Chemoenzymatic Synthesis of Chito-oligosaccharides with Alternating N- d -Acetylglucosamine and d -Glucosamine

Article abstract of DOI:10.1021/acs.biochem.0c00839

Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of N-acetylglucosamine (GlcNAc, A) and d-glucosamine (GlcN, D). Production of well-defined CHOS-mixtures, or even pure CHOS, with specific lengths and sugar compositions, is of great interest since these oligosaccharides have interesting bioactivities. While direct chemical synthesis of CHOS is not straightforward, chemo-enzymatic approaches have shown some promise. We have used engineered glycoside hydrolases to catalyze oligomerization of activated DA building blocks through transglycosylation reactions. The building blocks were generated from readily available (GlcNAc)2-para-nitrophenol through deacetylation of the nonreducing end sugar with a recombinantly expressed deacetylase from Aspergillus niger (AnCDA9). This approach, using a previously described hyper-transglycosylating variant of ChiA from Serratia marcescens (SmChiA) and a newly generated transglycosylating variant of Chitinase D from Serratia proteamaculans (SpChiD), led to production of CHOS containing up to ten alternating D and A units [(DA)2, (DA)3, (DA)4, and (DA)5]. The most abundant compounds were purified and characterized. Finally, we demonstrate that (DA)3 generated in this study may serve as a specific inhibitor of the human chitotriosidase. Inhibition of this enzyme has been suggested as a therapeutic strategy against systemic sclerosis.

Full text of DOI:10.1021/acs.biochem.0c00839

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Article
Chemoenzymatic Synthesis of Chito-oligosaccharides with
Alternating N‑D‑Acetylglucosamine and D‑Glucosamine
Rianne A. G. Harmsen, Berit Bjugan Aam, Jogi Madhuprakash, Anne Grethe Hamre,
Ethan D. Goddard-Borger, Stephen G. Withers, Vincent G. H. Eijsink, and Morten Sørlie*
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ABSTRACT: Chito-oligosaccharides (CHOS) are homo- or hetero-
oligomers of N-acetylglucosamine (GlcNAc, A) and D-glucosamine
(
GlcN, D). Production of well-defined CHOS-mixtures, or even pure
CHOS, with specific lengths and sugar compositions, is of great
interest since these oligosaccharides have interesting bioactivities.
While direct chemical synthesis of CHOS is not straightforward,
chemo-enzymatic approaches have shown some promise. We have
used engineered glycoside hydrolases to catalyze oligomerization of
activated DA building blocks through transglycosylation reactions.
The building blocks were generated from readily available
(
GlcNAc) -para-nitrophenol through deacetylation of the non-
2
reducing end sugar with a recombinantly expressed deacetylase
from Aspergillus niger (AnCDA9). This approach, using a previously
described hyper-transglycosylating variant of ChiA from Serratia
marcescens (SmChiA) and a newly generated transglycosylating variant of Chitinase D from Serratia proteamaculans (SpChiD), led to
production of CHOS containing up to ten alternating D and A units [(DA) , (DA) , (DA) , and (DA) ]. The most abundant
2
3
4
5
compounds were purified and characterized. Finally, we demonstrate that (DA) generated in this study may serve as a specific
3
inhibitor of the human chitotriosidase. Inhibition of this enzyme has been suggested as a therapeutic strategy against systemic
sclerosis.
1
. INTRODUCTION
suitable for various applications in agriculture, cosmetics, water
treatment, and medicine.
Chito-oligosaccharides (CHOS) are oligomers prepared
2
−4
Chitin, a crystalline water-insoluble biopolymer consisting of
β-1,4 linked 2-acetamido-2-deoxy-β-D-glucopyranosyl units
from chitosan and have several potential applications,
(
GlcNAc, A), is the second most abundant biopolymer in
5
especially in the food, medical, and agriculture fields.
nature and is a common structural component in the
exoskeletons of insects, crustaceans, and parasitic nematodes
and in the cell walls of yeast and fungi. Chitin harvested from
nature may be converted to chitosan by partial deacetylation.
Chitosan is a heteropolymer of GlcNAc and 2-amino-2-deoxy-
β-D-glucopyranosyl (GlcN; D) residues, which, unlike chitin, is
soluble in water under mildly acidic conditions that imparts
chitosan with a net positive charge.
Examples of biological activities of CHOS include antifungal
6
,7
8
9
activity, inhibition of tumor growth, antibacterial activity,
8
,10
and an immunoenhancing effect. As is the case for chitosan,
the bioactivity of CHOS depends on oligomer length, degree
11
of acetylation, and acetylation pattern. A recent example
includes induction of H O production in rice cells by CHOS
2
2
for which the effects were optimal at DP 4 and depended on
The physicochemical properties of chitosan depend on the
sequence, with an ADDD oligomer showing twice the activity
of a DDDA oligomer.
12
fraction of N-acetylated residues (F ), the degree of polymer-
A
ization (DP; also expressed as molecular weight, MW), the
molecular weight distribution (PD, for polydispersity), and the
pattern of N-acetylation (PA; also referred to as sequence).
The pK of chitosan is between 6.0 and 6.5, depending on F ,
Received: October 14, 2020
Revised: November 11, 2020
a
A
1
DP, and PA, which is beneficial for biological applications.
