Differential Recognition of Deacetylated PNAG Oligosaccharides by a Biofilm Degrading Glycosidase

Article abstract of DOI:10.1021/acschembio.9b00467

Exopolysaccharides consisting of partially de-N-acetylated poly-β-d-(1→6)-N-acetyl-glucosamine (dPNAG) are key structural components of the biofilm extracellular polymeric substance of both Gram-positive and Gram-negative human pathogens. De-N-acetylation

Full text of DOI:10.1021/acschembio.9b00467

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Article
Differential Recognition of Deacetylated PNAG
Oligosaccharides by a Biofilm Degrading Glycosidase
Shaochi Wang, Alexandra P. Breslawec, Elaine Alvarez, Michal Tyrlik, Crystal Li, and Myles B Poulin
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00467 • Publication Date (Web): 20 Aug 2019
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Differential Recognition of Deacetylated PNAG Oligosaccharides by
a Biofilm Degrading Glycosidase
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Shaochi Wang, Alexandra P. Breslawec, Elaine Alvarez, Michal Tyrlik, Crystal Li and Myles B. Poulin*
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States
ABSTRACT: Exopolysaccharides consisting of partially de-N-acetylated poly-β-D-(16)-N-acetyl-glucosamine (dPNAG) are
key structural components of the biofilm extracellular polymeric substance of both gram-positive and gram-negative human
pathogens. De-N-acetylation is required for the proper assembly and function of dPNAG in biofilm development suggesting
that different patterns of deacetylation may be preferentially recognized by proteins that interact with dPNAG, such as
Dispersin B (DspB). The enzymatic degradation of dPNAG by the Aggregatibacter actinomycetemcomitans native -
hexosaminidase enzyme DspB plays a role in biofilm dispersal. To test the role of substrate de-N-acetylation on substrate
recognition by DspB, we applied an efficient pre-activation based one-pot glycosylation approach to prepare a panel of dPNAG
trisaccharide analogs with defined acetylation patterns. These analogs served as effective DspB substrates and the rate of
hydrolysis was dependent on the specific substrate de-N-acetylation pattern, with glucosamine (GlcN) located +2 from the
site of cleavage being preferentially hydrolyzed. The product distributions support a primarily exoglycosidase cleavage
activity following a substrate assisted cleavage mechanism, with the exception of substrates containing a non-reducing GlcN
that were cleaved endo leading to the exclusive formation of a non-reducing disaccharide product. These observations provide
critical insight into the substrate specificity of dPNAG specific glycosidase that can help guide their design as biocatalysts.
biofilm forming bacteria.18,19 DspB is a member of the CAZy
INTRODUCTION
GH20 family of glycosyl hydrolase enzymes,20,21 and is one
Biofilms consisting of surface associated bacteria imbedded
of only two enzyme specific for dPNAG hydrolysis
in an extracellular polymeric substance (EPS), are
identified.22 The other, PgaB contains a C-terminal CAZy
responsible for approximately 65% of all human bacterial
GH153 family of glycosyl hydrolase that catalyzes the endo-
infections.1 The EPS is composed of exopolysaccharides,
glycosidic cleavage of dPNAG containing de-N-acetylated
proteins and extracellular DNA, that facilitate cell–cell
GlcN in the –3 binding site using a yet unidentified cleavage
attachment and provide
a protective barrier against
mechanism.22 Compared to PgaB, the specific interactions
required for dPNAG binding and recognition by DspB
remain poorly understood. dPNAG hydrolysis by DspB
likely follows a substrate assisted cleavage mechanism in
which the substrate 2-acetamido group facilitates glycoside
antibiotics and the host immune system.2-6 Many medically
important biofilm forming bacterial strains, including
Staphylococcus epidermidis,2 Staphylococcus aureus,3
Escherichia coli,4 Acinetobacter baumannii,5 Actinobacillus
pleuropneumoniae,6 Klebsiella pneumoniae,7 and Yersinia
pestis,8 produce a similar partially de-N-acetylated poly-β-
D-(1-6)-N-acetyl-glucosamine (dPNAG) as the key EPS
exopolysaccharide. In these organisms dPNAG functions as
a major virulence factor, and contributes to pathogenicity in
animal infection models.9 Partial deacetylation of PNAG by
specific carbohydrate esterase enzymes plays a role in the
proper export and function of dPNAG in biofilms of gram-
negative bacteria and is required for proper cell association
in gram-positive bacteria,9-12 yet the exact function of
deacetylation is poorly understood. Specifically, it is not
clear if deacetylation impacts binding and recognition of
dPNAG by other proteins.
hydrolysis through formation of
a
characteristic
oxazolinium ion intermediate(Figure 1A),23 which is
characteristic of GH20, 18, 25, 56, 84, and 85 family
enzymes.24 All studies of DspB activity have used either
isolated PNAG polysaccharides containing a heterogeneous
distribution of acetylated and deacetylated GlcNAc,17,19 or
synthetic PNAG oligosaccharides composed entirely of
acetylated GlcNAc.25,26 From these studies it is not clear how
DspB cleaves the 3-20% deacetylated GlcN present in native
dPNAG polysaccharides,27 which lack a 2-acetamido group
required for substrate assisted cleavage. Studying the role
of deacetylation for PNAG hydrolysis by DspB requires
PNAG oligosaccharides with chemically defined acetylation
patterns (Figure 1B).
