the glucosylation or acylation of anthocyanins. The enzymes, functionally expressed in Escherichia
coli, exhibit broad substrate specificity toward 21 structurally diverse types of phenolic acids, including (hydroxy)cinnamates,
vanillic acid, 3-hydroxycoumarin, and 7-hydroxyflavonoids. The catalytic characteristics of UGT75L25 and UGT75X1 were
exploited to generate the corresponding acyl-glucose-esters or glucosides with high efficiency. These findings demonstrate the
significant potential of acyl-glucose-esters in the further enzymatic synthesis of bioactive anthocyanins.
KEYWORDS: Erigeron breviscapus, enzymatic catalysis, glycosylation, glycosyltransferase, acyl-glucose-ester
of plant GT1 proteins. In comparison with the characterized
UGT84A enzymes (UGT84A1-A4,14 A6,15 A9-A10,16,17
A13,18 A17-A19,4 and A22-A2419,20), which mainly exhibit
specific substrate preference for hydroxycinnamate substrates,
UGT75L proteins can tolerate a number of structurally
different acceptors to form different 1-O-acyl-glucose-esters
or glucosides. In tobacco (Nicotiana tabacum L.), UGT75L3
(NtGT2) showed particular glucosylation activity against 7-
hydroxyflavonoids and 3-hydroxycoumarin.21 UGT75L6 iso-
lated from Gardenia jasminoides preferentially glucosylates the
carboxyl group yielding crocetin glucosyl-ester.22 Both of these
enzymes also exhibit slight glucosylation activity against
hydroxycinnamate substrates, which was mentioned in the
literature or within their respective Supporting Information
sections.21,22 The UGT75L4 from Osage orange (Maclura
pomifera) exhibited broad substrate recognition toward
isoflavonoids and flavonoids,23 while Rubus suavissimus
UGT75L20 and Angelica keiskei UGT75L21 were found to
be promiscuous toward ent-kaurene-19-oic acid-type sub-
strates.24 The hydroxycinnamate substrates are not mentioned
in these articles.
INTRODUCTION
■
Phenolic acids are versatile substances with various biological
activities in vitro and in vivo, such as antioxidative, anti-
inflammatory, antibacterial, and antitumor effects.1,2 They play
important roles in maintaining cell wall integrity through
esterification with the pectins and arabinoxylans3 and are
precursors to other more elaborate phenylpropanoid metabo-
lites, such as anthocyanins and flavonoids.4 Hydroxycinna-
mates, deriving from the precursor phenylalanine, always
accumulate in the form of ester or amide conjugates with
monosaccharides, lipids, and amines, and these conjugates
have been used for ultraviolet protection and pathogen
response.4−7 Apart from NDP-sugars and acyl-CoA as sugar
donor and acyl donor in the enzymatic glycosylation and
acylation reactions, respectively, 1-O-hydroxycinnamoyl-glu-
cose-esters (HCGEs) or 1-O-p-hydroxybenzoyl-glucose-ester
(pHBGE) serve as “zwitter” donors (bifunctional donors) in
the enzymatic modification of anthocyanins.8−10 Glucosylation
of anthocyanins in carnations (Dianthus caryophyllus) and
delphiniums (Delphinium grandiflorum) involves 1-O-acyl-
glucose-ester as sugar donor.8 In butterfly pea (Clitoria
ternatea), acyl-glucose-dependent acyltransferase modifies
anthocyanins with HCGEs as acyl donor substrates.9,11
Erigeron breviscapus is rich in diverse flavonoid glycosides,
hydroxybenzoyl or hydroxycinnamoyl glucose esters, and
caffeoyl quinine ester.25,26 With the purpose of seeking more
diverse glycosylation, we analyzed the transcriptome data of E.
breviscapus. In the present investigation, we used homology-
As an important bifunctional donor in the enzymatic
reaction, the biosynthesis of HCGEs depends on family 1
glycosyltransferases (GT1). GT1 catalyzes the transfer of
active UDP-sugars to small molecular acceptors,7 which is
generally regarded as a powerful tool in the glycoldiversifica-
tion of natural products.12,13 To date, only a handful of GT1s
have been detected to catalyze the formation of HCGEs, and
all belong to the UGT75L and UGT84A subfamily of group L
Received: December 25, 2018
Revised: February 21, 2019
Accepted: February 25, 2019
© XXXX American Chemical Society
A
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EbUGT was incubated at 25 °C overnight and was released from the
beads by cleaving off the chitin binding domain (CBD) with Buffer II.