These properties, combined with the fact that chitosan is
nontoxic, biocompatible, and biodegradable, make chitosan
©
XXXX American Chemical Society
https://dx.doi.org/10.1021/acs.biochem.0c00839
A
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2
2
Chitosan can be converted to CHOS by acid hydrolysis or
by enzymatic hydrolysis with glycoside hydrolases (GHs) such
as chitinases or chitosanases. While progress has been made in
encoding ChiA-D313N−F396W into the pET16b vector
(Novagen, Madison, WI, USA). The Chitinase fragments were
amplified by PCR using the following primers, 5′-
TCGAAGGTCGTCATATGGCCGCGCCGGGC-3′ (for-
ward) and 5′-CAGCCGGATCCTCGAGTTATTGAACGC-
CGGCGC-3′ (reverse) purchased from Life Technologies
(Carlsbad, CA, USA), using a method described by Hamre et
5
,13
producing reasonably well-defined CHOS samples,
such
preparations usually contain a wide range of oligomer species
of varying lengths and sequences. Chemical synthesis of fully
14
defined CHOS is challenging, whereas chemo-enzymatic
approaches have met with some success. One example
concerns a method for chemo-enzymatic synthesis of elicitor-
2
4
al. The resulting pET16b construct was sequenced using
GATC Biotech’s LIGHTrun sequencing service to confirm the
correct insert before it was transformed into E. coli BL21Star
(DE3) cells (Life Technologies). Expression and purification
of His -SmChiA-D313N/F396W was performed as described
active chitosan oligomers using (GlcNAc) as substrates in a
3
15
lysozyme-catalyzed transglycosylation reaction system. Fur-
thermore, having access to a library of chitin deacetylases and a
detailed knowledge concerning their specificity in removal of
the N-acetyl-groups allowed the production of 14 partially
1
0
2
4
by Hamre et al.
2.2.2. Construction, Expression, and Purification of
SpChiD-D151N/Y226W/Y154F. The triple mutant of SpChiD,
that is, D151N/Y226W/Y154F, was generated by site-directed
mutagenesis using pET-22b (+)-Y226W as template, and using
a single mutagenic primer and PCR amplification conditions as
described earlier. The primer used for site-directed muta-
genesis was 5′-GCATCGATCTCAACTGGGAATTCCCGG-
TTAACG-3′ (mutation sites are italicized and underlined).
The sequence of the mutated gene fragment was confirmed by
automated DNA sequencing, and the expression vector with
the desired mutations was transformed into E. coli BL21 (DE3)
for protein expression.
16
acetylated chitosan tetramers. Finally, the use of a GH18
glycosynthase” for the coupling of oxazoline-activated donors
to CHOS acceptors obtained from microbial fermentation has
“
1
7
been reported. Still, the chemo-enzymatic of CHOS with
alternating N-acetylglucosamine and D-glucosamines is to our
knowledge, yet to be achieved.
2
1
Interestingly, many retaining GHs, including certain family
8 chitinases, possess an innate ability to catalyze trans-
1
glycosylation reactions to form new glycosidic bonds between
18,19
donor and acceptor saccharides.
Recent work, for example,
on Chitinase A from Serratia marcescens (SmChiA) (CHIA_-
SERMA, UniProtKB - P07254) and Chitinase D from Serratia
proteamaculans (SpChiD) (A8GFD6_SERP5, UniProtKB
A8GFD6), has shown that the ratio of transglycosylation
versus hydrolysis can be increased through rational site-
Periplasmic fractions were prepared using the two-step
osmotic shock procedure as described in the pET expression
system instructions (Novagen), with minor modifications.
Prior to purification, the supernatant was passed through a 0.2-
μm sterile filter, and the periplasmic fraction was buffer-
exchanged to 50 mM NaH PO , 300 mM NaCl, 10 mM
2
0−22
directed mutagenesis.
In SmChiA, transglycosylating
activity was increased by exchanging a conserved Asp-residue
that interacts with the oxazolinium ion intermediate and the
catalytic acid with an Asn-residue. A further increase was then
obtained by improving the hydrophobicity of the glycosyl
acceptor binding site through the additional exchange of a Phe-
2
4
imidazole, pH 8.0. This same buffer was also used as the
equilibration buffer in further affinity purification steps, as
described previously. The purity of the protein in the
fractions was analyzed by 12% SDS-PAGE.
.2.3. Expression and Purification of AnCDA9. For protein
expression, E. coli TOP10 cells containing the appropriate
2
5
22
residue with a Trp-residue. Similar studies with SpChiD have
2
revealed several single point-mutations that improve trans-
20,21
glycosylation activity.
Here, we describe the use of tranglycosylating mutants of
plasmid were inoculated into 5 mL of TY medium containing
.1 mg/mL ampicillin and grown at 37 °C, 200 rpm for 16 h.
26
0
SmChiA and SpChiD to produce CHOS of specific F , DP,
A
The cell culture was then used to inoculate 500 mL of TY
medium containing 50 μg/mL ampicillin followed by
cultivation until the OD600 reached 0.8−1.0. At this point,
the temperature was decreased to 30 °C and gene expression
was induced by adding 0.001 volume of 20% (w/v) L-
arabinose, followed by incubation for 20 h at 28 °C. Next, the
cells were harvested by centrifugation (8000 rpm, 10 min, 4
C).
A cytoplasmic extract was prepared by first resuspending the
cell pellet in 250 mL of ice-cold PBS buffer (100 mM
and PA, starting with 4-nitrophenyl 2-acetamido-2-deoxy-4-O-
(
(
2-amino-2-deoxy-β-D-glucopyranosyl)-β-D-glucopyranoside
20,22,23
DA-pNP). On the basis of previous reports,
it was
anticipated that the CHOS thus obtained, with an alternating
acetylation pattern [(DA) ], had potential as inhibitors of the
n
human chitotriosidase. Here we show that this indeed is the
case. On the basis of the results of this chemo-enzymatic
approach to the production of CHOS, we discuss structural
features that may enable a GH18 to catalyze the coupling of
partially deacetylated oligomers.