Enzymes that breakdown the biofilm EPS, including
glycosidase, DNase and protease enzymes have emerged as
promising tools to disrupt existing biofilms.13-16 DispersinB
Unlike direct isolation from bacterial biofilms, chemical
synthesis provides access to deacetylated PNAG in
relatively large scale.32-35 Previous approaches to synthesize
fully acetylated PNAG oligosaccharides have employed
convergent approaches,25,28-32 acid reversion chemistry,33
one-
(DspB), specifically, is
a
native Aggregatibacter
actinomycetemcomitans -hexosaminidase enzyme specific
for the hydrolysis of dPNAG polysaccharides,17 and has been
used to catalyze biofilm dispersal in vitro for a number of
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Figure 1. Mechanism of dPNAG hydrolysis catalyzed by DspB. (A) dPNAG hydrolysis is proposed to involve substrate assisted
cleavage characterized by formation of an oxazolinium ion intermediate. (B) Structure of dPNAG trisaccharide analogs with defined
acetylation patterns synthesized to probe DspB specificity.
a general one-pot approach to oligosaccharide synthesis
that is independent of glycosyl donor reactivities.40 This
Scheme 1. Synthesis of Monosaccharide Building Blocks
Figure 2. Retrosynthetic analysis of trisaccharide 2. The
selectively protected trisaccharide 5 can be prepared using a
combination of glycosyl donor 7 with glycosyl acceptors 8 and
9
with orthogonal nitrogen protection. The remaining
glycosylation approach has been successfully employed to
prepare complex oligo- and polysaccharides.41,42 Here we
present the synthesis of PNAG trisaccharides (1–5) with
defined acetylation patterns using a pre-activation based
one-pot strategy. The product trisaccharides were tested as
substrates for DspB hydrolysis by evaluating both the rate
of hydrolysis and product distribution. The results support
a mechanism in which the dPNAG substrate acetylation
patterns influence their recognition and mechanism of
hydrolysis by DspB.
trisaccharide analogs are prepared in an analogous manner
using the same building blocks.
pot glycosylation reactions,34 automated synthesis,35-37 and
non-specific polymerization reactions.38 However, these
methods produced either uniformly acetylated PNAG
derivatives or dPNAG with random de-N-acetylation, with
only one example of dPNAG synthesis with defined de-N-
acetylation reported,39 and there the GlcN content was far
higher than in dPNAG samples isolated from bacterial
species. Thus, there is a need for efficient methods to
prepare deacetylated PNAG oligosaccharides as functional
probes for dPNAG–protein interactions.
RESULTS AND DISCUSSION
Retrosynthesis. The retrosynthesis of trisaccharide 2,
illustrated in Figure 2, demonstrates our general approach
to assemble PNAG analogs with defined acetylation
Compared to other methods, the pre-activation based
glycosylation reactions developed by Huang et al. provides
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patterns. The desired trisaccharide analogs 1–5 are
selective deprotection and N-acetylation sequence outlined
in scheme 2. Oxidative cleavage of the N-Troc protecting
groups using pre-activated zinc dust under acidic
conditions and in situ N-acetylation by acetic anhydride
followed by global deprotection using aqueous hydrazine
gave the product trisaccharides 2–5 in 58-62% yield over 2
steps, following recrystallization. Each trisaccharide analog
was obtained in >95% purity as estimated from NMR and
HPLC analysis and showed no evidence of N-Ac group
migration during the reaction and purification. This
approach provided rapid access to a panel of five PNAG
trisaccharide analogs with defined acetylation patterns
using the same glycosyl donors and acceptor molecules in
sufficient quantities to facilitate their evaluation as
substrates for DspB.
1
2
3
4
5
6
7
8
accessible using the same glucosamine derived glycosyl
donors, and glycosyl acceptors 8 and 9 with the primary
amine orthogonally protected as N-phthalamide or N-Troc.
Both serve as participating protecting groups allowing for
high -selectivity during glycosylation reactions. The N-
Troc of 6 can be selectively removed under reductive
conditions and acetylated in situ in the presence of N-Phth
protection resulting in trisaccharides with defined
acetylation patterns. An orthogonal protecting group (i.e.
fluorenylmethyloxycarbonyl (Fmoc)), was incorporated
onto the 6-OH of donor 7 to provide the flexibility to further
apply the trisaccharide products (i.e. 6) as glycosyl donors
or alternatively deprotect the Fmoc to obtain glycosyl
acceptors for preparing longer PNAG derivatives.
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Table 1. Pre-activation based iterative one-pot
glycosylation.
Building block synthesis. Based on this retrosynthetic
approach, two monosaccharide glycosyl acceptors 8 and 9
containing a free 6-OH group and two fully protected
glycosyl donors 7 and 14 (Scheme 1) were synthesized. The
O-acetyl groups of thioglycoside 10, prepared as previously
described,43 were first removed followed by a selective
protection of the primary hydroxyl as the tert-
butyldimethylsilyl ether and in situ O-acetylation to afford
the thioglycoside 12 in 72% yield over three steps. Silyl
ether hydrolysis for 12 under acidic conditions yielded
glycosyl acceptor 944 in a 93% yield. Donor 12 was initially
explored as a glycosyl donor, however, it’s activation with
p-TolSCl and AgOTf resulted in its quantitative conversion
to the 1,6-anhydrosugar (data not shown). Instead, Fmoc
protection of the primary alcohol of 9 afforded glycosyl
donor 14 in 70 % yield. Starting from thioglycoside 11,43 the
N-Troc protected thioglycoside donor 845 and glycosyl
acceptor 7 were synthesized in an analogous manner in
63% and 43% overall yields, respectively.
