The glycosyltransferase involved in thurandacin biosynthesis catalyzes both O- and S-glycosylation

Article abstract of DOI:10.1021/ja411159k

The S-glycosyltransferase SunS is a recently discovered enzyme that selectively catalyzes the conjugation of carbohydrates to the cysteine thiol of proteins. This study reports the discovery of a second S-glycosyltransferase, ThuS, and shows that ThuS catalyzes both S-glycosylation of the thiol of cysteine and O-glycosylation of the hydroxyl group of serine in peptide substrates. ThuS-catalyzed S-glycosylation is more efficient than O-glycosylation, and the enzyme demonstrates high tolerance with respect to both nucleotide sugars and peptide substrates. The biosynthesis of the putative products of the thuS gene cluster was reconstituted in vitro, and the resulting S-glycosylated peptides thurandacin A and B exhibit highly selective antimicrobial activity toward Bacillus thuringiensis.

Full text of DOI:10.1021/ja411159k

Communication
pubs.acs.org/JACS
The Glycosyltransferase Involved in Thurandacin Biosynthesis
Catalyzes Both O- and S‑Glycosylation
Huan Wang, Trent J. Oman, Ran Zhang, Chantal V. Garcia De Gonzalo, Qi Zhang,
and Wilfred A. van der Donk*
Howard Hughes Medical Institute and Roger Adams Laboratory, Department of Chemistry, University of Illinois at
Urbana−Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, United States
*
S Supporting Information
ABSTRACT: The S-glycosyltransferase SunS is a recently
discovered enzyme that selectively catalyzes the con-
jugation of carbohydrates to the cysteine thiol of proteins.
This study reports the discovery of a second S-
glycosyltransferase, ThuS, and shows that ThuS catalyzes
both S-glycosylation of the thiol of cysteine and O-
glycosylation of the hydroxyl group of serine in peptide
substrates. ThuS-catalyzed S-glycosylation is more efficient
than O-glycosylation, and the enzyme demonstrates high
tolerance with respect to both nucleotide sugars and
peptide substrates. The biosynthesis of the putative
products of the thuS gene cluster was reconstituted in
vitro, and the resulting S-glycosylated peptides thurandacin
A and B exhibit highly selective antimicrobial activity
toward Bacillus thuringiensis.
lycosylation represents one of the most common post-
Figure 1. (A) Biosynthetic gene clusters of sublancin and glycocin F and
the putative biosynthetic gene cluster that includes thuS in B.
thuringiensis BGSC 4AW1. (B) Sequence of the ThuA precursor
peptide. Leader sequence is in bold, and the double-glycine-type
proteolytic cleavage site is in red. The Ser and Cys residues glycosylated
by ThuS are in green. The predicted helical segments are underlined.
1
−6
G
translational modifications of proteins.
Naturally
occurring glycopeptides most commonly involve an O- or N-
glycosidic linkage between the saccharide moiety and the side
chain of an appropriate amino acid residue. In contrast, examples
of naturally occurring S-linked glycopeptides or glycoproteins are
(
C) MALDI-TOF MS spectra of His -ThuA (10 μM) before (black)
6
7
−11
very rare.
To date, only two bacterial S-linked glycopeptides
and after (red) incubation with His -ThuS (2 μM), UDP-Glc (1 mM),
6
and Mg2 (1 mM) in 50 mM Tris buffer, pH 7.5 for 10 h at room
+
have been reported, which both display antimicrobial activities,
sublancin 168 produced by Bacillus subtilis 168 and glycocin F
temperature.
12,13
produced by Lactobacillus plantarum KW30.
Sublancin
contains a glucose moiety linked to Cys, which is installed by
were shown to be highly selective in their antimicrobial activity,
targeting only B. thuringiensis.