The eluted fraction was replaced with 50 mM phosphate buffer (pH
6.0) using ultrafiltration membranes (Millipore, United States).
Protein purity was confirmed by SDS-PAGE to be >90%, and the
protein concentrations for all studies were determined using a Protein
Quantitative Kit (Bradford) (Bio-Rad, United States).31
Functional Analysis of Recombinant EbUGTs. To obtain a
comprehensive knowledge of the resultant EbUGTs, a systematic
investigation of the qualitative and quantitative biochemical character-
istics was performed. The sugar donor specificity of purified EbUGT
enzymes was characterized with three sugar donors: uridine
diphosphate glucose (UDPG), UDP-glucuronic acid (UDPGA), and
UDP-galactose (UDP-Gal). To assay for the substrate specificity of
EbUGTs, a compound library including phenylpropanoids (1−9, 22−
26), flavonoids (10−20, 27−37), aromatic phenols and phenolic
acids (21, 38−47), and triterpenoid acids (48−49) were used as
sugar acceptors.
Standard enzymatic assays (200 μL total volume) were conducted
in a 1.5 mL tube containing reaction buffer (50 mM phosphate buffer,
pH 6.0), UDP-sugar (0.3 mM UDPG-Na or UDPGA-Na or UDPGal-
Na), substrates (0.1 mM of diverse substrates dissolved in DMSO),
and purified enzyme (20 μg EbUGTs). The reaction mixture was
incubated at 40 °C for 120 min. The reactions were initiated by the
addition of glycosyltransferase and terminated by adding 400 μL of
ice-cold methanol. Subsequently, the samples were centrifuged at
16 000g for 10 min, and aliquots were analyzed by high-performance
liquid chromatography-UV absorption/mass spectrometry (HPLC-
UV/MS). The preparative-scale reactions of acyl-glucose-esters and
glucosides are recorded in the Supporting Information.
Determination of Temperature, pH, and Metal Ion Effects
on Enzyme Activity. To test the optimal reaction temperature, the
reaction mixtures were incubated at different temperatures (0−70 °C)
with a 10 °C interval. To assay for the optimal pH, the enzymatic
reaction was performed in various reaction buffers with pH values in
the range of 5.0−5.5 (citric acid-sodium citrate buffer), 6.0−8.0
(phosphate buffer), and 8.5−9.0 (Tris-HCl buffer). To study the
necessity of divalent metal ions for EbUGT activity, CaCl2, CoCl2,
CuCl2, MgCl2, MnCl2, ZnCl2, and EDTA were used individually at
the final concentration of 5 mM. The assays were performed with
UDPG as a donor and p-coumaric acid (1) as an acceptor.
based cloning to isolate and identify the UGT75L25 enzyme
and a new subgroup of UGT75X1 enzyme producing phenolic
1-O-acyl-glucose-esters and flavonoid glucosides. The broad
substrate recognition and functionality of UGT75L25 and
UGT75X1 are also described. Our findings not only shed light
on characterization of 1-O-acyl-glucose-ester forming glucosyl-
transferase but also provide new “bifunctional donor”
substrates for glucosylation and acylation of anthocyanins.
MATERIALS AND METHODS
■
Chemicals and Media. All chemicals and reagents were obtained
from Sigma-Aldrich (St Louis, United States), J&K Scientific Ltd.