acceptor
donor
14
Product (% yield)
1
2
9
9
7
7
8
9
8
9
9
8
8
8
Iterative one-pot glycosylation. The one-pot assembly of
fully protected trisaccharide analogs was performed as
outlined in Table 1, using a pre-activation based one-pot
sequential glycosylation approach.40 Briefly, stoichiometric
pre-activation of donors 7, 10 or 14 with freshly distilled p-
TolSCl in the presence of AgOTf (3 eq) at –78℃ was followed
by addition of the first acceptor (8 or 9) and the
temperature raised to 0℃ over 20 min. After complete
consumption of the acceptor, as judged by TLC analysis, the
reaction mixture was returned to –78℃ followed by
sequential addition of AgOTf, p-TolSCl, and a second
acceptor (8 or 9). Warming to 0℃ over 20 min gave
protected trisaccharides 6 and 15–18 in isolated yields of
49–58% after flash column chromatography. The remaining
material largely consisted of hydrolyzed donors. It is
noteworthy that the reaction efficiency was not significantly
affected by differences in the anomeric reactivity for each
glycosyl donor. Thus, all orthogonally protected
trisaccharide analogs could be obtained via the same
approach by simply varying the order of substrate addition
with little impact on product yield.
14
10
Scheme 2. Selective Deprotection and N-Acetylation
Selective deprotection. Per-N-acetylated trisaccharide 1
was obtained by treating 15 with ethylenediamine in
refluxing ethanol followed by per-acetylation resulted in
the isolation of trisaccharide 19 in 90% yield (scheme 2).
This was followed by Zemplén deacetylation to give 1 in
95% yield. For access to analogs 2–5, we developed a
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Figure 3. Time course for the hydrolysis of PNAG trisaccharide analogs 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E). Lines were added to aid
identification of the disappearance of the trisaccharide (black), appearance of the disaccharide (red) and appearance of the
monosaccharide (blue). The concentration of remaining trisaccharide substrate and reducing-end disaccharide and monosaccharide
products were determined from the relative peak areas as determined by HPLC using the S-tolyl aglycone absorbance at 254nm.
Table 2. Rate of trisaccharide hydrolysis catalyzed by
DspB
nitrophenyl-GlcNAc, which contains a better leaving group
as the aglycone, is a poor substrate for DspB.23
Analysis of time points within the first 60 min for the
hydrolysis of fully acetylated trisaccharide 1 by DspB
showed a decrease in trisaccharide and the predominant
formation of a disaccharide product (Figure 3A). This is
followed by the appearance of monosaccharide and a
decrease in the rate of disaccharide formation over the
course of the reaction. The product distribution is
consistent with primarily exo cleavage in which the non-
reducing GlcNAc residues are cleaved in succession (Figure
S3A). This is also consistent with product distributions that
have been previously reported for the hydrolysis of fully
acetylated PNAG oligosaccharide substrates by DspB.25,26
Analyzing the product distribution by MALDI-TOF MS
showed only the reducing-end disaccharide product. We
observed no evidence for endoglycosidic cleavage of
trisaccharide 1, which would lead directly to the formation
of a nonreducing-end disaccharide product.
Trisaccharide
kobs (min-1)a
3.8  0.1  10–3
11.2  0.2  10–3
4.0  0.2  10–3
4.6  0.2  10–3
--b
1
2
3
4
5
a
b
Values are shown  the 95% confidence interval. No
hydrolysis products were observed over 6 h.
Product distributions from enzymatic hydrolysis by
DspB. Trisaccharides 1–5 were each independently
evaluated as substrates for hydrolysis by recombinant A.
actinomycetemcomitan DspB. Substrate hydrolysis as a
function of time was monitored through separation of the
substrate and reducing-end products by reversed phase
HPLC and monitoring the absorbance at 254 nm stemming
from the S-tolyl aglycone. Thus, the concentration of
substrate and reducing-end hydrolysis products were
quantified based on their relative peak areas in HPLC
chromatograms (Figure 3, Figure S1). For each reaction the
product distribution was also analyzed directly by MALDI-
TOF mass spectrometry to confirm product identities and
identify any non-reducing end hydrolysis products lacking
the S-tolyl aglycone (Figure S2). No evidence for aglycone
cleavage from any substrate was observed by either MS or
HPLC analysis. This is not surprising as thioglycosides are
known to be resistant to glycosidase cleavage and 4-
Next, we sought to analyze how deacetylation of the PNAG
trisaccharide substrate might impact the observed product
distributions. The hydrolysis of trisaccharide 2 resulted in
the same product distribution observed for the hydrolysis
of 1 (Figure 3B). This is again consistent with two
successive cleavage of the non-reducing GlcNAc residue
resulting in a GlcN-S-Tol monosaccharide product. With
trisaccharide 3, however, only disaccharide product
formation was observed with no further hydrolysis to the
monosaccharide (Figure 3C). This is in agreement with the
proposed substrate assisted hydrolysis mechanism for
DspB, where an N-Ac is required at the site of hydrolysis.20,23
Similarly, incubations with trisaccharide 5 containing two
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nonreducing GlcN produced no hydrolysis products over
DspB with trisaccharide 2 resulted in an observed rate of
hydrolysis ~2.5-fold greater than observed for any other
trisaccharide analog. This demonstrates a preferential
turnover of substrates with GlcN located in the +2 binding
site of DspB. To verify this observed substrate preference,
internal competition experiments in which both
trisaccharide 1 and 2 were co-incubated with DspB were
performed (Figure S5). Here, the same substrate preference
was observed with the rate of trisaccharide 2 consumption
being ~ 3-fold faster than trisaccharide 1 co-incubated in
the same hydrolysis reaction. Taking this data together it is
clear that DspB shows a preference for GlcN in the +2-
position relative to the site of cleavage, but that this is not a
strict requirement for substrate hydrolysis. It is possible
that the preference for GlcN in the +2 binding site allows for
more efficient recognition of dPNAG polysaccharides.