°
Na HPO , 18 mM KH PO , 137 mM NaCl, 27 mM KCl) after
2
4
2
4
which the cells were harvested by centrifugation (8000 rpm, 10
min). The supernatant was removed, and the cell pellet was
resuspended in 20 mM Na HPO buffer, pH 7.4 containing
2
. MATERIALS AND METHODS
.1. Chemicals and Reagents. 4-Nitrophenyl 2-acetami-
2
2
4
do-2-deoxy-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-β-
D-glucopyranoside (AA-pNP) was purchased from Toronto
Research Chemicals (Toronto, Canada). All remaining
reagents and solvents were obtained from Sigma-Aldrich and
used without further purification. The enzymes used in the
different synthetic procedures were engineered and produced
in our own laboratories using the protocols described below.
150 mM NaCl, 50 mM imidazole, and 10% glycerol (the
amount of liquid added corresponded to approximately six-
times the weight of the pellet as determined by weighing
centrifuge tubes before centrifugation and after removal of the
supernatant). The cells were sonicated for 12 min with 5 s
pulses at 33% amplitude, using a VibraCell Ultrasonic Liquid
Processor VCX500/VCX750 (Sonics, Newtown, Connecticut,
USA). The cell debris was removed by centrifugation (8000
rpm, 10 min), and the supernatant was passed through a 0.2
μm sterile filter prior to protein purification.
2
.2. Enzymes. 2.2.1. Construction, Expression, and
Purification of His -SmChiA-D313N−F396W. His -ChiA-
1
0
10
D313N−F396W was obtained by cloning the gene fragment
B
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The protein was purified using a Ni-NTA agarose column
1.5 cm diameter, 5 mL stationary phase in total) using a flow
20 mM ammonium acetate, pH 6.1, and BSA (0.1 mg/mL
reaction mixture) at 37 °C. The reaction was followed by
regularly taking 5 μL samples that were mixed with 5 μL of
acetonitrile to inactivate the enzyme. These samples were
analyzed using MALDI/TOF-MS. After completion, the
reaction was quenched by adding acetonitrile to a final
concentration of 70% (v/v), after which the reaction mixture
was centrifuged and the precipitate was removed by filtration.
Separation of the soluble products using SEC-column
chromatography gave the following products: DA, (DA)2,
(DA) -pNP, (DA) , (DA) -pNP, and (DA) .
(
rate of 2.5 mL/min. The column was equilibrated in buffer
containing 20 mM Na PO buffer, 150 mM NaCl, 50 mM
2
4
imidazole, and 10% glycerol, pH 7.4. The cytoplasmic extract
was loaded directly onto the column and the column was then
washed with 3 column volumes starting buffer. AnCDA9 was
eluted from the column with a buffer containing 20 mM
Na PO buffer, 150 mM NaCl, 500 mM imidazole, and 10%
2
4
glycerol, pH 7.4. Enzyme purity was verified by SDS-PAGE,
and the purified proteins were concentrated and transferred to
2
3
3
4
1
20 mM Tris-HCl, 20 mM NaCl, pH 8.0, using an Amicon
2.4.2.1. DA. H NMR (400 MHz, D O) δ 5.21 (d, J = 2.2
2
Ultra Centrifugal Filter (MW 10 000, Millipore). The protein
concentration was determined using the Bradford Protein
Assay from Bio-Rad using BSA as a standard.
Hz, 1 H), 4.89−4.77 (m, 1 H), 4.74 (d, J = 7.0 Hz, 1 H),
4.09−3.99 (m, 1 H), 3.99−3.70 (m, 7 H), 3.69−3.61 (m, 2 H),
3.57−3.44 (m, 2 H), 3.22−3.00 (m, 1 H), 2.05 (s, 3 H) ppm.
1
3
2
.3. Isolation and Characterization of Saccharide
C NMR (101 MHz, D O) δ 175.6, 99.3, 95.6, 91.4, 78.2,
2
Products. Retention factor (R ) values were determined by
77.2, 75.3, 73.4, 70.8, 69.7, 61.2, 60.2, 57.4, 56.8, 54.8, 24.0
ppm.
f
thin layer chromatography using Supelco analytical aluminum
foil-precoated silica gel with fluorescence indicator UV254 from
Sigma-Aldrich. Spots were visualized under UV light and by
basic aqueous solution (10% w/w K CO in water) of KMnO .
MALDI-TOF-MS: m/z [M + Na]+ calculated for
C H N NaO , 405.1485; found, 405.1462.
1
4
26
2
10
1
2.4.2.2. (DA) . H NMR (400 MHz, D O) δ 5.21 (d, J = 2.2
2
3
4
2
2
Flash column chromatography was carried out using silica gel
from Merck KGaA (Silica gel 60, 0.040−0.063 mm). Reversed
phase chromatography was performed using Sep-Pak C18
cartridges from Waters. Purification by size-exclusion chroma-
tography (SEC) was conducted using three Superdex 30
columns coupled in series (overall dimensions of 2.60 cm ×
Hz, 1 H), 4.93−4.82 (m, 2 H), 4.74 (d, J = 7.7 Hz, 1 H),
4.10−4.00 (m, 1 H), 3.97−3.72 (m, 14 H), 3.72−3.61 (m, 3
H), 3.57−3.47 (m, 4 H), 3.28−2.96 (m, 2 H), 2.05 (s, 6 H)
ppm.