(Beijing, China), and BioBioPha (Kunming, China) unless otherwise
stated. The pTWIN1 vector, together with all restriction enzymes and
DNA ligase, were purchased from New England Biolabs (Ipswich,
United States). Analyses of substrate specificity and determinations of
conversion rates were performed on a Thermo U3000 series HPLC
system. Details of substrates and product analyses are supplied in the
Supporting Information.
Transformed Escherichia coli cells were grown on Luria−Bertani
(LB) medium supplemented with antibiotics (100 mg/L ampicillin).
Transcriptome Sequencing and RNA-seq Data Analysis. The
fresh leaves of E. breviscapus were collected and frozen with liquid
nitrogen immediately and stored at −80 °C until use. The E.
breviscapus transcriptome was then analyzed by high-throughput RNA
sequencing (RNA-seq). The resulting transcriptome data were
analyzed to retrieve nucleotide sequences showing high identity
with UDP-glycosyltransferase (UGT). Specifically, these resulting
unigenes based on transcriptome assembly were aligned by Blast X to
protein databases including NCBI nr, Swiss-Prot, KEGG, and COG
(e-value <0.00001), resulting in proteins with high sequence similarity
with the given unigenes along with their functional annotations. These
unigenes showing high identity with UGT genes were selected for
further investigation.
Full-Length cDNA Isolation and Construction of a Plasmid
with the EbUGTs Fusion Fragment. The total RNA was extracted
from the sterile leaves of E. breviscapus using a RNeasy Plant Mini Kit
(Qiagen). First-strand cDNA was synthesized by reverse transcription
using a GeneRacer RACE Kit (Invitrogen, United States) with a
sequence-specific primer, EbUGT-5, designed according to the
resultant transcriptome data of E. breviscapus. The full-length
glycosyltransferase DNA homologues were generated by nested-
PCR with the primers EbUGT-1 to EbUGT-4. The sequences were
verified by DNA sequencing and then subcloned into a derivative of
the plasmid pTWIN1 (designated as pTWINB) as reported in our
previous paper,27 resulting in the plasmid pTWINB-EbUGT. All
primers used for plasmid construction are listed in Table S1.
Phylogenetic Tree Analysis and Multiple Sequence Align-
ment. The phylogenetic analysis was colored for visualization by the
online ornament tool (Evolview: http://www.evolgenius.info/
evolview/),28 and the phylogenetic tree was constructed using
MEGA 6.0 software29 with the neighbor-joining method based on
ClustalW multiple alignments. Multiple alignment of proteins was
performed on Clustal X software, and the picture presenting the
sequence with secondary structure elements was generated by the
online ESPript software (http://espript.ibcp.fr/ESPript/cgi-bin/
ESPript.cgi).30
Determination of Kinetic Parameters of EbUGTs. To
determine the initial velocity of EbUGTs, the assays were performed
in a final volume of 200 μL of 50 mM phosphate buffer (pH 6.0) at 40
°C. The reaction mixtures contained certain concentrations of enzyme
and excessive amount of UDPG (800 μM) with varying
concentrations (0−800 μM) of p-coumaric acid, ferulic acid, or
sinapic acid. After incubation at 40 °C for 40 min, the reaction was
terminated by addition of 400 μL of ice-cold methanol, and the
samples were centrifuged at 16 000g for 10 min. Supernatants were
analyzed by analytical reverse-phase HPLC as described above. All
experiments were performed in triplicate. The calculation of Km, Vmax
,
and kcat values were achieved through nonlinear regression software
GraphPad Prism 5.01.
One-Pot Reaction Coupled by EbUGTs. The model one-pot
reactions (200 μL total volume) were performed with 2-nitrophenyl-
β-D-glucopyranoside (50, 100 μM) as a “sugar donor” and
phenylpropanoids (1−9, 50 μM), flavonoids (10−20, 50 μM) or
vanillic acid (21, 50 μM) as “sugar acceptors” together with UDP (10
μM) as a “cofactor”. The reactions were initiated by addition of
purified EbUGTs (0.5 mg) and incubated at 40 °C for 12 h. Reactions
were stopped by adding 400 μL of methanol, and results were
analyzed by HPLC as described in the Supporting Information.