1
2
3
4
5
6
7
8
the entire 6-hour time period evaluated (Figure 3E). This
provides further support for a substrate assisted exo-
glycosidic cleavage mechanism, which requires GlcNAc at
the site of cleavage common among other GH20 enzymes.46
Given the strict requirement for an N-Ac at the site of
cleavage, we did not anticipate much if any hydrolysis of
trisaccharide 4 containing a non-reducing GlcN. However,
the hydrolysis reactions resulted in a single product
corresponding to a reducing-end monosaccharide (Figure
3D). The structure of this product was confirmed by co-
injection with authentic monosaccharide standard (data not
shown). The MALDI-TOF MS results also showed direct
formation of a non-reducing end -D-GlcN-(16)--D-
GlcNAc disaccharide (Figure S2).
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These results afford a reasonable hypothesis for how DspB
efficiently catalyzes the hydrolysis of native dPNAG biofilm
polysaccharides. The enzyme functions predominantly as
an exoglycosidase cleaving the non-reducing terminal
GlcNAc from dPNAG polysaccharides. When a non-reducing
GlcN residue is encountered, DspB is able to bypass and
facilitate endo-glycosidic cleavage at the next available
GlcNAc (Figure S3A). It is not clear if this activity is limited
to the cleavage of a single non-reducing terminal -D-GlcN-
(16)--D-GlcNAc disaccharide, or if DspB is capable of
accommodating longer oligosaccharides down from the site
of cleavage. Further studies with longer dPNAG analogs are
still required to differentiate these possibilities.
DspB and PgaB utilize different mechanism for dPNAG
hydrolysis. Hydrolysis of dPNAG by PgaB proceeds
exclusively via an endo-glycosidic cleavage mechanism and
requires GlcN, and GlcNAc in the –3 and –1 binding sites,
respectively. 22 In this case de-N-acetylation is essential for
PgaB hydrolytic activity. By comparison, the results from
hydrolysis of 1–5 by DspB support a mechanism where
DspB is capable of hydrolyzing dPNAG through either exo-
or endo-glycosyl hydrolysis mechanisms depending on the
substrate acetylation pattern.
The different mechanisms employed by PgaB and DspB may
relate to their different biological roles in the biofilm
lifecycle. PgaB is localized in the periplasm as part of the
biosynthetic machinery required for dPNAG assembly and
export.10 It contains both the C-terminal GH153 domain and
Differential rate of trisaccharide hydrolysis by DspB.
We attempted to measure steady state kinetic parameters
to gain more mechanistic insight into the DspB catalyzed
hydrolysis of trisaccharide analogs 1 and 2. For all
concentrations tested (0.1–5 mM), we observed a linear
increase in the initial velocity with concentration
suggesting that even 5 mM trisaccharide is well below KM
(Figure S4A). This was unexpected as related GH20
enzymes have KM values in the 0.1–1 mM range.47,48 Instead,
the DspB catalytic efficiencies for 1–5 were determined
from the observed rate constants (kobs) for disappearance of
trisaccharide by fitting reaction progress curves to a single
exponential (eq 1). Here [S] is the trisaccharide substrate
concentration, [So] is the initial trisaccharide concentration,
and t is time. Under conditions where [So]KM a single
exponential fit to the reaction progress curve (kobs) is
proportional to the enzyme specificity constant V/K, but
a
N-terminal carbohydrate esterase domain, which
12,22,50
catalyzes the de-N-acetylation of PNAG.
It is still not
clear what biological role this hydrolytic activity plays, if
any, during dPNAG biosynthesis. DspB, on the other hand, is
a
secreted
protein
that
functions
in
A.
actinomycetemcomitans biofilm dispersal. A transposon
insertion mutant of A. actinomycetemcomitans that disrupts
DspB expression displays a phenotype deficient in biofilm
cell detachment.17 The ability of DspB to effectively catalyze
both exo- and endo-glycosidic cleavage reactions would
allow for more efficient dPNAG hydrolysis during biofilm
dispersal.
DspB has
a charged substrate binding surface.
Additional insight into the origin of this observed specificity
was gained by comparing the binding site of DspB to other
GH20 family glycosyl hydrolase enzymes. An analysis of the
DspB structure using the DALI server identified the tandem
GH20 domains of StrH as the closest structural
homologues.51 The GH20 domains of StrH catalyze the
hydrolysis of terminal GlcNAc residues of N-glycan and
functions as a major virulence factor in Pneumococcal
species.47 The overlay in Figure 4A shows the apo structure
of DspB compared to that of StrH bound to a N-glycan
tetrasaccharide substrate.20,47 StrH and DspB share 23%
overall sequence identity and all of the active site residues
involved in recognition of the non-reducing GlcNAc in the –
1 binding site are conserved with the exception of Asp56
and Trp216, which correspond to Asn670 and Phe849 in
StrH. Therefore, it is likely that the GlcNAc residue in the –1
binding site is bound in a similar conformation in both
does not allow for unambiguous determination of KM and
49
Vmax
.
Thus, kobs represents a pseudo-first-order rate
constant for hydrolysis of the trisaccharide that can be used
to directly compare DspB catalytic efficiency with different
substrates.