1
3
C NMR (101 MHz, D O) δ 175.3, 99.0, 95.6, 91.4, 78.1,
2
77.8, 77.2, 75.3, 73.2, 72.7, 70.8, 70.4, 69.6, 61.3, 61.2, 61.2,
57.4, 56.7, 54.8, 22.7 ppm.
1
4
80 cm; GE Healthcare) with 0.15 M ammonium acetate, pH
.15, as the mobile phase. Four milliliter fractions were
MALDI-TOF-MS: m/z [M + H]+ calculated for
C H N O , 747.3148; found, 747.3148.
collected and analyzed.
All NMR spectra were recorded using a Bruker Ascend 400
2
8
51
4
19
1
2.4.2.3. (DA) -pNP. H NMR (400 MHz, D O) δ 8.26 (d, J
2
2
spectrometer with D O as solvent. The chemical shifts are
= 9.2 Hz, 2 H), 7.20 (d, J = 9.3 Hz, 2 H), 5.35 (d, J = 8.4 Hz, 1
H), 4.89−4.77 (m, 2 H), 4.60 (d, J = 7.3 Hz, 1 H), 4.14−4.04
(m, 1 H), 4.03−3.84 (m, 9 H), 3.84−3.74 (m, 5 H), 3.73−3.57
(m, 5 H), 3.57−3.46 (m, 2 H), 3.15−2.99 (m, 2 H), 2.08 (s, 3
H), 2.03 (s, 3 H) ppm.
2
reported in parts per million relative to TMS and the coupling
constants are given in Hz. Matrix-assisted laser desorption/
ionization-time-of-flight-mass spectrometry (MALDI-TOF
MS) was carried out using an Ultraflex TOF/TOF mass
spectrometer (Bruker Daltonik GmbH, Bremen, Germany)
with gridless ion optics under control of Flexcontrol, version
1
3
C NMR (101 MHz, D O) δ 177.8, 177.5, 164.5, 145.6,
2
129.0, 119.4, 104.2, 101.3, 81.3, 79.7, 79.2, 77.9, 77.6, 77.3,
75.5, 74.3, 72.4, 63.2, 63.1, 63.0, 62.6, 58.8, 58.4, 58.1, 25.2,
25.0 ppm.
4
1
.1. Samples were prepared by mixing 1 μL of the sample with
μL of 10% 2,5-dihydroxybenzoic acid (DHB) and spotted on
a MALDI-plate. Known concentrations of (GlcNAc) were
MALDI-TOF-MS: m/z [M + H]+ calculated for
C H N O , 869.3390; found, 869.3374.
4
added as an internal standard to estimate product concen-
trations. The MALDI-TOF-MS experiments were conducted
using an accelerating potential of 20 kV in the reflector mode.
3
4
56
5
21
1
2.4.2.4. (DA) . H NMR (400 MHz, D O) δ 5.20 (d, J = 7.7
3
2
Hz, 1 H), 4.90−4.67 (m, 3 H), 4.59 (d, J = 6.9 Hz, 2 H), 4.14
(d, J = 10.9 Hz, 1 H), 4.04−3.99 (m, 1 H), 3.98−3.46 (m, 31
H), 3.16−3.05 (m, 3 H), 2.07 (s, 9 H) ppm.
2
.4. Synthesis. 2.4.1. DA-pNP. AA-pNP (50 mg, 3.7 mM)
was incubated with AnCDA9 (0.31 mg, 1.5 μM) in 9 mL of
1
3
Tris-HCl, pH 8.0 (25 mM) and 0.9 mL of CoCl (aq) (0.1
C NMR (101 MHz, D O) δ 175.5, 102.2, 98.8*, 98.6*,
2
2
mM) at 37 °C. The reaction was monitored using TLC
77.5, 75.1*, 73.1*, 72,5*, 71.8*, 77.2, 75.9, 75.2, 72.3 70.4,
62.3, 61.1, 60.2, 56.4, 56.2*, 55.7* ppm.
(
EtOAc:MeOH:H O; 7:2:1) and MALDI-TOF-MS. The
2
1
3
product was isolated using reversed phase chromatography
Sep-Pak C18; gradient from 100% H O to 100% MeOH).
C peaks marked with ∗ were identified from cross peaks in
(
HSQC spectra.
2
Freeze-drying yielded GlcN-β1,4-GlcNAc-pNP (DA-pNP) as a
MALDI-TOF-MS: m/z [M + H]+ calculated for
C H N O , 1112.4708; found, 1112.4682.
colorless solid (45.2 mg, 0.090 mmol, 98%).
4
2
76
6
28
1
+
H NMR (400 MHz, D O) δ 8.26 (d, J = 9.3 Hz, 2 H), 7.20
2.4.2.5. (DA) -pNP. MALDI-TOF-MS: m/z [M + H]
2
3
(
d, J = 9.3 Hz, 2 H), 5.35 (d, J = 8.4 Hz, 1 H), 4.54 (d, J = 8.1
calculated for C H N O , 1233.4871; found, 1233.4824.
48 79 7 30
+
Hz, 1 H), 4.09 (t, J = 9.1 Hz, 1 H), 4.03−3.93 (m, 2 H), 3.93−
2.4.2.6. (DA) . MALDI-TOF-MS: m/z [M + H] calculated
4
3
2
.83 (m, 6 H), 3.76 (dd, J = 12.3, 5.8 Hz, 1 H), 3.55−3.49 (m,
H), 3.44−3.39 (m, 2 H), 2.72 (t, J = 8.8 Hz, 1 H), 2.02 (s, 3
for C H N O , 1477.6268; found, 1477.6248.
