Positional specifity of acetylxylan esterases on natural polysaccharide: An NMR study

Article abstract of DOI:10.1016/j.bbagen.2013.01.011

Background Microbial degradation of acetylated plant hemicelluloses involves besides enzymes cleaving the glycosidic linkages also deacetylating enzymes. A detailed knowledge of the mode of action of these enzymes is important in view of the development of efficient bioconversion of plant materials that did not undergo alkaline pretreatment leading to hydrolysis of ester linkages. Methods In this work deacetylation of hardwood acetylglucuronoxylan by acetylxylan esterases from Streptomyces lividans (carbohydrate esterase family 4) and Orpinomyces sp. (carbohydrate esterase family 6) was monitored by ¹H-NMR spectroscopy. Results The ¹H-NMR resonances of all acetyl groups in the polysaccharide were fully assigned. The targets of both enzymes are 2- and 3-monoacetylated xylopyranosyl residues and, in the case of the Orpinomyces sp. enzyme, also the 2,3-di-O-acetylated xylopyranosyl residues. Both enzymes do not recognize as a substrate the 3-O-acetyl group on xylopyranosyl residues α-1,2-substituted with 4-O-methyl-d-glucuronic acid. Conclusions The ¹H-NMR spectroscopy approach to study positional and substrate specificity of AcXEs outlined in this work appears to be a simple way to characterize catalytic properties of enzymes belonging to various CE families. Significance The results contribute to development of efficient and environmentally friendly procedures for enzymatic degradation of plant biomass.

Full text of DOI:10.1016/j.bbagen.2013.01.011

Biochimica et Biophysica Acta 1830 (2013) 3365–3372
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
Positional specifity of acetylxylan esterases on natural polysaccharide:
An NMR study
Iveta Uhliariková a, Mária Vršanská a, Barry V. McCleary b, Peter Biely a,
⁎
a
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovakia
b
Megazyme International Ireland, Bray Business Park, Bray, Co. Wicklow, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: Microbial degradation of acetylated plant hemicelluloses involves besides enzymes cleaving the
glycosidic linkages also deacetylating enzymes. A detailed knowledge of the mode of action of these enzymes
is important in view of the development of efficient bioconversion of plant materials that did not undergo al-
kaline pretreatment leading to hydrolysis of ester linkages.
Methods: In this work deacetylation of hardwood acetylglucuronoxylan by acetylxylan esterases from
Streptomyces lividans (carbohydrate esterase family 4) and Orpinomyces sp. (carbohydrate esterase family 6)
was monitored by 1H-NMR spectroscopy.
Received 5 December 2012
Received in revised form 15 January 2013
Accepted 17 January 2013
Available online 30 January 2013
Keywords:
Acetylxylan esterase
Positional specificity
NMR
Natural substrate
Acetylglucuronoxylan
Results: The 1H-NMR resonances of all acetyl groups in the polysaccharide were fully assigned. The targets of
both enzymes are 2- and 3-monoacetylated xylopyranosyl residues and, in the case of the Orpinomyces sp. en-
zyme, also the 2,3-di-O-acetylated xylopyranosyl residues. Both enzymes do not recognize as a substrate the
3-O-acetyl group on xylopyranosyl residues α-1,2-substituted with 4-O-methyl-D-glucuronic acid.
Conclusions: The 1H-NMR spectroscopy approach to study positional and substrate specificity of AcXEs outlined
in this work appears to be a simple way to characterize catalytic properties of enzymes belonging to various CE
families.
Significance: The results contribute to development of efficient and environmentally friendly procedures for en-
zymatic degradation of plant biomass.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
The microbial degradation of acetyl glucuronoxylans involves be-
sides enzymes cleaving the β-1,4-glycosidic linkages of the main chain
Hardwood glucuronoxylans are partially acetylated hetero-
polysaccharides. Xylopyranosyl (Xylp) residues linked β-1,4-gly-
cosidically form the polysaccharide main chain and this is
substituted with α-1,2-linked 4-O-methyl-D-glucuronic acid (MeGlcA)
residues. The ratio of Xyl:MeGlcA varies, but the most frequently
reported values are around 10. In native state, i.e. in plant cell walls,
the polysaccharide is partially acetylated [1–3]. More than a half of
Xylp residues of the main chain is monoacetylated at position 2 or 3,
or 2,3-di-O-acetylated. 3-O-Acetylation is to a high degree observed
on Xylp residues substituted with MeGlcA [4–6]. In summary, the acetic
acid in hardwood glucuronoxylans is esterified by hydroxyl groups at
positions 2 and 3, however, the acetyl groups occur on the xylan main
chain in five structural variants due to substituents on the vicinal
hydroxyl groups (see the formula in Fig. 1).
also α-glucuronidase and enzymes deacetylating the polymeric sub-
strate or acetylated xylooligosaccharides generated by endoxylanases.
Regarding the fact that hardwood acetylglucuronoxylan contains five
different acetyl groups, a question arises whether the acetylxylan ester-
ases, classified in several CE families [7] and reviewed recently [8], are
specialized for one particular acetyl group or behave as more general
acetyl esterases. Previous studies of the positional specificity of AcXEs
on methyl β-D-xylopyranosides indicated that esterases deacetylate
both positions 2 and 3 [9,10]. This positional “non-specificity” was
ascribed to the formation of two productive complexes differing in
the 180° orientation of the substrate [11,8]. However, all typical
acetylxylan esterases exhibited a strong preference for deacetylation
of the position
2 in monoacetyl derivatives of 4-nitrophenyl
β-D-xylopyranoside [12]. Recent work indicated that AcXEs belong-
ing to families CE1 and CE5 deacetylate in the polysaccharide and ol-
igosaccharides both positions 2 and 3 [13]. It became clear that the
catalytic properties of these enzymes should be investigated on nat-
ural partially acetylated xylan.
Abbreviations: AcXE, acetylxylan esterase; Xylp, D-xylopyranose or D-xylopyranosyl;
CE, carbohydrate esterase
One of the approaches to verify the positional specificity of AcXEs
⁎
Corresponding author. Tel.: +421 2 59410275; fax: +421 2 59410222.
on natural substrates could be the monitoring of the 1H-NMR signal
E-mail address: chempbsa@savba.sk (P. Biely).
0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbagen.2013.01.011