[S] = [S ∘ ] × 푒( ― 푘
표푏푠푡)
(1)
Surprisingly, the hydrolysis of trisaccharide 1, 3 and 4
showed little variation in the observed rates (Table 2,
Figure S4B) despite the differences in product distribution
that were obtained in HPLC and MS measurements. These
rates are of similar magnitude to those reported previously
for DspB hydrolysis of fully acetylated PNAG oligomers
ranging in size from disaccharides to pentasaccharides.26
Thus, it appears as though the rate of hydrolysis is not
significantly impacted by de-acetylation of the substrate in
in either the +1 or –2 binding sites (Figure S3B). Reaction of
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proteins. Compared to StrH, the predicted binding surface
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product to be converted into both glycosyl donors or
acceptors for further glycosylation reactions. All
trisaccharide products synthesized served as effective
probes for DspB hydrolytic activity. From the results of
these studies it is clear that DspB activity and product
distributions are dependent on substrate acetylation
patterns. Specifically, de-N-acetylation in the +2 position of
dPNAG trisaccharide resulted in an increase in substrate
hydrolysis by DspB. The enhanced rate of hydrolysis was
not observed for deacetylation at other positions, indicating
that dPNAG with specific acetylation patterns are
preferentially recognized. The hydrolysis product
distributions observed are consistent with primarily exo-β-
D-N-acetylglucosaminidase activity using substrate assisted
hydrolysis for substrates containing non-reducing GlcNAc.
However, DspB can also catalyze endo-glycosidic cleavage
at nearly identical rate for substrates containing GlcN at the
–2 binding site. These results imply that DspB hydrolysis of
dPNAG polysaccharides can proceed via either exo- or
endoglycosidase mechanisms depending on the specific
substrate acetylation pattern, but further studies are
required to characterize the substrate binding interactions
in greater detail. As DspB is one of only a few enzymes
known to degrade dPNAG, understanding its activity and
specificity is crucial for our understanding of biofilm
dispersal.
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2
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of DspB is more open and displays a net negative charge
(Figure 4B–C). This anionic surface of DspB may function in
recognition of positively charged GlcN
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Figure 4. Structural comparison of DspB with other GH20
enzymes. (A) Overlay of DspB apo structure (PDB 1YHT, green)
with StrH (PDB 2YL9, grey). The GlcNAc in the –1 site of StrH is
shown (cyan). Select active site residues of DspB are
highlighted. Electrostatic surface maps for StrH (B) and DspB
(C) showing positive surface charge in blue and negative
surface charge in red. The GlcNAc at the –1 binding site of StrH
is shown and is modeled in the active site of DspB (cyan).
MATERIALS AND METHODS
DspB-catalyzed hydrolysis of PNAG trisaccharide
analogs.
Hydrolysis
reactions
containing
final
concentrations of 15 M His6-DspB and 1 mM trisaccharide
(1–5) in a buffer of 50 mM potassium phosphate, pH 6.0,
with 100 mM NaCl in a 50 L final volume. The reaction was
initiated through the addition of His6-DspB, and incubated
at 22C. Aliquots of 5 L were removed after 0 min, 10 min,
30 min, 60 min, 90 min, 150 min, 240 min and 360 min
incubation and quenched through addition of 5 L of 100
mM trifluoroacetic acid (TFA). Quenched fractions were
centrifuged at 17,000  g for 2 min to pellet any insoluble
material and 5 L of supernatant was diluted to a final
volume of 50 L with MQ water for HPLC analysis. Samples
were separated on a Kinetex C18 column (150  4.6 mm, 5
m) at 1 mL/min flow rate using a linear gradient elution
from 0.1% formic acid (buffer A) to 50% acetonitrile (buffer
B) over 12 min. The column was washed with buffer B for 4
min and then re-equilibrated with buffer A for 4 min before
subsequent injections. Analytes were observed using the
absorbance at 254 nm resulting from the S-tolyl aglycone.
Concentrations of residual substrate and reducing-end
hydrolysis products were determined from their relative
peak areas, which were plotted as a function of time to
obtain the reaction time course. Analyte concentrations
from two independent experiments were averaged to
obtain reaction time course data.
present in native dPNAG and explain the preferential
recognition of substrates with GlcN in the +2 binding site. A
similar charged surface is also present in the binding site of
PgaB indicating that this may be a general feature of dPNAG
specific GH enzymes.22 It is not clear from this study if the
preference for recognition of GlcN over GlcNAc limited to
the +2 subsite or if there are additional binding sites for
recognition of GlcN. Thus, further work is required to
specifically characterize the conformation of bound dPNAG
in the active site of DspB and identify specific interactions
of the +2 and –2 binding sites. Understanding how
glycosidase enzymes like DspB interact with their
exopolysaccharide substrates is not only critical for their
development as biocatalysts but will provide critical insight
into the process of EPS breakdown during the natural
process of biofilm dispersal and may lead to the
identification of novel dPNAG specific -hexosaminidase
enzymes.
CONCLUSIONS
A
robust one-pot glycosylation approach has been
Initial velocity measurements were carried out using the
same activity assay but with varying trisaccharide
concentrations between 0.1–5 mM. Reaction aliquots were
removed and quenched at 5, 10, 20 and 30 min time points
and initial velocities were determined from the linear
portion of the reaction progress curve.
developed to assemble dPNAG trisaccharides with specific
acetylation patterns from easily accessible building blocks.
Trisacharides with defined acetylation patterns can be
obtained in good yield by simply varying the order of
reagent addition during the one-pot glycosylation. Inclusion
of a 6-O-Fmoc protecting group allows the trisaccharide
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encodes the production of poly--1-6-N-acetylglucosamine, which
The direct competitive hydrolysis of 1 and 2 was measured
using a reaction mixture of 15 M His6-DspB containing
both 1 mM trisaccharide 1 and 1 mM trisaccharide 2 in a
final volume of 50L of 50 mM potassium phosphate, pH
6.0, 100 mM NaCl. The reactions were analyzed as described
above.
is critical for biofilm formation. J. Bacteriol. 191, 5953–5963.