5
6
99
8
37
2.5. Incubation of (DA) with Human Chitotriosidase.
3
1
3
H) C NMR (101 MHz, D O) δ 174.00, 160.74, 141.80,
(DA) (100 μM) were incubated with 0.5 μM of the catalytic
2
3
1
6
25.19, 101.69, 97.54, 96.54, 76.96, 75.30, 74.58, 74.09, 70.97,
8.66, 59.71, 59.08, 55.76, 54.08, 21.17.
domain of the human chitotriosidase (HCHT) (isolated as
27
described in Stockinger et al.) in 50 mM citrate/phosphate
buffer pH 5.2, at 37 °C and 300 rpm. Aliquots of 1 μL were
withdrawn at several time points up to 2 h, and reactions were
2
.4.2. Enzymatic Polymerization of DA-pNP. DA-pNP (1
mM) was incubated with ChiA D313N/F369W (0.5 μM) in
C
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Scheme 1. Deacetylation of AA-pNP Using CDA9
Scheme 2. Polymerization of DA-pNP Using SmChiA-D313N/F396W
quenched directly on the MALDI target plate by addition of an
equal volume of the matrix solution containing 2,5-
dihydroxybenzoic acid (DHB) dissolved in water (30%) and
acetonitrile (70%).
deacetylated specifically at the nonreducing end and that
contained a good leaving group. The partially deacetylated
disaccharide derivative would then act as both a donor and an
acceptor in a transglycosylation reaction. Para-nitrophenol
(pNP) was chosen as the leaving group since this is widely
used in a plethora of GH assays. AA-pNP can be synthesized as
described by Noguchi et al. through generation of a GlcNAc
oxazoline from GlcNAc using a dehydrative condensing agent
2
.6. Determination of IC50 for (DA)3 with Human
Chitotriosidase (HCHT). The IC value for the inhibition of
50
HCHT catalyzed hydrolysis of the (A) analog 4-methyl-
3
umbelliferyl-2-acetamido-2-deoxy-4-O-(2-acetamido-2-deoxy-
29
β-D-glucopyranosyl)-β-D-glucopyranoside (AA-4-MU) by
followed by an enzymatic coupling to A-pNP. For practical
reasons, we started with commercially available AA-pNP.
The chitin deacetylase AnCDA9 was used for the
deacetylation of the nonreducing end GlcNAc moiety yielding
28
(
DA) was determined as described by Cederkvist et al.,
3
with minor modifications.
In a standard assay (no inhibitor added), 100 μL of a
mixture containing 5 nM enzyme, 20 μM substrate, 50 mM
citrate/phosphate buffer pH 5.2, and 0.1 mg/mL bovine serum
albumin was incubated at 37 °C in a linear shaking water bath
26
acetic acid and DA-pNP in 98% yield (Scheme 1). Complete
deacetylation to DD-pNP was not observed, indicating good
selectivity in the enzymatic action of AcCDA9.
(
Grant Instruments OLS200 from Fisher Scientific, Hampton,
3.2. Synthesis of CHOS with an Alternating Pattern of
New Hampshire, USA). After 5 min, the reaction was stopped
by addition of 1.9 mL of glycine/NaOH pH 10.6. The amount
of 4-MU released was determined using a DQ300 Fluorometer
Deacetylation Using a Transglycosylating Variant of
3
13
SmChiA. Previously, we showed that the exchange of Asp
,
the middle residue in the conserved catalytic DxDxE motif of
3
96
(
Hoefer, Holliston, Massachusetts, USA). The experiments
were performed in triplicate parallels. The IC50 value was
determined by adding varying concentrations of (DA) (150,
family GH18s, to Asn and exchange of Phe in the +2 subsite
to Trp in SmChiA resulted in a hyper-transglycosylating
22
3
mutant with suppressed hydrolytic activity. DA-pNP (2), as
both glycosyl donor and acceptor, was incubated with
SmChiA-D313N/F396W allowing the production of CHOS
with alternating units of D and A (Scheme 2). Reaction
progress was monitored by MALDI-TOF-MS and revealed
chain elongation of DA-pNP as shown in Figure 1.
1
00, 50, 25, 10, and 5 μM) to the mixture before starting the
enzyme reaction. The IC -values were calculated by nonlinear
50
fitting to eqs 1 or 2, where [I] is the concentration of (DA)3,
and Bmax is 100% inhibition (see below for further details):
Bmax[I]
IC50 + [I]
%
%
Inhibition =
Inhibition =
DA-pNP can only productively bind from subsites −2 to +1
in the active site of SmChiA. The binding of this substrate in
the subsites −1 to +2 would place the glucosamine moiety in
the −1 subsite, which would prevent hydrolysis due to the lack
of the N-acetyl group that is essential for substrate-assisted
(1)
(2)
Bmax(1)[I]
IC50(1) + [I]
Bmax(2)[I]
IC50(2) + [I]
+
3
0,31
catalysis.
would yield a DA-oxazolinium ion intermediate in the −2 and
1 subsites that can either react with a water molecule in a
For this reason, productive binding of DA-pNP
3
. RESULTS AND DISCUSSION
.1. Synthesis of DA-pNP. The synthesis strategy was
based on using a disaccharide of GlcNAc that could be
−
3
hydrolysis reaction to produce DA or a second DA-pNP to
produce (DA) -pNP in a transglycosylation reaction. The same
2
D
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3
site.
The catalytic domain and the N-terminal carbohy-
drate-binding FnIII domain of SmChiA may simultaneously
34−36
interact with as many as 17 sugar units.
Attempts were
undertaken to optimize production of (DA) for inhibition
3
studies (see below) by varying the enzyme/substrate ratio (0.1
μM to 10 μM for SmChiA-D313N/F396W) without achieving
higher concentrations than the maximum level of 130 μM
when 0.5 μM of the enzyme was used.
