Two trifunctional leloir glycosyltransferases as biocatalysts for natural products glycodiversification

Article abstract of DOI:10.1021/acs.orglett.9b03040

Two promiscuous Bacillus licheniformis glycosyltransferases, YdhE and YojK, exhibited prominent stereospecific but nonregiospecific glycosylation activity of 20 different classes of 59 structurally different natural and non-natural products. Both enzymes transferred various sugars at three nucleophilic groups (OH, NH₂, SH) of diverse compounds to produce O-, N-, and S-glycosides. The enzymes also displayed a catalytic reversibility potential for a one-pot transglycosylation, thus bestowing a cost-effective application in biosynthesis of glycodiversified natural products in drug discovery.

Full text of DOI:10.1021/acs.orglett.9b03040

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Two Trifunctional Leloir Glycosyltransferases as Biocatalysts for
Natural Products Glycodiversification
Ramesh Prasad Pandey,†,‡ Puspalata Bashyal,‡ Prakash Parajuli,‡ Tokutaro Yamaguchi,†,‡,§
and Jae Kyung Sohng*,†,‡
†Department of Pharmaceutical Engineering and Biotechnology and ‡Department of Life Science and Biochemical Engineering, Sun
Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 31460, Republic of Korea
§Genome-based BioIT Convergence Institute, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 31460, Republic of
Korea
S
* Supporting Information
ABSTRACT: Two promiscuous Bacillus licheniformis glyco-
syltransferases, YdhE and YojK, exhibited prominent stereo-
specific but nonregiospecific glycosylation activity of 20
different classes of 59 structurally different natural and non-
natural products. Both enzymes transferred various sugars at
three nucleophilic groups (OH, NH2, SH) of diverse
compounds to produce O-, N-, and S-glycosides. The enzymes
also displayed a catalytic reversibility potential for a one-pot
transglycosylation, thus bestowing a cost-effective application
in biosynthesis of glycodiversified natural products in drug
discovery.
lycodiversification is developing as one of the most
Based on the genome sequences of 5349 complete bacterial
Gprominent biocatalyst tools to create glycoside libraries genome sequences available from 2192 unique species in the
of bioactive small molecules.1 In place of hazardous, time-
dbCAN2 meta-server database (http://bcb.unl.edu/dbCAN2/
blast.php), a total of 572 269 CAZymes homologues are
reported.6 From the database, we found a total of 3100 GT1
family proteins in the genomes of 1335 unique bacterial strains
(see Figure S1B in the Supporting Information). However, few
of the GT1 family proteins (∼400) are functionally
characterized and a considerably low number of structures
have been resolved by crystallography so far. Herein, we
focused on GT1 family proteins from the industrially
important nonpathogenic Bacillus licheniformis DSM 13 strain
whose genome sequence (NC_006270.3) has been com-
pleted.7
According to the CAZy database, B licheniformis genome
contains 146 CAZyme-related protein encoding genes. Of
these, 38 GTs fall into nine different GT families (Figure S1C
in the Supporting Information). However, only three GTs
(YjiC, YdhE, YojK) belong to the GT1 family of proteins.
Since CAZymes are displayed in modular architecture in
genomes with other noncatalytic modules, they often appear in
modular fashion. To study the genomic organization of
CAZymes, we performed a CAZyme gene clusters (CGC)
analysis of the genome sequence of B. licheniformis, using the
open web server dbCAN2 meta server. The genome contains
seven CGCs; however, among 146 CAZymes, most of the
enzymes were present out of the CGC clusters, whereas none
consuming, and costly chemical synthesis approaches to
generating natural product (NP) glycosides, the search for
novel glycosyltransferases (GTs) (EC 2.4.1) with wider
tolerance of donor and acceptor substrates with permissive
catalysis is of enormous importance for producing various
glyco-functionalized NPs in an eco-friendly and cost-effective
manner.2
GTs are the gatekeepers for NPs glycosylation, which is well-
known to influence the physical, chemical, and biological
properties of the parent molecules.3 The process of
glycosylation is well-characterized in cells, involved in
metabolism, cell integrity, molecular recognition, pathogenic-
ity, and post-modification of secondary metabolites during
biosynthesis.4 Since glycans are virtually present in every cells,
GTs are ubiquitous in nature and can transfer sugar moieties
from activated nucleotide diphosphate sugars (NDP-sugars) to
acceptor molecules at nucleophilic OH, NH2, SH, or CH.