Expression and Purification of Recombinant Proteins.
Transformants harboring the pTWINB-EbUGT plasmids were
cultured in E. coli according to a previously published method.27
The recombinant EbUGT was induced with 0.5 mM isopropyl-β-D-
thiogalactopyranoside (IPTG) at 16 °C for 18 h. All subsequent
purification procedures were conducted at 4 °C. Cells were
centrifuged, and pellets were lysed by using high-pressure
homogenization in Buffer I (20 mM HEPES, 500 mM NaCl, pH
8.5) and then centrifuged at 25 000g for 16 min to remove debris. The
supernatant containing recombinant proteins was subjected to affinity
chromatography on a chitin column and eluted with 100 mL of Buffer
II (20 mM HEPES, 500 mM NaCl, pH 6.5). The target protein
RESULTS AND DISCUSSION
■
cDNA Isolation and Sequence Analysis of EbUGT
Gene Families. The species E. breviscapus is rich in various
types of polyphenolic glycosides, suggesting that it may contain
a diversity of glycosyltransferases;25,26 transcriptome sequenc-
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Figure 1. Partial sequences and phylogenetic analyses of the deduced EbUGTs and other known UGTs. (A) Multiple sequence alignments of the
PSPG boxes of the 2 deduced EbUGT proteins. The amino acids which were inconsistent with those of EbUGT75L25 are highlighted by red, blue,
and purple boxes. (B) Phylogenetic analyses of the 2 deduced EbUGT proteins with functionally known UGTs from other plant species. Protein
sequences were aligned with Clustal W, and a neighbor-joining tree was constructed by using MEGA 6.0. Scale bar indicates the number of amino
acid substitutions. (C) SDS-PAGE analysis of the purified recombinant EbUGT proteins. Lane 1, purified EbUGT75X1 protein; lane 2, purified
EbUGT75L25 protein.
ing can greatly accelerate full-length gene isolation. To
characterize UGT genes in E. breviscapus, we performed
transcriptome sequencing with leaf tissue. The assembled
sequences were further analyzed by BLAST X and open
reading frame (ORF) Finder for their ORF identification with
the aim of discovering acyl-glucose-ester forming glucosyl-
transferases. Thus, two unigenes, comp29311_c0_seq1 and
comp5142_c0_seq1, were retrieved for further character-
ization. Moreover, ORF Finder results showed that these two
unigenes both contained a complete ORF of 1344 bp and 1482
bp, respectively. The ORF sequences of these two unigenes,
comp29311_c0_seq1 and comp5142_c0_seq1, were desig-
nated as UGT75X1 and UGT75L25, according to the UGT
Naming Committee,32 and then deposited into the GenBank
database (accession numbers: MH853831 for UGT75X1 and
MH853832 for UGT75L25).
Further analysis using publicly available protein databases
for functional annotations showed that these two proteins
(EbUGT75X1 and EbUGT75L25) display a 46% identity with
each other at the amino acid level, 47% and 62% identity to
MdF7GT from apple (Malus x domestica), 46% and 61%
identity to UGT75L21 from A. keiskei, and 28−35% and 27−
36% identity to UGT75L6 from G. jasminoides and UGT84A6
from Strawberry (Fragaria ananassa), respectively (Table S2
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Figure 2. Catalytic promiscuity of EbUGT75L25 and EbUGT75X1 in glucosylation with UDP-glucose as donor. (A) The relative percent
conversions of glucosylated products catalyzed by EbUGT75L25 and EbUGT75X1. The black and white columns of luteolin (16) represent the
conversions of 7-O-glucosides (16a) and 3′-O-glucoside (16b), respectively. The specific activity on baicalein catalyzed by EbUGT75L25 is set as
100%. Error bars represent the standard deviation from three replicates. N.D. indicates “not detected”. (B) The structures of the substrate members
(R = H) and corresponding glucosylated products (R = Glc).