At the given concentrations of DA-pNP (1000 μM) and
SmChiA-D313N/F396W (0.5 μM), concentrations of approx-
imately 200 μM and 130 μM are expected for (DA) and
2
(
DA) , respectively, after 210 min turnover (from Figure 1).
3
An initial effort for upscaling the procedure, without any
attempts to optimize this procedure, was undertaken to
observe the amount of (DA) and (DA) that could be
2
3
Figure 1. Product formation upon incubation of 0.5 μM SmChiA-
D313N/F396W with 1 mM DA-pNP. Up to four continuous DA
isolated and to be used for the studies described below. When
using a reaction volume of 50 mL, totals of 3.5 mg and 1.5 mg
of (DA) and (DA) , respectively, were isolated. For (DA) ;
units were observed. For clarity, only the formations of (DA) -pNP
2
2
3
2
(
red), (DA) (black), (DA) -pNP (green), and (DA) (blue) are
2
3
3
this yield is approximately 21% of what is expected from what
is observed from Figure 1 and 8% of theoretical yield if all DA-
pNP was converted to (DA) . For (DA) , the yield is about
shown. Small amounts of octamers [(DA) , max 30 μM, and (DA) -
4
4
pNP, max 16 μM] were also observed as well as, transiently, small
amounts of (DA) . Yields of product were estimated relative to an
2
3
5
internal standard in MALDI-TOF MS analysis, as described in the
Materials and Methods. All experiments were done in triplicate giving
standard deviations amounting to 2−10% of the value. Error bars are
omitted for clarity.
10% of what is estimated from what is observed from Figure 1
and 4% of theoretical yield if all DA-pNP was converted to
(
DA)3.
.3. Synthesis of CHOS Using SpChiD. Literature
indicates that SpChiD and several of its engineered variants
3
20,21
have good transglycosylating activity.
Therefore, to test the
rational applies for (DA) -pNP to form either (DA) or
2
2
universality of using GH18s to produce CHOS, experiments
with this enzyme were conducted. First, DA-pNP, again as
both glycosyl donor and acceptor, was incubated with wild-
type SpChiD since this enzyme possesses innate TG activity.
However, only hydrolysis products were observed. A single
mutant, SpChiD-D151N, and a double mutant, SpChiD-
D151N/Y226W, where the latter is mutation-wise analogous
to Sm-ChiA-D313N/F396W (Figure 2), displayed both
improved transglycosylating activity and reduced hydrolytic
(
DA) -pNP and (DA) -pNP to form (DA) or (DA) -pNP,
3 3 3 4
and so on. In addition, with these reaction mixtures, (DA)n
oligomers can also be formed from hydrolysis of longer CHOS
produced from transglycosylation reactions, with and without
pNP at the reducing end. The absence of accumulation of
longer CHOS is likely because these are better substrates than
the shorter CHOS, as binding affinity to GHs with extended
substrate binding surfaces, such as SmChiA, increases with
increasing number of sugar moieties up to eight in the active
34
37
Figure 2. Selected residues for SmChiA (wild-type in A, double mutant in B, pdb ehn) and SpChiD (wild-type in C, triple mutant in D, pdb
4
nzc) important for catalytic activity and transglycosylation. The substrate shown was positioned based on the crystal structure of SmChiA
34
complexed with (GlcNAc) using structural superposition (only sugar moieties in subsites −1, +1, and +2 are shown) (pdb 1ehn). See text for
more details.
8
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2
0
activity when using (A)4 as a substrate; however, no
transglycosylation products were observed for our substrate.
Members of the GH18 family differ in terms of endo- versus
exoactivity, processive versus nonprocessive action, and the
and SpChiD in the experiments described above, although the
causal relationship between these differences and the efficiency
of (DA) production from DA-pNP remains unclear.
n
Results from our own work and work by others had
indicated that increased TG activity may be obtained by either
increasing the hydrophobicity of acceptor subsites, binding
strength to individual subsites, or by changing residues
important for stabilizing the transition state/intermedi-
38
directionality of processivity. These differences are reflected
in different active site architectures, which may also affect the
TG potential. Comparison of the crystal structure of SpChiD
with the structures of SmChiA, which is exoprocessive acting
from the reducing end, and SmChiB, which is exoprocessive
suggested that SpChiD
resembles SmChiB more than SmChiA (Figure 3). Both
2
0−22,44,45
ate.
With this in mind, we examined differences in
3
9,40
from the nonreducing end,
154
the active sites of SpChiD and SmChiA and noticed that Tyr
in SpChiD is a phenylalanine, Phe , in SmChiA (Figure 3).
This residue is adjacent to the catalytic acid (Glu
SpChiD and Glu for SmChiA; Figure 2) and interacts with
the C6 OH-group of the +1-sugar moiety (Figure 2). Mutating
Tyr into Phe in SpChiD would yield both an increase in
hydrophobicity and a likely perturbation of the stability of the
transition state/intermediate. Interestingly, the SpChiD-
D151N/Y154F/Y226W triple mutant was able to catalyze
316
1
53
for
3
15
oligomerization of DA-pNP to (DA) -pNP and traces of
2
(
DA) -pNP (products were not quantified) but did not
3
produce (DA) or (DA) (Figure 4A).