According to the recent CAZy classification (http://www.cazy.
org/), GTs are classified into 107 different families. Among
them, GT1 family proteins are inverting enzymes having a GT-
B type 3D structure.5
A total of more than 19 000 GT1 family proteins are
distributed across five domains of life. However, one-third of
these proteins are present in bacteria (see Figure S1A in the
Supporting Information). The number of these genes in the
database is increasing day by day, because the number of
genome sequences of new bacterial strains is rapidly increasing.
Received: August 26, 2019
© XXXX American Chemical Society
A
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Scheme 1. Phenolic Hydroxyl Glucosylation Reaction by YdhE and YojK
of the GT1 family proteins were present in all of these seven
CGCs (see Table S4 in the Supporting Information). A
glycogenomics approach has recently been used to characterize
several glycosylated natural products (GNPs); the biosynthesis
gene clusters including GTs involved in their biosynthesis.8 To
study the possibility of GT1 family proteins involved in
secondary metabolites glycosylation in B. licheniformis, we
analyzed the genome sequence by anti-SMASH bacterial
version.9 However, none of the cluster showed GT1 family
proteins involved in their biosynthesis pathways (see Table S5
in the Supporting Information). None of these in silico genome
analyses provided clues on the involvement of these GTs in
NPs glycosylation. However, our previous experiences showed
that one of the GT1 family proteins, YjiC, from the same strain
exhibited high promiscuity to glycosylate wide range of
secondary metabolites from plants and microbes.10
The amino-acid sequence alignment of YjiC, YdhE, and
YojK showed comparatively low sequence identity and
similarity with each other (see Figure S2 and Table S6 in
the Supporting Information). Although all three GTs fell into
the same clade, they are reasonably divergent from each other
in the phylogenetic tree analysis. YjiC is more similar to GTs
from Streptomyces, whereas YdhE and YojK are more similar to
Bacillus GTs (see Figures S3 and S4 in the Supporting
Information). These two genes were cloned in pET28a(+)
vector (see Figure S5 in the Supporting Information) and
functionally expressed in E. coli BL21 (DE3) for biochemical
studies (see Figure S6 in the Supporting Information).
To investigate the glycosylation property of YdhE and YojK,
50 μg/mLthe final concentrationof Ni-affinity purified
His6-tagged fusion pure recombinant enzymes (YdhE 48 kDa
and YojK 47 kDa) were reacted with 1 mM 3-hydroxyflavone
(3-HF, 1) in 100 mM Tris-HCl buffer (pH 7.5) containing 2
mM UDP-α-D-glucose (UDP-Glc), and 10 mM MgCl2
(Scheme 1) at 37 °C for 3 h. The high-performance liquid
chromatography-photo diode array (HPLC-PDA) showed the
conversion of 1 (retention time (tR = 13 min)) to 1a (tR = 9.4
min) by YdhE, but YojK produced an additional product 1b
(tR = 9 min) in small amounts, both of them showing a similar
ultraviolet−visible light (UV-vis) spectrum (Figure S7 in the
Supporting Information). High-resolution quadruple time-of-
flight electrospray ionization mass spectrometry (HRqTOF-
ESI/MS) in positive mode showed 1a with a proton adduct
mass of 401.1221 Da, whereas 1b displayed a mass of 563.1765
Da, which were exactly 162 and 324 amu greater than the ion
peak of 1 (239.0687 Da), respectively, resembling one and two
glucose-conjugated derivatives of 1 (see Figure S7). Product 1a
was isolated from YdhE and 1b was isolated from YojK-
catalyzed reactions separately to the purity of 98% using prep-
HPLC for NMR studies. The 1D and 2D NMRs of both
products identified 1a as 3-HF-3-O-β-D-glucopyranoside, and
1b as 3-HF 3-O-β-D-glucopyranosyl (1−3)-β-D-glucopyrano-
side. The large coupling constant values (>7.5 Hz) showed
conjugation of glucose in β-configuration (see Table S7 in the
Supporting Information).