(6) Izano, E. A., Sadovskaya, I., Vinogradov, E., Mulks, M. H.,
Velliyagounder, K., Ragunath, C., Kher, W. B., Ramasubbu, N.,
Jabbouri, S., Perry, M. B., and Kaplan, J. B. (2007) Poly-N-
acetylglucosamine mediates biofilm formation and antibiotic
resistance in Actinobacillus pleuropneumoniae. Microb. Pathog. 43,
1–9.
(7) Chen, K.-M., Chiang, M.-K., Wang, M., Ho, H.-C., Lu, M.-C., and
Lai, Y.-C. (2014) The role of pgaC in Klebsiella pneumoniae
virulence and biofilm formation. Microb. Pathog. 77, 89–99.
(8) Jarrett, C. O., Deak, E., Isherwood, K. E., Oyston, P. C., Fischer,
E. R., Whitney, A. R., Kobayashi, S. D., DeLeo, F. R., and Hinnebusch,
B. J. (2004) Transmission of Yersinia pestis from an infectious
biofilm in the flea vector. J. Infect. Dis. 190, 783–792.
1
2
3
4
5
6
7
8
Product analysis by MALDI-TOF mass spectrometry.
DspB reaction mixtures were prepared as described above.
Quenched reaction aliquots for MALDI-TOF MS analysis
were directly spotted as a 1:1 mixture with DHB matrix.
Spectra were recorded in positive reflectron mode using a
Bruker Autoflex Speed mass spectrometer. Laser power
was adjusted to be above threshold to obtain good
resolution and signal/background ratios. Spectra were
gathered for 3000 shots total to ensure the normalization of
peak ratios during data collection.
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(9) Vuong, C., Kocianova, S., Voyich, J. M., Yao, Y., Fischer, E. R.,
DeLeo, F. R., and Otto, M. (2004)
A crucial role for
exopolysaccharide modification in bacterial biofilm formation,
immune evasion, and virulence. J. Biol. Chem. 279, 54881–54886.
(10) Itoh, Y., Rice, J. D., Goller, C., Pannuri, A., Taylor, J., Meisner,
J., Beveridge, T. J., Preston, J. F., and Romeo, T. (2008) Roles of
pgaABCD genes in synthesis, modification, and export of the
Escherichia coli biofilm adhesin poly--1,6-N-acetyl-D-
glucosamine. J. Bacteriol. 190, 3670–3680.
(11) Little, D. J., Milek, S., Bamford, N. C., Ganguly, T.,
DiFrancesco, B. R., Nitz, M., and Howell, P. L. (2015) The protein
BpsB is a poly--1,6-N-acetyl-D-glucosamine deacetylase required
for biofilm formation in Bordetella bronchiseptica. J. Biol. Chem.
290, 22827–22840.
(12) Little, D. J., Bamford, N. C., Pokrovskaya, V., Robinson, H.,
Nitz, M., and Howell, P. L. (2014) Structural basis for the de-N-
acetylation of poly--1,6-N-acetyl-D-glucosamine in gram-positive
bacteria. J. Biol. Chem. 289, 35907–35917.
(13) Baker, P., Hill, P. J., Snarr, B. D., Alnabelseya, N., Pestrak, M.
J., Lee, M. J., Jennings, L. K., Tam, J., Melnyk, R. A., Parsek, M. R.,
Sheppard, D. C., Wozniak, D. J., and Howell, P. L. (2016)
Exopolysaccharide biosynthetic glycoside hydrolases can be
utilized to disrupt and prevent Pseudomonas aeruginosa biofilms.
Sci. Adv. 2, e1501632.
(14) Kaplan, J. B., Mlynek, K. D., Hettiarachchi, H., Alamneh, Y. A.,
Biggemann, L., Zurawski, D. V., Black, C. C., Bane, C. E., Kim, R. K.,
and Granick, M. S. (2018) Extracellular polymeric substance (EPS)-
degrading enzymes reduce staphylococcal surface attachment and
biocide resistance on pig skin in vivo. PLoS ONE 13, e0205526.
(15) Fey, P. D. (2010) Modality of bacterial growth presents
unique targets: how do we treat biofilm-mediated infections? Curr.
Opin. Microbiol. 13, 610–615.
(16) Asker, D., Awad, T. S., Baker, P., Howell, P. L., and Hatton, B.
D. (2018) Non-eluting, surface-bound enzymes disrupt surface
attachment of bacteria by continuous biofilm polysaccharide
degradation. Biomaterials 167, 168–176.
(17) Kaplan, J. B., Ragunath, C., Ramasubbu, N., and Fine, D. H.
(2003) Detachment of Actinobacillus actinomycetemcomitans
biofilm cells by an endogenous -hexosaminidase activity. J.
Bacteriol. 185, 4693–4698.
(18) Kaplan, J. B., Ragunath, C., Velliyagounder, K., Fine, D. H., and
Ramasubbu, N. (2004) Enzymatic detachment of Staphylococcus
epidermidis biofilms. Antimicrob. Agents Chemother. 48, 2633–
2636.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Methods for synthesis of trisaccharides 1–5, experimental
protocols for protein production, supplemental figures, and
spectral data for all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
* mpoulin@umd.edu
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We are grateful to the University of Maryland College Park for
startup funds. We thank F. Chen and Y. Li for help with
analytical NMR and mass spectrometry services. We thank D.