12
the S-glycosyltransferase SunS. Glycocin F contains two sugars,
an N-acetylglucosamine β-O-linked to Ser18 and an N-
acetylglucosamine S-linked to the C-terminal Cys43 (Figure
S1). The glycocin F biosynthetic gene cluster contains one gene
encoding a putative glycosyltransferase GccA suggesting that
perhaps the enzyme installs both sugars (Figure 1A), but its
As part of our efforts to mine genomes for new antibiotics, we
detected a gene cluster in the genome of B. thuringiensis BGSC
4AW1 that showed similarity to the biosynthetic gene clusters of
sublancin and glycocin F (Figure 1A). This gene cluster contains
genes for a putative precursor peptide ThuA, a glycosyltransfer-
ase ThuS, an ABC-transporter ThuT, two thiol-disulfide
oxidoreductaseses BtdbA and BtdbB, and a putative immunity
protein ThuI. Bioinformatic analysis shows that ThuS shares
13
function has not been confirmed. In this study, we identified
the glycosyltransferase ThuS from Bacillus thuringiensis serovar
andalousiensis BGSC 4AW1 and reconstituted its enzymatic
activity in vitro. We show that ThuS glycosylates its peptide
substrate ThuA at both Ser19 and Cys28, and therefore ThuS
represents the first glycosyltransferase that catalyzes both O- and
S-glycosylation of proteins in vitro. ThuS also displays high
promiscuity toward peptide substrates and nucleotide sugars.
The biosynthesis of thurandacin A and B, the putative products
of the biosynthetic gene cluster that includes thuS, was
reconstituted in vitro, and the resulting glycosylated products
3
9% sequence identity with SunS and belongs to the
glycosyltransferase family A. ThuA consists of a 38-residue
leader sequence and a 42-residue core peptide, separated by a
Gly-Ser motif, which is a double-glycine type proteolytic
cleavage site (Figure 1B). Similar to the sublancin precursor
1
4
Received: November 4, 2013
Published: December 10, 2013
©
2013 American Chemical Society
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Communication
peptide SunA, ThuA contains five Cys residues in its core
peptide. Secondary structure prediction tools (PSIPRED)
lead to more bisglucosylated product with ThuA-S19C
compared to the assay with wild type ThuA (Figures 1C, S4a).
Interestingly, glucose was present on either Cys19 or Cys28 in
the monoglucosylated ThuA-S19C, as determined by tandem
MS (Figure S4b). Although the MSMS data could not quantify
the selectivity of ThuS for Cys19 or Cys28 in this mutant, the
intensity of the fragment ions indicated that the enzyme did not
display high preference for either. Collectively, these results show
that ThuS is chemoselective for the thiol of cysteines over the
hydroxyl groups in serines but displays relatively relaxed site-
selectivity.
15
suggest that the peptide contains two α-helical segments
spanning residues 3−14 and 32−41 (Figure 1B). The prediction
that four of the five Cys residues reside in helical structures is
16
consistent with the NMR structure of glycocin F.
To investigate the function of ThuS, the thuS and thuA genes
were cloned and expressed in Escherichia coli as N-terminal fusion
proteins with a hexahistidine tag (His -ThuS and His -ThuA).
6
6
Upon purification by immobilized metal affinity chromatog-
raphy, His -ThuS was incubated with the purified precursor
6
peptide His -ThuA. Addition of uridine diphosphate α-D-glucose
To investigate the substrate tolerance of ThuS toward sugar
6
2
+
2+
(
UDP-Glc) and Mg resulted in conversion of ThuA to two
donors, the enzyme was incubated with His -ThuA and Mg in
6
products with mass increases of 162 and 324 Da, as determined
by matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS) (Figure 1C), suggesting
mono- and bisglucosylation of ThuA. Tandem MS analysis
revealed that both Ser19 and Cys28 were glucosylated in the
bisglucosylated ThuA (Figure S2), but no glycosylation was
observed for Cys7, Cys14, Cys35, or Cys42. The lack of
glycosylation of these cysteine residues that are located in the
likely helical regions is similar to the site-selectivity that is
the presence of UDP-α-D-N-acetylglucosamine (UDP-GlcNAc),
UDP-α-D-galactose (UDP-Gal), and guanosine diphosphate α-D-
mannose (GDP-Man). All three nucleotide sugars proved to be
good substrates (K values <10 μM), but the glycosyl transfer
m
reaction with UDP-Glc was at least 5-fold more efficient than
reactions with other sugar donors, as indicated by MALDI-TOF-
MS analysis and the enzymatic coupled assay (Figures S5, S6,
Table S4). Hence, ThuS likely installs a glucose onto its substrate
peptide under physiological conditions.