and Figure S1). Subsequent sequence analysis (Figure 1A)
showed that the two deduced EbUGT proteins shared 29
identical amino acids within the conserved plant secondary
product glycosyltransferase (PSPG) motif and that the last
glutamine (Q) residue within the PSPG motif is implicated to
grant specificity for UDP-glucose as sugar donor.33 Comparing
the PSPG motif of EbUGT75L25 with AkUGT75L21 (ent-
kaurene-19-oic acid 1-glucose-ester glucosyltransferase) and
MdF7GT or MpUGT75L4 (flavonoid-7-O-glucosyltrans-
ferases), only 5−8 amino acids differ. Moreover, the identities
of the PSPG motif between EbUGT75L25 and GjUGT75L6
(crocetin 1-glucose-ester glucosyltransferase), or among the
EbUGT75X1 with AkUGT75L21, MdF7GT, MpUGT75L4,
or GjUGT75L6, were below 50%. These results indicated that
the protein EbUGT75L25 might display a broader substrate
preference for flavonoids than that of protein EbUGT75X1.
The phylogenetic tree containing the deduced amino acid
sequences encoded by EbUGTs and other reported plant
UGTs was constructed. The GenBank accession numbers of
the sequences used in the phylogenetic analysis are shown in
Table S3. Phylogenetic analysis (Figure 1B) showed that the
UGTs used in this study can be divided into six clusters:
flavonoid 3-O-glucosyltransferase (3-O-UGT), flavonoid 7-O-
glucosyltransferase (7-O-UGT), flavonoid multisite glucosyl-
transferase, C-glucosyltransferase, 1-O-acyl-glucose-ester form-
ing glucosyltransferase and salicylic acid 1-glucosyltransferase
(SA 1-O-glucoside UGT). The two EbUGT proteins were
grouped into the flavonoid 7-O-UGT cluster. Moreover, a
portion of the proteins in this cluster also exhibit glucosylation
activity against 3-hydroxycoumarin (NtUGT75L3)21 and ent-
kaurene-19-oic acid type substrates (AkUGT75L21),24 sug-
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gesting that EbUGT75X1 and EbUGT75L25 are likely to
exhibit promiscuity of acceptor substrates.
tin), flavanone (naringenin), and isoflavonoids (genistein and
biochanin A) and showed obvious preferences for baicalein
(10) and scutellarein (11), which were higher than that for
hydroxycinnamate substrates. However, the recombinant
UGT75X1 protein displayed a narrow range and poorer
activity toward flavonoids. HPLC analysis (Figures S12−S22)
showed that a new product peak was detected compared to the
control. These enzymatic products were then further analyzed
by HPLC-MS/MS, revealing that these products lost one
glucose moiety (m/z 162) to yield the corresponding flavonoid
aglycone. Further comparison to authentic reference standards,
or NMR spectroscopic analyses, indicated that the products are
flavonoid 7-O-glucosides (details in the Supporting Informa-
tion). Notably, two product peaks appeared in the HPLC
chromatogram in the case of the reactions with recombinant
UGT75L25 protein and substrate luteolin (16) (Figure S18),
whereas no product peak was observed from control reactions
without recombinant UGT75L25 protein. The ESI-MS spectra
of compounds 16a and 16b showed an [M-H]− peak at m/z
447.1 and [M-H-162]− (m/z 285.1) fragment ions in the MS/
MS detection, indicating monoglucosylated products. These
metabolites were then assigned to luteolin 7-O-glucoside (16a)
and luteolin 3′-O-glucoside (16b) based on their NMR
spectral data compared with their authentic reference stand-
ards. Taken together, EbUGT75L25 exhibited a preference for
flavonoids, which was in accordance with the fact that
EbUGT75L25 showed a relatively higher identity with
MdF7GT and UGT75L4 at the amino acid level of the full
sequence and PSPG motif than that of EbUGT75X1.
Heterologous Expression and Purification of EbUGTs.