To understand the characteristics of YdhE and YojK,
identical reactions were performed with both enzymes using
1 and UDP-Glc at 37 °C. The HPLC profile of the reaction
mixture was monitored at different time points, from 5 min to
60 min. Both enzymes achieved maximum conversion of 1
after 30 min (see Figure S8 in the Supporting Information).
YojK has ∼50% relative conversion in 5 min incubation, which
was 85% with YdhE at the same time. Interestingly, the
conversion rate was subtly decreasing in both reactions after 30
min. The optimum activity of the enzymes was found to be
observed at 30 °C. Although YojK has relatively low tolerance
to temperature variations, YdhE was found to be more stable
and active, even below 15 °C and above 37 °C. Similarly, we
found the optimal pH of the reaction buffer to be pH 10.0 and
8.8 for YdhE and YojK, respectively. Interestingly, YojK had a
higher conversion at a low pH range (pH 3.0−6.5) than YdhE.
The divalent metal ion profiling showed that both enzymes are
catalytically active, even in the absence of metal ions. However,
some metal ions, such as Hg2+, Ni2+, Pb2+, Zn2+, Fe2+, and
Cu2+, decreased the enzymes’ activity considerably. The
inhibitory effect of some divalent metal ions on enzyme
activities of UGTs toward Cu2+ and Co2+ has been previously
reported.11 The overall results showed that none of the GTs
require divalent cations for enzyme activities, but the presence
of Mg2+, Mn2+, and Ca2+ enhanced the catalytic activity (see
Figure S8 in the Supporting Information). The enzyme kinetics
of both enzymes using 1 under optimal conditions showed
similar Km values. But the Vmax, kcat, and kcat/Km values of
YdhE are higher than those of YojK (see Table S8 in the
Supporting Information), revealing a higher catalytic efficiency
of YdhE than of YojK.
We then investigated the enzymes for donor substrate
flexibility using all five natural nucleotide diphosphate glucoses
(NDP-Glcs). The reaction with 1 and the five NDP-Glcs
showed that YdhE can accept all five different NDP-Glcs as
donor substrates, whereas YojK can accept only ADP-α-D-
glucose (ADP-Glc), TDP-α-D-glucose (TDP-Glc), and UDP-
Glc (Figure 1). The time profile conversion of 1 to 1a with
different NDP-Glcs showed conversion increment in the order
of UDP-Glc > CDP-Glc > TDP-Glc > ADP-Glc > GDP-Glc
with YdhE, which was in the order of UDP-Glc > ADP-Glc >
TDP-Glc for YojK. The study revealed that both GTs are
flexible toward all types of NDP-Glcs as donor substrates, thus
increasing the possibility of employing diverse types of sugars
as donor substrates for the glycodiversification reactions.
Since GTs are known to synthesize N-, C-, and S-glycosidic
linkages,12 we performed reactions using 3,4-dichlorophenol
(2), 3,4-dichloroaniline (3), and 3,4-dichlorobenzenethiol (4)
as acceptor substrates and UDP-Glc as the glucose donor
substrate (see Scheme 2). As seen in the aforementioned
reaction with 1, both YdhE and YojK catalyzed the conversion
B
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analysis of reaction mixtures of 3 and 4 showed a prominent
product peak in all four reaction mixtures. The products (3a
and 4a) also had an UV/vis absorbance similar to that of their
parent molecules 3 and 4 and HR-qTOF ESI/MS confirmed
the conjugation of a glucose moiety to each substrate (see
Figures S9B and S9C in the Supporting Information). To
further confirm the products by NMR studies, 3a and 4a were
produced in large volume and purified by prep-HPLC. The
NMR analysis confirmed the conjugation of a glucose at the
amino functional group of 3 and thiol functional group of 4 in
β-configuration, thus, producing 3,4-dichlorobenzyl 1-N-β-D-
glucopyranoside (3a) and 3,4-dichlorobenzyl 1-S-β-D-gluco-
pyranoside (4a), respectively. Neither of the reaction mixtures
showed C-glucosylated products in either enzyme reaction.