2
3
DA-pNP may not be an optimal substrate to demonstrate
the TG ability of SpChiD and its mutants. First, several studies
3
4
Figure 3. (A) Crystal structures of SmChiA (left, pdb code 1ctn),
37
SpChiD (middle, pdb code 4nzc), and SmChiB (right, pdb code
e6n). Surface-exposed aromatic amino acids are highlighted in
43
1
blue. (B) Close-up look at the active sites with a focus on positive
subsites. The “extra” aromatic amino acids compared to SmChiA in
1
60
290
the +3, +4, and +5 subsites of SpChiD (Trp (+3) and Trp (+5))
190
and SmChiB (Phe (+3) and Tyr240 (+4)) are labeled. Subsite −1
containing Trp3 in SpChiD is hidden by a loop in the active site. In
contrast to SpChiD, SmChiA, and SmChiB have a CBM.
95
SmChiB and SpChiD have aromatic residues in subsites +3
1
90
290
(
Phe and Trp , respectively), whereas (putative) inter-
actions between SmChiA and a +3 sugar moiety would take
place through hydrogen bonds, directly or via water
3
4,41
molecules.
A certain similarity between SmChiB and
SpChiD is supported by the observation that the thermody-
namic signature of ligand binding by SmChiD is more similar
42
to that of SmChiB than SmChiA. In SpChiD, the path of
exposed aromatic residues on the acceptor side continues to
subsite +5, while this path extends all the way down to the
carbohydrate-binding module for SmChiB (Figure 3; note that
in exoprocessive enzymes, this extended stretch of exposed
aromatic residues interacts with the polymeric part of the
chitin molecule, the “substrate”, which is being processively
degraded). In SmChiA, a similar path of aromatic residues
appears on the donor side, providing negative subsites for
substrate binding (Figure 3). Thus, while in ChiA, the glycosyl
acceptor interacts with subsites designed for product binding,
in SpChiD, the acceptor interacts with subsites that seem
optimized for substrate binding, as in SmChiB. These
differences may underlie the different performances of SmChiA
Figure 4. MALDI-TOF-MS analysis of products generated upon
incubation of DA-pNP (A) or (DA)2 (B) with SpChiD-D151/
Y154F/Y226W.
F
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Figure 5. Plots of % inhibition of HCHT with concentration of (DA) . The curve in panel A represents the fit of a single productive substrate
3
binding and inhibition mode, while the curve panel B shows the fit for a two productive substrate binding and inhibition modes.
show that binding affinity to GHs increases as number of
The mammalian genome codes for eight different putative
chitinases of which two, human chitotriosidase (HCHT) and
acidic mammalian Chitinase (AMCase) have shown enzymatic
activity and are believed to be part of the innate immune
system. Recently, it has been shown that levels of HCHT
activity are significantly increased in the circulation and lungs
32,33
carbohydrate moieties increases.
Second, Km increases
(
(
4
lower substrate affinity) when pNP is the leaving group
binding in subsite +1) compared to, for example, GlcNAc or
46
50
-methylumbelliferone during GH turnover. To address the
size dependency of the starting substrate and the nature of the
leaving group, SpChiD-D151N/Y154F/Y226W was incubated
of systemic sclerosis patients and for this reason inhibition of
51
HCHT is a suggested therapeutic strategy. Both Gorzelanny
et al. and Eide et al. have observed a strong preference for a
GlcNAc-moiety in the −2 subsite of HCHT, leading to the
hypothesis that CHOS with alternating D and A units (i.e.,
with the tetrameric (DA) , produced from the reaction of DA-
2
pNP with SmChiA-D313N/F396W, and oligomers with
alternating A and D of up to ten units were detected (Figure
4
B, products were not quantified), further confirming the
(
DA) or (DA) ) would bind strongly but nonproductively
transglycosylating potential of SpChiD and its variants.
The inability of SpChiD-D151N/Y154F/Y226W, unlike
SmChiA-D313N/F396W, to remove pNP from a (DA)n
oligomer may be explained by the unique active site
architecture of SpChiD. From previous studies, it is clear
that SpChiD prefers to bind CHOS with its nonreducing end
2 3
with a GlcNAc-moiety situated in the −2 subsite and a GlcN-
4
8,49
moiety situated in the −1 subsite.
A detailed kinetic analysis of HCHT-catalyzed hydrolysis of
a water-soluble chitosan with an acetylation degree of 62%
yielded an apparent catalytic rate constant of 102 s (pH 5.2
and t = 37 °C). Although the kinetic parameters for HCHT
catalyzed degradation of (GlcNAc)6 have not been fully
determined, it is clear that HCHT rapidly breaks down this
fully acetylated compound (incubation of 100 μM (GlcNAc)6
in the presence of 1 nM HCHT resulted in 50% degradation
−
1
48
21
in subsite −2 or −1 where the −1 subsite is preferred. This is
in part explained by the presence of a loop in the active site
37
that occludes the −3 subsite and partly the −2 subsite.
Moreover, a crystal structure of SpChiD with the inhibitor
allosamidin, which binds to subsites −3 to −1, showed that
there are more interactions between the enzyme and the sugar
moieties in subsite −1 than −2, explaining the preference for
52
within 1 min at pH of 4.2 and t = 37 °C). As a first approach
to assess the inhibitory potential of (DA) , (DA) (50 μM)
3
3
was incubated with HCHT (0.25 μM) at pH 5.2 and t = 37 °C
for up to 2 h. For all time points, a MALDI-TOF-MS analysis
42
the binding of the nonreducing end in the −1 subsite.
Interestingly, there are no interactions between the sugar
moiety and the protein in subsite −3, and there is no −4
subsite. Even though the loop can be displaced upon ligand
showed the presence of (DA) , indicating that this is a poor
3
substrate for HCHT and may act as an inhibitor.