To further investigate the biochemical characteristics of the
two candidate UGTs, they were screened by functional
expression in E. coli independently. Generally, two full-length
EbUGTs were cloned into E. coli BL21 (DE3) in the form of
the plasmid pTWINB-EbUGT, which allowed them to be
expressed as respective CBD-EbUGT fusion proteins. As
shown in Figure 1C, EbUGT75X1 and EbUGT75L25 could be
released from the beads by cleaving off the chitin binding
domain, and they were expressed as soluble proteins with
expected sizes (51 and 56 kDa, respectively).
In Vitro Functional Characterization of Recombinant
UGTs from E. breviscapus. To characterize the substrate
specificities of EbUGT75X1 and EbUGT75L25, each member
of the acceptor library (1−49, Figures 2 and S2) was tested
using purified EbUGT enzymes. Because these two deduced
EbUGT proteins shared the final glutamine (Q) residue within
the PSPG motif, UDP-glucose was first selected as sugar
donor. The reaction products were analyzed by HPLC-UV/
MS.
Of the 14 phenylpropanoids (1−9, and 22−26), eight
cinnamates and one coumarin were observed to be
glucosylated by EbUGT75X1 and EbUGT75L25. Under the
standard enzymatic reactions with the above-mentioned
phenylpropanoids substrates, EbUGT75X1 exhibited a sub-
strate preference only for p-coumaric acid (1), while
EbUGT75L25 showed a preference for o-coumaric acid (5)
and 3-hydroxycoumarin (9). The glucosylated products were
isolated from the preparative-scale reactions, and their
structures, including the glucosylated positions and anomeric
stereochemistry, were identified by comparison of their HPLC,
Among 11 aromatic phenols and phenolic acids and two
triterpenoid acids, only vanillic acid was glucosylated by
EbUGT75L25 (Figure S23). The product was identified as
vanillyl-glucose-ester by mass and NMR spectroscopic analyses
with an authentic reference standard, which can be used as a
“zwitter donor” for the modification of anthocyanins.10
1
MS, or H NMR and 13C NMR spectra with reported data
(details are listed in the Supporting Information) or
commercially available standards. The mass spectra of the
products presented an [M-H]− ion peak in the negative mode
and [M-H-162]− fragment ions in the MS/MS detection,
suggesting that a glucose group had been introduced into the
skeleton of phenylpropanoids aglycone (Figures S3−S11). The
observed large anomeric proton-coupling constants (J = 6.8−
7.8 Hz) indicated the formation of the β-anomers and an
inverting mechanism for EbUGT75X1 and EbUGT75L25.
The elucidation and comparison of 1H, 13C NMR, and HMBC
data of the products confirmed that the EbUGT75X1 and
EbUGT75L25 react on the carboxyl group of hydroxycinna-
mates (the long-range correlations between H-1′ and C-9) and
the 3-hydroxy group of coumarin (the long-range correlations
between H-1′ and C-3). In particular, 2-hydroxy and 4-hydroxy
as well as 2-methyl, 2-chlorine, and 3-chlorine cinnamic acids
were glucosylated, yielding glucose esters, and that was distinct
from UGT84A6 which can tolerate substrates containing a
carboxylic group and an aromatic ring structure, except for the
substitution at the aromatic ring located in the ortho
position.15 Moreover, UGT75X1 and UGT75L25 showed a
preference for the carboxyl groups of hydroxycinnamates, and
the substituents at the aromatic ring were tolerated. Among the
above-mentioned 1-O-acyl-glucose-ester forming glucosyltrans-
ferases, only UGT84A17 and UGT75L3 can accept 2-
coumaric acid as substrate.4,21
Because diverse flavonoid glycosides (glucosides, galacto-
sides, glucuronides, and galacturonides) are present in E.