To expand the possibility of use of YdhE and YojK toward
O-, N-, and S-glycosidic linkage formation with various NDP
sugars for O-, N-, and S-functionalized natural products, we
reacted the same set of three acceptor substrates 2, 3, and 4
with eight different NDP-D/L sugars. NDP-D sugars, including
UDP-α-D-galactose (UDP-Gal), UDP-α-D-glucuronic acid
(UDP-GlcA), UDP-α-D-N-acetyl-galactosamine (UDP-Gal-
NAc), UDP-α-D-N-acetyl-glucosamine (UDP-GluNAc),
TDP-α-D-2-deoxyglucose (TDP-2dGlc), TDP-α-D-viosamine
(TDP-Vio), and NDP-L sugars (TDP-α-L-rhamnose (TDP-
Rhm), GDP-α-L-fucose (GDP-Fuc)) were reacted using YdhE
and YojK. The HPLC-PDA and HR-qTOF ESI/MS results
were fascinating that both YdhE and YojK accepted all three
acceptor substrates and most of the donor NDP-D/L sugars and
converted them to their respective glycosides (2a−2i, 3a−3i,
4a−4i) (see Figure 2, as well as Figures S10−S33 in the
Supporting Information). Interestingly, both enzymes con-
verted 4 to diverse glycosides with a relatively higher
Figure 1. Donor substrate flexibility of YdhE and YojK for the
reaction of 1 and NDP-glc at different time intervals.
Scheme 2. O-, N-, and S-Glucosylation Reactions by YdhE
and YojK
of 2 (tR 14.6 min) to its O-glucopyranoside derivative (tR 10.8
min), which we further characterized by UV-vis, HR-qTOF
ESI/MS (Figure S9A in the Supporting Information), and 1D
NMR analyses, identifying the glucosylated product of 2 as 3,4-
dichlorophenyl O-β-D-glucopyranoside (2a). Similarly, the
Figure 2. Catalytic promiscuity of YdhE and YojK at the OH, NH2, and SH functional groups containing compounds with various NDP-D/L sugars.
(A) Structures of the O-, N-, and S-glycosides generated. (B) Conversion percentage of each glycosylated molecule by YdhE and YojK. (C)
Structures of different sugar moieties conjugated to compounds 2, 3, and 4.
C
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Figure 3. Acceptor substrate promiscuity study of YdhE and YojK with a set of various compounds. The upper panel shows the percent conversion
of each compound catalyzed by YdhE and YojK. The number just above the bars represents the number of products generated from the reaction.
The numbers of the molecules are listed as shown in the structures of the molecules presented in the lower part of the figure. The symbol “D”
indicates that the compound was detected by HR-ESIMS. The compounds used for the reaction, but not accepted by both enzymes, are listed in
Figure S90 in the Supporting Information.
conversion percentage than 2 and 3 (see Figure 2). This result
showed that the newly identified GTs preferred S-glycosylation
with flexible activity toward O- and N-glycosylation. Neither
enzymes could conjugate glucuronic acid, rhamnose, or fucose
to 2 and 3.