To test inhibitory activity, (DA)3 was incubated at
concentrations from 0 to 150 μM in the presence of HCHT
4
2
binding, we speculate that a potential energetic penalty of a
displacement of the loop and the lack of a “pull” toward the
substrate from subsites “−4” and −3 and low binding affinity of
pNP in the +1 subsite precludes the productive binding of
(
5 nM) and AA-4-MU (20 μM), a concentration where
HCHT predominantly performs hydrolysis and not trans-
19
glycosylation, and initial rates of hydrolysis were determined
(
DA) -pNP to yield (DA) . The same argument can be made
2
2
by measuring the concentrations of released 4-methyl-
umbelliferyl alcohol over time. The initial rates were used to
calculate percentage inhibition, which was plotted against the
concentration of (DA) to obtain an IC value by nonlinear
for (DA) -pNP explaining why (DA) is not observed.
3
3
3
.4. Biological Activity of (DA) . All family 18 chitinases
3
employ a substrate-assisted catalytic mechanism involving the
N-acetyl group of the sugar bound in the −1 subsite, resulting
in an absolute preference for a GlcNAc-moiety in this subsite
3
50
fitting using eq 1 outlined in the Materials and Methods. By
using this approach, a fit was obtained with an IC value of 27
5
0
30,43,47
during productive binding.
Moreover, several members
μM with a Bmax of 100%, meaning that 100% inhibition could
be obtained. An inspection of the fit (Figure 5A) suggested
that there is a deviation between the model and the
experiment. To address this, we attempted to fit the data to
two productive binding modes of the substrate (i.e., hydrolysis
and transglycosylation) and two binding modes for the
of this family display strong preferences for GlcNAc-moieties
in other individual subsites due to specific substrate−protein
2
8,48,49
interactions.
It is thus conceivable that (DA) can bind
3
productively to some chitinases, whereas it may bind
nonproductively and thus inhibit other chitinases.
G
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inhibitor each competing with the binding modes of the
substrate by nonlinear fitting using eq 2 outlined in the
Materials and Methods. Indeed, fitting the data to eq 2 gave a
better fit (Figure 5B) yielding an IC50(1) value of 2.5 μM
Authors
Rianne A. G. Harmsen − Department of Chemistry,
Biotechnology and Food Science, Norwegian University of Life
Sciences, N-1432 Ås, Norway
Berit Bjugan Aam − Department of Chemistry, Biotechnology
and Food Science, Norwegian University of Life Sciences, N-
(Bmax(1) = 39%) and IC50(2) value of 90 μM (Bmax(2) = 61%),
respectively. Bmax(1) is the % of the activity stemming from the
substrate binding mode 1, and this is inhibited by the inhibitor
1432 Ås, Norway
Jogi Madhuprakash − Department of Chemistry,
Biotechnology and Food Science, Norwegian University of Life
Sciences, N-1432 Ås, Norway; Department of Plant Sciences,
School of Life Sciences, University of Hyderabad, Hyderabad,
India
Anne Grethe Hamre − Department of Chemistry,
Biotechnology and Food Science, Norwegian University of Life
Sciences, N-1432 Ås, Norway
Ethan D. Goddard-Borger − Walter & Eliza Hall, Institute of
Medical Research, Parkville, Victoria 3052, Australia;
Department of Medical Biology, University of Melbourne,
Parkville, Victoria 3010, Australia; Department of
Chemistry, University of British Colombia, Vancouver, British
Columbia V6T 1Z1, Canada; orcid.org/0000-0002-
binding mode 1 with the strength represented by IC50(1)
Bmax(2) is the % of the activity stemming from the substrate
binding mode 2, and this is inhibited by the inhibitor binding
.
mode 2 with the strength represented by IC50(2)
.
Both SmChiA and SmChiB, two bacterial chitinases, easily
degrade both (DA) and (DA) under the same conditions
2
3
(
results not shown). Previously, it has been shown that
SmChiB has a strong preference for an acetylated unit in the
2
8,53
−3 subsite.
With this preference, (DA) will preferentially
3
bind to subsites −4 to +2 or subsites −2 to +4, either of which
results in productive binding. Regardless of which of the
models used in Figure 5 represents the data most correctly, it is
clear that CHOS with alternating D and A units indeed display
specific inhibition of HCHT, as hypothesized by Gorzelanny et
8181-9733
48,49
al. and Eide et al.
Stephen G. Withers − Department of Chemistry, University of
British Colombia, Vancouver, British Columbia V6T 1Z1,
Canada; orcid.org/0000-0002-6722-5701
Vincent G. H. Eijsink − Department of Chemistry,
Biotechnology and Food Science, Norwegian University of Life
Sciences, N-1432 Ås, Norway; orcid.org/0000-0002-
4
. CONCLUSION
In this proof-of-concept study, we have developed a chemo-
enzymatic synthesis route of CHOS with a highly defined
pattern of deacetylation. We have isolated and characterized
CHOS with up to four repeating DA units and we present the
first reported isolation and thorough chemical characterization
of (DA) , (DA) -pNP, and (DA) . During the trans-
9220-8743
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.0c00839
2
2
3
glycosylation reactions, CHOS containing up to five repeating
DA units were observed by MALDI-TOF analysis. Further
optimization of the current methods is feasible and necessary
and could be achieved through the engineering of better
enzyme variants, optimizing reaction kinetics, and exploring
different substrates. Although likely far away, an ability to
enzymatically engineer polymeric chitosans with defined
acetylation patterns will be of considerable interest to the
material science field. In addition, well-defined CHOS, such as
those generated here, are likely to have interesting bioactivities
against a range of medically important GHs and lectins, as
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Grant Nos. 197388 and 221576
from the Norwegian Research Council of Norway and by
funding from the Canadian Institutes for Health Research.
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