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Xyl-3Ac
Xyl-2Ac
Xyl-2,3Ac
Xyl-3Ac-2-MeGlcA
O
O
O
R
O
O
O
O
O
O
O
R1
O
O
O
HO
O
O
O
O
OH
OH
O
O
O
OH
H3C
H3C
CH3
O
O
OH
O
CH3
CH3
OH
CH3O
COOH
Fig. 1. Five types of acetylation of xylopyranosyl residues in hardwood acetylglucuronoxylans and selective HMBC spectrum of the polysaccharide showing two sets of four distinct signals
and their correlation. The two acetyl groups esterifying the same Xylp residue could not be distinguished in both anomeric and acetyl methyl group region. The following designations are
used: Xyl-3Ac, 3-O-acetylated Xylp; Xyl-2Ac, 2-O-acetylated Xylp; Xyl-2,3Ac, 2, 3-di-O-acetylated Xylp; Xyl-3Ac-2MeGlcA, MeGlcA 2-O-substituted and 3-O-acetylated Xylp. The H/C cor-
relations are marked with vertical and horizontal lines (see also Table 2). For better clarity the 1H-NMR signals of the acetyl methyl groups are connected to the formula.
intensity of individual acetyl groups in acetylglucuronoxylans. The
corresponding signals in the anomeric region of the spectrum have
already been assigned [4–6], however, the complete assignment of
the signals of methyl group protons of the acetyl groups is one of
the subjects of the present work. These signals of the methyl groups
are of much higher intensity that the relevant signals in the anomeric
region, and therefore they enable an easy study of the positional spec-
ificity of the deacetylases. This is demonstrated here on example of
AcXEs from Streptomyces lividans [14] and Orpinomyces sp. [15]. The
results obtained in NMR experiments are complemented with the
data on ability of the enzymes to precipitate acetylglucuronoxylan
from solution and on their positional specificity on artificial substrates.
The precipitation of acetylglucuronoxylan due to deacetylation by SlCE4
and the mode of action of SlCE4 on artificial substrates have been
published earlier [12,8]. The SlCE4 showed negligible activity on fully
acetylated and 2,3-di-O-acetylated MeXylp, and 2,4-di-O-acetyl and
3,4-di-O-acetyl-MeXylp were deacetylated at positions 2 and 3 several
orders faster that the above mentioned compounds with acetyl group
at both positions 2 and 3 [16]. Replacement of the free hydroxyl
group in 2,4-di-O- and 3,4-di-O-acetyl derivatives by hydrogen or
fluorine resulted in dramatic reduction of the rate of deacetylation
[16]. These data pointed out that the SlCE4 requires free vicinal
hydroxyl group for efficient deacetylation of position 2 or 3. This con-
clusion found strong support in structural studies [17]. The enzyme
behaved differently on monoacetyl derivatives of 4-nitrophenyl
β-D-xylopyranoside, showing a high preference for the 2-acetyl
derivative [12]. In this respect the Orpinomyces AcXE was examined
here for the first time.
2. Materials and methods
2.1. Acetylated carbohydrates
Beechwood acetylglucuronoxylan was isolated from delignified
pulp by DMSO extraction [18]. Birchwood acetylglucuronoxylan pre-
pared by steam explosion of birch saw dust was supplied by Dr. Henry
Schneider (NRCC, Ottawa, Canada). The steam explosion leads to reduc-
tion of the molecular mass of the polysaccharide which makes it more
suitable for the NMR study. The 13C-NMR spectrum of this material be-
fore and after chemical deacetylation was published earlier [19]. In the
present work the chemical deacetylation of the polysaccharide was
done in D2O solution of 0.1 M sodium hydroxide at room temperature
overnight. The alkaline solution of the deacetylated polysaccharide
was neutralized with 1 M solution of deuterized acetic acid (Aldrich
Chemicals, USA) in D2O before the NMR measurement.
2-O-acetyl-, 3-O-acetyl and 4-O-acetyl 4-nitrophenyl β-D-
xylopyranoside were synthetized as described [20]. Di-O-acetates
(2,3-, 2,4- and 3,4-) of Me-β-Xylp were generous gifts from Dr. P.
Kovac (National Institute of Health, Bethesda, MD, USA), Dr. J. Hirsch
(Institute of Chemistry, Slovak Academy of Sciences) and Dr. A.
Fernandes-Mayorales (Instituto de Quimica Organica General, CSIC,
Madrid, Spain).
2.2. Enzymes
AcXE of CE4 family from S. lividans (SlCE4) [14] was supplied with
Dr. Dieter Kluepfel (Institute of Armand Frappier, Laval, Canada). CE6