Fushman, and J. Davis for helpful discussion.
REFERENCES
(1) Joo, H.-S., and Otto, M. (2012) Molecular basis of in vivo
biofilm formation by bacterial pathogens. Chem. Biol. 19, 1503–
1513.
(2) Mack, D., Fischer, W., Krokotsch, A., Leopold, K., Hartmann,
R., Egge, H., and Laufs, R. (1995) The intercellular adhesin involved
in biofilm accumulation of Staphylococcus epidermidis is a linear -
1,6-linked glucosaminoglycan: purification and structural analysis.
J. Bacteriol. 178, 175–183.
(3) Cramton, S. E., Gerke, C., Schnell, N. F., Nichols, W. W., and
Götz, F. (1999) The intercellular adhesion (ica) locus is present in
Staphylococcus aureus and is required for biofilm formation. Infect.
Immun. 67, 5427–5433.
(4) Wang, X., Preston, J. F., and Romeo, T. (2004) The pgaABCD
locus of Escherichia coli promotes the synthesis of a polysaccharide
adhesin required for biofilm formation. J. Bacteriol. 186, 2724–
2734.
(19) Itoh, Y., Wang, X., Hinnebusch, B. J., Preston, J. F., and Romeo,
T. (2005) Depolymerization of -1,6-N-acetyl-D-glucosamine
disrupts the integrity of diverse bacterial biofilms. J. Bacteriol. 187,
382–387.
(20) Ramasubbu, N., Thomas, L. M., Ragunath, C., and Kaplan, J.
B. (2005) Structural analysis of Dispersin B, a biofilm-releasing
glycoside hydrolase from the periodontopathogen Actinobacillus
actinomycetemcomitans. J. Mol. Biol. 349, 475–486.
(5) Choi, A. H. K., Slamti, L., Avci, F. Y., Pier, G. B., and Maira-
Litran, T. (2009) The pgaABCD locus of Acinetobacter baumannii
(21) Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P.
ACS Paragon Plus Environment

ACS Chemical Biology
Page 8 of 9
M., and Henrissat, B. (2013) The carbohydrate-active enzymes
(37) Nokami, T., Hayashi, R., Saigusa, Y., Shimizu, A., Liu, C.-Y.,
Mong, K.-K. T., and Yoshida, J.-I. (2013) Automated solution-phase
synthesis of oligosaccharides via iterative electrochemical
assembly of thioglycosides. Org. Lett. 15, 4520–4523.
(38) Kanno, K. I., and Hatanaka, K. (2005) Synthesis of 1,6-
Anhydro-4-O-benzyl-2-deoxy-3-O-p-methoxybenzoyl-2-
phalimido--D-glucopyranose and its oligomers. Polym. J. 30, 678–
680.
database (CAZy) in 2013. Nucl. Acids Res. 42, D490–D495.
(22) Little, D. J., Pfoh, R., Le Mauff, F., Bamford, N. C., Notte, C.,
Baker, P., Guragain, M., Robinson, H., Pier, G. B., Nitz, M., Deora, R.,
Sheppard, D. C., and Howell, P. L. (2018) PgaB orthologues contain
a glycoside hydrolase domain that cleaves deacetylated poly-
β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.
PLoS Pathog. 14, e1006998.
1
2
3
4
5
6
7
8
9
(23) Manuel, S. G. A., Ragunath, C., Sait, H. B. R., Izano, E. A.,
Kaplan, J. B., and Ramasubbu, N. (2007) Role of active-site residues
of Dispersin B, a biofilm-releasing β-hexosaminidase from a
periodontal pathogen, in substrate hydrolysis. FEBS J. 274, 5987–
5999.
(24) Greig, I. R., Zahariev, F., and Withers, S. G. (2008)
Elucidating the nature of the Streptomyces plicatus β-
hexosaminidase-bound intermediate ssing ab initio molecular
dynamics simulations. J. Am. Chem. Soc. 130, 17620–17628.
(25) Fekete, A., Borbás, A., Gyémánt, G., Kandra, L., Fazekas, E.,
Ramasubbu, N., and Antus, S. (2011) Synthesis of -(1,6)-linked N-
acetyl-D-glucosamine oligosaccharide substrates and their
hydrolysis by Dispersin B. Carbohydr. Res. 346, 1445–1453.
(26) Fazekas, E., Kandra, L., and Gyémánt, G. (2012) Model for -
1,6-N-acetylglucosamine oligomer hydrolysis catalysed by
Dispersin B, a biofilm degrading enzyme. Carbohydr. Res. 363, 7–
13.
(39) Yudina, O. N., Gening, M. L., Tsvetkov, Y. E., Grachev, A. A.,
Pier, G. B., and Nifantiev, N. E. (2011) Synthesis of five nona--(1,6)-
D-glucosamines with various patterns of N-acetylation
corresponding to the fragments of exopolysaccharide of
Staphylococcus aureus. Carbohydr. Res. 346, 905–913.
(40) Huang, X., Huang, L., Wang, H., and Ye, X.-S. (2004) Iterative
one-pot synthesis of oligosaccharides. Angew. Chem. Int. Ed. 43,
5221–5224.
(41) Dulaney, S. B., Xu, Y., Wang, P., Tiruchinapally, G., Wang, Z.,
Kathawa, J., El-Dakdouki, M. H., Yang, B., Liu, J., and Huang, X.
(2015) Divergent synthesis of heparan sulfate oligosaccharides. J.
Org. Chem. 80, 12265–12279.
(42) Wu, Y., Xiong, D.-C., Chen, S.-C., Wang, Y.-S., and Ye, X.-S.