Inspired by the broad catalytic activity of enzymes with
NDP-D/L sugars, we investigated the promiscuity of YdhE and
YojK toward 84 different natural and non-natural small
molecules. Both enzymes were reacted with 50 hydroxyl
groups containing compounds (1, 2, 5−42, 60−69) mostly
belonging to plant-originated secondary metabolites; 27 amino
functional groups containing metabolites (3, 43−55, 70−82),
of which most are synthetic compounds and used as human
drugs; and seven thiol groups containing synthetic molecules
(4, 56−59, 83, 84) in the Tris-HCl buffer containing MgCl2
and UDP-Glc. The reaction mixtures were analyzed by HPLC-
D
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PDA followed by HR-qTOF ESI/MS. Both enzymes showed
unprecedented substrate promiscuity of O-, N-, and S-
glycosylation of various classes of compounds.
synthesized p-nitro-β-D-glucoside (85),12d,13 the catalytic
deglycosylation property of YdhE and YojK were exploited
in the presence of UDP. The system was eventually coupled
with intermolecular transglycosylation reactions with 2, 3, and
4 (see Figure 4). The system showed efficient conversion of 2
Interestingly, the enzymes showed multiple glycosylations at
phenolic hydroxyl groups of 23 different structural types of
flavonoids, including flavonols (1, 5−8), flavones (9−16),
flavanols and flavanones (17−20), isoflavones (21−24), trans-
stilbene (25), and dihydrochalcone (26) (see Figure 3). The
glucosides were identified as O-glucosides based on the
HRqTOF ESI/MS analysis showing a characteristic mass ion
after loss of glucose unit with 162 amu (see Figures S34−S53
in the Supporting Information). For most of the flavonoids
compounds, the conversion rate was >75% with both enzymes,
except for compounds 8, 9, 11−14, 22, and 23 for only YojK.
It is noteworthy that both enzymes exhibited a catalytic
potential of >95% with flavonols (5−7), flavones (10, 15, 16),
flavanols (17−20), stilbene (25), and dihydrochalcone (26)
(see Figures S54 and S55 in the Supporting Information). Both
enzymes accepted hydroxyl groups containing alkaloid 8-
hydroxy quinoline (27), acetanilide (28), monoterpenes (29−
32), phenylpropanoic acids (33 and 34), coumarin (35),
napthoquinone (36), anthraquinones (37−41), and curcumin
(42) (see Figure 3, as well as Figures S56−S71 in the
Supporting Information). In addition, both enzymes demon-
strated N- and S-glycosylation of various classes of compounds.
The compounds containing amino-functional groups, including
aminocoumarin (43, 44), anthraquinone (45), unique
synthetic compound (46), acridine (47), aminobenzene
(48−51), piperidine (52), antipyrine (53), indole alkaloid
(54), and anthracene (55) were accepted by both enzymes
(see Figures S72−S84 in the Supporting Information). The
conversion rate of most of the amino functional groups
containing molecules tested in this study was <20%, except for
aminocoumarin (43, 44), aminobenzene (48, 49), and
anthracene (55). Similarly, we tested the substrates tolerance
capacity of YdhE and YojK with different thiol groups
containing molecules, including benzothiol (4, 56), mercaptan
(57, 83, 84), dimercapto triazine (58), and benzothiazole (59)
(see Figure 3, as well as Figures S85−S88 in the Supporting
Information). Both enzymes showed robust capabilities to
convert 56−59 into their respective thiol-glucoside derivatives
with the conversion rate of 80% for 56, followed by conversion
rates of >40% with 57 and 59. Altogether, both enzymes
showed broad substrate promiscuity by glycosylating more
than 20 different classes of compounds (see Figure 3, as well as
Figure S89 in the Supporting Information). However, none of
them catalyzed steroid groups of compounds (60−64), 16-
membered macrolactone (65), or tetracycline antibiotics (66−
69), five nitrogen bases (70−74), aliphatic NH2 group
containing compounds (75, 76, 79), other classes of synthetic
molecules with NH2 (77, 78, 80−82), or compounds with
aliphatic SH functional groups into glucopyranosides (see
Figure S90 in the Supporting Information).
Figure 4. Catalytic reversibility and transglycosylation of compounds
2, 3, and 4 by YdhE and YojK. The conversion percent of each
substrate is shown at different time intervals.
(>80%), 3 (∼40%), and 4 (>50%) into their respective O-, N-,
and S-glucosides in the absence of UDP-Glc within 15 min,
suggesting a possible application of enzymes for one-pot
transglycosylation using various synthetic glucosides without
adding UDP-Glc.