We next attempted to reconstitute the biosynthesis of the
product(s) encoded by the gene cluster in vitro. First, a factor Xa
cleavage site was engineered into the ThuA substrate at the
junction of the putative leader and core peptide. The resulting
12
observed with SunS. The glycosylation of Ser19, however, was
surprising since SunS displays high chemo- and regioselectivity
toward Cys22 of its peptide substrate SunA and does not modify
12
Ser22 in the SunA-C22S mutant. To the best of our knowledge,
ThuS is the first glycosyltransferase that catalyzes both S-
glycosylation of cysteine and O-glycosylation of the hydroxyl
group of serine in proteins in vitro. This finding also provides
support that GccA may install both N-acetylglucosamine groups
in glycocin F.
His -ThuA-Xa peptide was expressed, purified from E. coli, and
6
glucosylated by ThuS, yielding a mixture of mono- and
bisglucosylated peptides. These peptides were then treated
with a mixture of oxidized and reduced glutathione and EDTA to
18
form disulfide cross-links. Finally, the leader peptide was
removed by the addition of the protease factor Xa, and each of
the resulting products was purified by RP-HPLC (Figures 2A, S7,
S8). Mass spectrometric analysis showed that the mass of both
mono- and bisglucosylated products were 4 Da lower than the
mass of the corresponding glucosylated ThuA-Xa core peptides.
Furthermore, incubation with triscarboxyethyl phosphine
increased the mass of both peptide products by 4 Da, indicating
the successful formation of two disulfide bonds in both samples.
Trypsin digestion of the purified bisglucosylated product yielded
a peptide fragment spanning residues 12−39 with one disulfide
bond as well as a peptide fragment containing residues 1−11 and
residues 40−41 connected by one disulfide bond, as determined
by MALDI-TOF MS (Figure S9a), indicating that one disulfide
cross-link was between Cys14 and Cys35 and a second disulfide
was formed between Cys7 and Cys42. Similarly, trypsin digestion
of the monoglucosylated product also resulted in a peptide
fragment spanning residues 12−39 with one disulfide bond,
indicating the formation of the same disulfide pattern (Figure
S9b).
We next investigated the efficiency of the O- and S-
glycosylation reactions catalyzed by ThuS. Incubation of
equimolar amounts of ThuA and UDP-Glc (20 μM) in the
2
+
presence of ThuS and Mg resulted in glycosylation of only
Cys28, as determined by MALDI-TOF MS and tandem MS
(Figure S3), suggesting that S-glycosylation is favored. Indeed,
installation of a second glucose on Ser19 of the monoglucosy-
lated peptide (hereafter termed ThuA-Cys28-Glc) was signifi-
cantly slower than the glycosylation of Cys28 in unmodified
ThuA, as indicated by a coupled enzymatic assay (Supporting
17
Information and Table S2). In this assay, the nucleotide
diphosphate generated upon glycosyl transfer reactions was
coupled to NADH oxidation via pyruvate kinase (PK) and lactate
dehydrogenase (LDH) (Scheme S1). Under appropriate
conditions such that reactions catalyzed by PK and LDH are
much faster than nucleotide diphosphate release catalyzed by
ThuS, the observed rate of NADH consumption equals the rate
of nucleotide diphosphate generation and can be continuously
monitored at 340 nm.