breviscapu,25,26,34 we then detected the activities of UGT75X1
and UGT75L25 with commercially available UDP-galactose
and UDP-glucuronic acid as sugar donors. However,
UGT75X1 and UGT75L25 only displayed sugar donor
specificity for UDP-glucose, which indicates a sugar donor
specificity for UDPG, as previously suggested by the results
from other UGTs.33
Enzymatic Properties of Recombinant EbUGTs
Protein. Previous research reports neglected the character-
ization and substrate preference for hydroxycinnamates of the
UGT75L group. Equally, the enzymatic characterization of
new UGT75X group (starting with UGT75X1) merited a
complete investigation. As shown in Figure 3A, the optimum
temperature of EbUGT75X1 is in the range of 30−40 °C, and
it showed low activity at 50 °C, which was similar to that of
CsUGT84A22,19 but the EbUGT75L25 optimum was in the
range of 40−50 °C. The optimal pH values of EbUGT75X1
and EbUGT75L25 were determined to be 6.0, while
CsUGT84A22 was reported to have a lower pH optimum of
5.5.19 In addition, the pH range of good enzymatic activity was
narrow, ranging only from pH 6.0 to 6.5 (Figure 3B). The
addition of Cu2+, Co2+, and Zn2+ ions significantly reduced the
glucose esterification activity of EbUGT75X1 and
EbUGT75L25 (Figure 3C). Interestingly, similar effects were
reported for the F. ananassa UGT84A6 for Cu2+ and Zn2+
ions.15 EDTA did not reduce or inhibit EbUGTs activity,
Recombinant UGT75L25 protein displayed a broad range of
activities toward flavones (baicalein, scutellarein, oroxylin A,
wogonin, chrysin, apigenin, and luteolin), flavonols (querce-
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Table 1. Kinetic Parameters of EbUGT75X1 and
EbUGT75L25
substrate
kcat (min−1
Wild-Type EbUGT75X1
)
Km (μM)
kcat/Km (mM−1 min−1
)
p-coumaric acid
ferulic acid
sinapic acid
1.99 0.07
3.03 0.29
2.03 0.27
88.0 3.4
253.8 28.5
329.3 43.0
22.6
11.9
6.1
Wild-Type EbUGT75L25
p-coumaric acid
ferulic acid
sinapic acid
0.58 0.01
0.75 0.03
0.88 0.05
69.9 5.7
128.8 13.7
220.8 24.8
8.3
5.8
4.0
was obvious, and the affinity was higher than that of UGT84A6
and UGT84A17, whose specific substrate preferences for
hydroxycinnamate substrates have been reported.
One-Pot Reaction Coupled by EbUGT75L25. GTs are
generally regarded as unidirectional catalysts that drive the
formation of glycosidic bonds from NDP-sugar donors and
aglycon acceptors according to a nucleophilic substitution
mechanism (SN2).35 An SN2 attack by the aromatic hydroxyl or
carboxylate oxygen to the anomeric carbon of UDP-glucose
was performed in the aromatic O-glucoside or acyl-glucose-
ester forming reactions.36 However, a number of reversible and
bidirectional GTs were observed in the coupling reactions, in
which GTs could be employed for deglycosylation in certain
cases or to transfer sugars from glycoside scaffolds to
aglycons.37−39 However, none of the GTs that participated
in the glucose esterification have been reported on the
reversible reactions. On the basis of this background, the
optimized model reactions were performed with 2-nitrophenyl-
β-D-glucopyranoside (50) as a sugar donor and diverse
substrates (1−21) as acceptors under the catalysis of
UGT75X1 and UGT75L25 (Figures 4 and S26−S28).
Interestingly, the three desired products (12a, 13a, and 14a)
catalyzed by UGT75L25 with yields of 58%, 20%, and 37%,
respectively, were generated only in the presence of an
exceedingly small amount of UDP (1/10 molar of 12−14).
Even if we enhanced the amount of 2-nitrophenyl-β-D-
glucopyranoside (50) and UDP or decreased that of
cinnamates, unfortunately, none of the cinnamoyl-glucose-
esters were obtained. We attempted to find a reasonable
explanation from the structures of substrates and products or
the substrate preference of the enzyme, but it remains elusive.
Above all, without adding the expensive additional UDPG, this
one-pot reaction provides an economic and environmentally
approach to the in vitro glucosylation of certain substrates.