(2017) Total synthesis of mycobacterial arabinogalactan
containing 92 monosaccharide units. Nat. Commun. 8, 1–7.
(43) Grann Hansen, S., and Skrydstrup, T. (2007) Studies
directed to the synthesis of oligochitosans – preparation of
building blocks and their evaluation in glycosylation studies. Eur. J.
Org. Chem. 2007, 3392–3401.
(44) Ren, B., Cai, L., Zhang, L.-R., Yang, Z.-J., and Zhang, L.-H.
(2005) Selective deacetylation using iodine–methanol reagent in
fully acetylated nucleosides. Tetrahedron Lett. 46, 8083–8086.
(45) Lu, S.-R., Lai, Y.-H., Chen, J.-H., Liu, C.-Y., and Mong, K.-K. T.
(2011) Dimethylformamide: An unusual glycosylation modulator.
Angew. Chem. Int. Ed. 50, 7315–7320.
(46) Mark, B. L., Vocadlo, D. J., Knapp, S., Triggs-Raine, B. L.,
Withers, S. G., and James, M. N. G. (2001) Crystallographic evidence
for substrate-assisted catalysis in a bacterial β-hexosaminidase. J.
Biol. Chem. 276, 10330–10337.
(47) Jiang, Y.-L., Yu, W.-L., Zhang, J.-W., Frolet, C., Di Guilmi, A.-
M., Zhou, C.-Z., Vernet, T., and Chen, Y. (2011) Structural basis for
the substrate specificity of a novel β-N-acetylhexosaminidase StrH
protein from Streptococcus pneumoniae R6. J. Biol. Chem. 286,
43004–43012.
(48) Ito, T., Katayama, T., Hattie, M., Sakurama, H., Wada, J.,
Suzuki, R., Ashida, H., Wakagi, T., Yamamoto, K., Stubbs, K. A., and
Fushinobu, S. (2013) Crystal structures of a glycoside hydrolase
family 20 lacto-N-biosidase from Bifidobacterium bifidum. J. Biol.
Chem. 288, 11795–11806.
(49) Stroberg, W., and Schnell, S. (2016) On the estimation
errors of KM and V from time-course experiments using the
Michaelis–Menten equation. Biophys. Chem. 219, 17–27.
(50) Little, D. J., Poloczek, J., Whitney, J. C., Robinson, H., Nitz, M.,
and Howell, P. L. (2012) The structure- and metal-dependent
activity of Escherichia coli PgaB provides insight into the partial de-
N-acetylation of poly--1,6-N-acetyl-D-glucosamine. J. Biol. Chem.
287, 31126–31137.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(27) Whitfield, G. B., Marmont, L. S., and Howell, P. L. (2015)
Enzymatic modifications of exopolysaccharides enhance bacterial
persistence. Front. Microbiol. 6, 7350–21.
(28) Yang, F., and Du, Y. (2003) A practical synthesis of a (1→6)-
linked β-D-glucosamine nonasaccharide. Carbohydr. Res. 338, 495–
502.
(29) Chen, Q., Yang, F., and Du, Y. (2005) Synthesis of a C3-
symmetric (1→6)-N-acetyl-β-D-glucosamine octadecasaccharide
using click chemistry. Carbohydr. Res. 340, 2476–2482.
(30) Gening, M. L., Tsvetkov, Y. E., Pier, G. B., and Nifantiev, N. E.
(2007) Synthesis of β-(1→6)-linked glucosamine oligosaccharides
corresponding to fragments of the bacterial surface polysaccharide
poly-N-acetylglucosamine. Carbohydr. Res. 342, 567–575.
(31) Gening, M. L., Tsvetkov, Y. E., Titov, D. V., Gerbst, A. G.,
Yudina, O. N., Grachev, A. A., Shashkov, A. S., Vidal, S., Imberty, A.,
Saha, T., Kand, D., Talukdar, P., Pier, G. B., and Nifantiev, N. E. (2013)
Linear and cyclic oligo--(1,6)-D-glucosamines: Synthesis,
conformations, and applications for design of a vaccine and
oligodentate glycoconjugates. Pure Appl. Chem. 85, 175–14.
(32) Weaver, L. G., Singh, Y., Blanchfield, J. T., and Burn, P. L.
(2013) A simple iterative method for the synthesis of -(1,6)-
glucosamine oligosaccharides. Carbohydr. Res. 371, 68–76.
(33) Leung, C., Chibba, A., Gómez-Biagi, R. F., and Nitz, M. (2009)
Efficient synthesis and protein conjugation of β-(1→6)-D-N-
acetylglucosamine oligosaccharides from the polysaccharide
intercellular adhesin. Carbohydr. Res. 344, 570–575.
(34) Fridman, M., Solomon, D., Yogev, S., and Baasov, T. (2002)
One-pot synthesis of glucosamine oligosaccharides. Org. Lett. 4,
281–283.
(35) Melean, L. G., Love, K. R., and Seeberger, P. H. (2002)
(51) Holm, L., and Laakso, L. M. (2016) Dali server update. Nucl.
Acids Res. 44, W351–W355.
Toward
the
automated
solid-phase
synthesis
of
oligoglucosamines: systematic evaluation of glycosyl phosphate
and glycosyl trichloroacetimidate building blocks. Carbohydr. Res.
337, 1893–1916.
(36) Ko, K.-S., Park, G., Yu, Y., and Pohl, N. L. (2008) Protecting-
group-based colorimetric monitoring of fluorous-phase and solid-
phase synthesis of oligoglucosamines. Org. Lett. 10, 5381–5384.
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