To further investigate the selectivity of ThuS, Cys28 in the
ThuA peptide was mutated to Ser to investigate whether the
preferential glucosylation of Cys28 over Ser19 is the result of site-
selectivity or chemoselectivity. In the presence of 0.5 μM ThuS,
To examine the stereochemistry of the glycosidic linkages,
bisglucosylated ThuA-C28S peptide, which contains a glucose
moiety on both Ser19 and Ser28, was treated with β-glucosidase.
Subsequent MALDI-TOF MS analysis revealed that two
glucoses were released by β-glucosidase, indicating that both
glucose moieties on ThuA-C28S were β-linked (Figure S10).
Therefore, ThuS is an inverting glycosyltransferase. Collectively,
these results show that the in vitro generated glycopeptides have
β-linked glucose moieties and a nested disulfide pattern similar to
the S-glycopeptide antibiotics sublancin 168 and glycocin F
100 μM UDP-Glc and 30 μM peptide substrate (maximum
peptide solubility), the modification rate of ThuA-C28S was
about 25% of the modification rate of ThuA (Table S3),
suggesting that Cys glucosylation is faster than Ser glucosylation.
This increased activity with thiols was also observed when
another ThuA mutant, ThuA-S19C, was incubated with ThuS
and UDP-Glc. The first glucosylation occurred with similar rates
for ThuA and ThuA-S19C, as determined by the coupled
enzymatic assay (Table S3). However, extended incubation time
1
2,13,16
(Figures 2A, S1).
The glucosylated and oxidatively folded peptides were then
tested for antimicrobial activity against a number of Gram-
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Figure 3. (A) Sequence alignment of ThuA and SunA peptides. Leader
sequences are in bold, and putative double glycine type proteolytic
cleavage sites are in red. Numbering shown is for the ThuA peptide. (B)
MALDI-TOF MS spectra of His -SunA before (black) and after (red)
6
2+
incubation with His -ThuS, UDP-Glc, and Mg .
6
Glucosylation at Cys28 and removal of the leader peptide were
strictly required for bioactivity of thurandacin A (Figure 2C).
Thurandacin A analogs with different sugar moieties were also
prepared by using GDP-Man, UDP-Gal, and UDP-GlcNAc in
the ThuS catalyzed glycosylation following the in vitro
reconstitution procedure described above. Interestingly, all
thurandacin analogs displayed similar growth inhibitory activity,
as indicated by agar diffusion assay (Figure 2C). Thus, the
stereochemistry of the hexose does not appear to be critical for
bioactivity.
Figure 2. (A) In vitro reconstitution of thurandacin biosynthesis. GSSG:
oxidized glutathione; GSH: reduced glutathione. (B) Agar diffusion
assay of thurandacin A and B against B. thuringiensis BGSC 4CC1.
Samples were spotted on LB agar in a volume of 5 μL: (1) 3 μM
thurandacin A; (2) 3 μM thurandacin B. (C) Agar diffusion assay of
thurandacin A and its analogs against B. thuringiensis BGSC 4CC1. The
following samples were spotted on LB agar in a volume of 5 μL: (1) 3
μM thurandacin A; (2) 3 μM thurandacin A-Man analog; (3) 3 μM
thurandacin A-GlcNAc analog; (4) 3 μM thurandacin A-Gal analog; (5)
Agar diffusion assays suggest that thurandacin A is more
potent than sublancin 168 against B. thuringiensis BGSC 4CC1
(
Figure S13). Sublancin from B. subtilis 168 displayed
19
antimicrobial activities mainly against B. subtilis, and glycocin
F produced by L. plantarum KW30 has inhibitory activity limited
to other lactobacilli. Therefore, S-glycopeptide antibiotics
discovered so far, while similar in structure (Figure S1), all
display narrow antimicrobial spectrum against bacteria that are
closely related to their producing organisms.