Furthermore, engineered cells with enhanced UDPG bio-
synthetic pathways can represent an effective approach to the
in vivo glucosylation.
Figure 3. Effects of different temperature (A), pH value (B), divalent
metal ions, and ethylenediaminetetraacetic acid (EDTA) (C) on the
enzymatic activities of EbUGT75L25 and EbUGT75X1. p-Coumaric
acid (1) and UDPG were used as aglycon acceptor and sugar donor,
respectively. CK represents the enzymatic reaction performed without
metal ions or EDTA. N.D. means not detected. Error bars represent
the standard deviations calculated from three replicates.
which indicated that these metal ions were not required in the
catalytic mechanism of the enzyme. However, Mg2+ ion
exhibited a strong positive impact on the enzymatic activity of
EbUGT75L25, which suggests the efficiency of preparative-
scale reactions can be improved at the existence of Mg2+ ion.
Kinetic analyses of EbUGT75X1 and EbUGT75L25
revealed a relatively higher affinity with lower Km values of
88.0 and 69.9 μM for p-coumaric acid, which were
approximately 14% of the values of UGT84A6 (603 μM)15
and Populus UGT84A17 (620 μM),4 respectively (Table 1 and
Figures S24 and S25). The specificity constants (i.e., the kcat/
Km values) for p-coumaric acid in the reaction of EbUGT75X1
and EbUGT75L25 were similar to that of UGT84A17, and all
were within 10% of that for p-coumaric acid of UGT84A6. The
kcat values of UGT75X1 and UGT75L25 for p-coumaric acid
were, to some extent, lower than those for ferulic acid and
sinapic acid, but the specificity constant was higher than the
latter two substrates. Overall, in terms of the kinetic parameters
of the tested hydroxycinnamate substrates (1−3), the
preference of UGT75X1 and UGT75L25 for p-coumaric acid
In this work, de novo transcriptome sequencing of E.
breviscapus leaves was performed and two acyl-glucose-ester
forming UGT genes were selected, analyzed, cloned and
expressed with the specific objective of gaining more
bifunctional donors. The biochemical characterizations and
in vitro one-pot reaction coupled with EbUGTs were also
investigated. With a relatively broad substrate promiscuity,
diverse 1-O-acyl-glucose-esters and glucosides were produced
by the EbUGTs. Furthermore, the synthetic intermediates of
2-methyl and 2- and 3-chlorine cinnamic acid can also be
glucosylated, which might provide new donors for the
acylation and glucosylation of anthocyanin. Although no
anthocyanins or acylated or glycosylated derivatives have
F
DOI: 10.1021/acs.jafc.8b07215
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
I2M-1-011) and National Mega-project for Innovative Drugs
(2018ZX09711001-006).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
CBD, chitin binding domain; GT, glycosyltransferase; HCGEs,
hydroxycinnamoyl-1-O-glucose-esters; HPLC-UV/MS, high-
performance liquid chromatography-UV absorption/mass
spectrometry; IPTG, isopropyl-β-D-thiogalactopyranoside;
pHBGE, p-hydroxybenzoyl-1-O-glucose-ester; ORF, open
reading frame; UDP, uridine diphosphate; UDPG, UDP-
glucose; UDPGA, UDP-glucuronic acid; UDP-Gal, UDP-
galactose; UGT, UDP-dependent glycosyltransferase.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.8b07215.
■
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(PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: wwang@imm.ac.cn.
ORCID
Wei Wang: 0000-0003-4192-0674
Author Contributions
Y.Y., M.L., and W.Z. conceived and performed the experi-
ments. Y.Y., M.L., and Y.C. analyzed the data, and Y.Y. drafted
the manuscript. C.L. assisted on the LC/MS analysis. W.W.
coordinated the study and revised the manuscript. All authors
read and approved the final manuscript.
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Funding
This work was supported by the CAMS Innovation Fund for
Medical Science (CAMS-2016-I2M-3-012 and CAMS-2017-
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