In summary, we identified two new highly promiscuous
GTs, YdhE and YojK, from B. licheniformis DSM 13 with
unprecedented catalytic efficiency toward different NDP-D/L
sugars and above 20 different classes of natural and non-natural
compounds to form glycosides. Moreover, both enzymes could
synthesize O-, N-, and S-glycosidic linkage with different sugars
with a higher activity toward the thiol nucleophilic group than
did hydroxyl and amine groups containing compounds. Both
GTs found to be superior to previously characterized GTs such
as OleD from Streptomyces antibioticus,1g,2a,c in terms of their
wide donor and acceptor substrates promiscuity and catalytic
potential to transfer multiple sugars to amine and thiol
functional groups without optimizing enzymes’ catalytic sites.
This study highlights the first Bacillus GTs to catalyze
numerous druglike compounds to produce O-, N-, and S-
glycosides. In addition, the enzymes reacted nonregiospecifi-
cally to produce multiple glucosides, along with catalytic
reversibility for one-pot transglycosylation reaction at −OH,
−NH2, and −SH groups. Cumulatively, these GTs could serve
as potential biocatalysts for the glycodiversification of various
druglike scaffolds.
To elucidate the structure of glucosides, we purified 19
selected glucosides (1a, 1b, 2a, 3a, 4a, 20a−20c, 21a−21c,
1
29a, 30a, 31a, 39a, 40a, 43a, 49a, 56a, 58a) and used H
13
NMR as well as NMR. To elucidate the structures of a few
selected molecules, various 2D NMRs were performed (see
Figures S91−S161 in the Supporting Information). The NMR
analysis showed the same beta-stereochemistry of conjugated
glucose in all molecules (see Table S7).
To develop an eco-friendly and cost-effective chemo-
enzymatic system to synthesize UDP-glc from chemically
E
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ASSOCIATED CONTENT
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The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b03040.
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Experimental procedures, HPLC-HRESIMS character-
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(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: sohng@sunmoon.ac.kr.
ORCID
(6) Zhang, H.; Yohe, T.; Huang, L.; Entwistle, S.; Wu, P.; Yang, Z.;
Busk, P. K.; Xu, Y.; Yin, Y. dbCAN2: a meta server for automated
carbohydrate-active enzyme annotation. Nucleic Acids Res. 2018, 46
(W1), W95−W101.
Jae Kyung Sohng: 0000-0003-1583-5046
Author Contributions
(7) Veith, B.; Herzberg, C.; Steckel, S.; Feesche, J.; Maurer, K. H.;
R.P.P., P.B., and P.P. performed experiments, analyzed data.
R.P.P. wrote the manuscript. T.Y. helped in analyzing NMR
data. J.K.S. supervised the work. All authors have given
approval to the final version of the manuscript.
̈
Ehrenreich, P.; Baumer, S.; Henne, A.; Liesegang, H.; Merkl, R.;
Ehrenreich, A.; Gottschalk, G. The complete genome sequence of
Bacillus licheniformis DSM13, an organism with great industrial
potential. J. Mol. Microbiol. Biotechnol. 2004, 7, 204−211.
Notes
(8) Kersten, R. D.; Ziemert, N.; Gonzalez, D. J.; Duggan, B. M.;
Nizet, V.; Dorrestein, P. C.; Moore, B. S. Glycogenomics as a mass
spectrometry-guided genome-mining method for microbial glycosy-
lated molecules. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, E4407−
4416.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant from the Next-Generation
BioGreen 21 Program (SSAC, Grant No. PJ013137), Rural
Development Administration, Republic of Korea.
(9) Blin, K.; Shaw, S.; Steinke, K.; Villebro, R.; Ziemert, N.; Lee, S.
Y.; Medema, M. H.; Weber, T. antiSMASH 5.0: updates to the
secondary metabolite genome mining pipeline. Nucleic Acids Res.
2019, 47, W81−W87.
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