3
μM oxidatively folded ThuA-Xa-Cys28-Glc peptide; (6) 3 μM ThuA-
Xa-Cys28-Glc; (7) 3 μM ThuA-Xa core peptide; (8) 10 mM Tris buffer,
pH 7.5.
20
16
positive bacteria including Lactococcus lactis HP, Bacillus subtilis
ATCC 6633, Bacillus halodurans C125, and B. thuringiensis
serovar pulsiensis BGSC 4CC1. The antimicrobial activities of in
vitro generated peptides were determined by agar diffusion
assays, and their potency was estimated from the diameter of the
inhibition zone, assuming that the number of sugar modifications
do not significantly alter their diffusion behavior in agar.
We next explored the substrate specificity of ThuS with respect
to peptide substrates. ThuA shares high homology with SunA at
the N-terminus of the leader peptide but low sequence similarity
in the core peptide (Figure 3A). Surprisingly, ThuS was able to
2
+
modify the SunA peptide in the presence of UDP-Glc and Mg ,
efficiently generating its bisglucosylated product (Figure 3B). In
contrast, SunS was not able to modify the ThuA peptide,
demonstrating that the peptide sequence selectivity of ThuS is
more relaxed. Tandem mass spectrometry analysis revealed that
Cys22 of bisglycosylated SunA was modified by ThuS, with the
second glucosylation occurring on Ser16, Thr19 or Cys29 in a
mutually exclusive manner (Figure S14). ThuS was also able to
glucosylate the SunA core peptide without the leader peptide
attached, however the modification was less efficient than that of
full length SunA and only monoglycosylation was observed by
MALDI-TOF MS (Figure S15). These results suggest that the
leader peptide of SunA contributes to substrate recognition by
ThuS but is not essential.
Both mono- and bis-glycosylated peptides exhibited potent
inhibitory activity against B. thuringiensis BGSC 4CC1 (Figure
2B), however, very low or no inhibitory activity toward other
strains (Table S6, Figure S11a). Hence, we have named the in
vitro generated mono- and bis-glycosylated peptides from the
gene cluster of B. thuringiensis serovar andalousiensis 4AW1
thurandacin A and B, respectively (Figure 2A). The minimum
inhibitory concentration (MIC) of thurandacin A against B.
thuringiensis BGSC 4CC1 was determined to be 0.6 μM in liquid
LB medium (Figure S11b); the quantities of thurandacin B were
insufficient for MIC determination in liquid culture. Thuranda-
cin A displayed somewhat higher potency than thurandacin B in
agar diffusion growth inhibition assays (Figures 2B, S12).
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Communication
Table 1. Partial Sequences of ThuA and SunA Mutant
Peptides (Residues 11−34)
AUTHOR INFORMATION
Corresponding Author
vddonk@illinois.edu
■
a
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the U.S. National Institutes of
Health (GM58822 to W.A.v.d.D.) and fellowships from the
NIGMS-NIH Chemistry-Biology Interface Training Grant
Program (5T32-GM070421 to C.G.D.G) and the Cellular and
Molecular Biology Training Program (T32 GM007283 to
T.J.O.). Mass spectra were recorded in part on an instrument
purchased with grant S10RR027109-01 from the U.S. National
Institutes of Health.
a
Sequences of CREB and Syn replacements of the native loop
sequences in SunA and ThuA are in bold and underlined. Target Cys
residues are highlighted in red.
Since ThuS was able to glucosylate SunA despite the
significant differences in the sequences of SunA and ThuA, we
decided to investigated whether ThuS could modify ThuA and
SunA peptides with non-natural sequences inserted in the loop
between the internal disulfides. S-glycosidic linkages have been
shown to have significantly improved chemical and biological
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ASSOCIATED CONTENT
Supporting Information
Description of procedures, purifications, and figures. This
information is available free of charge via the Internet at
http://pubs.acs.org.
■
*
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