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3367
2,3-di-O-Ac
2,4-di-O-Ac
3,4-di-O-Ac
2,3,4-tri-O-Ac
3-O-Ac
4-O-Ac
de-O-Ac
0
2
5
10 20 30
0
2
5
10 20 30
0
2
5
10 20 30
0
2
5 10 20 30
Time (min)
Fig. 2. Deacetylation of Me-Xylp diacetates and triacetate with Orpinomyces sp. AcXE followed by TLC.
AcXE from Orpinomyces sp. was from Megazyme Int. (Ireland). It is a
recombinant enzyme free of other xylanolytic enzymes and, in con-
trast to the SlCE4, active on 4-nitrophenyl acetate. Before use in the
NMR experiment, the enzymes were desalted by membrane filtration
with two changes of D2O followed by twofold lyophilization from
D2O. The auxiliary β-xylosidase was the Aspergillus niger GH3 enzyme
produced in recombinant Saccharomyces cerevisiae as reported [21].
DB NB probe head and on VNMRS 600 MHz Varian spectrometer
equipped with 5 mm 1H–19 F/15 N–31P PFG One NMR Probe with an au-
tomatic chemical shifts calibration. Advanced techniques from Varian
pulse sequence library of 2D homo- and hetero-correlated spectroscopy
including sequences with selective excitations were used for the signal
assignments.
The following NMR spectra were obtained on 400 MHz spectrometer:
1H-NMR spectra (acquired using PRESAT sequence, presaturation delay
2 s, r.f. 90°pulse, acquisition time 2.5 s, number of transients 32), 2D
1H–1H homonuclear COSY spectrum (relaxation delay 1 s, acquisition
time 0.35 s, the data size of 1024 in both, t1 and t2), TOCSY spectra
(τmix =0.08 s and τmix =0.15 s, relaxation delay 1 s, acquisition time
2.3. Action of AcXEs on acetylated carbohydrates
The ability of the AcXEs to precipitate beech acetylglucuronoxylan
from solution (0.5%, w/v) in 0.1 M sodium phosphate buffer (pH 6.0)
was tested at 25 °C. The positional specificity of the CE6 enzyme was
examined on the monoacetates of 4-nitrophenyl β-D-xylopyranoside
in the β-xylosidase-coupled assay [21]. The deacetylation of the triac-
etate and diacetates of Me-β-Xylp at 10 mM concentration in 0.1 M
sodium phosphate buffer (pH 6.0) with the enzyme (0.4 mg/ml) at
40 °C was followed by TLC on silica gel (Merck, Germany) in ethyl
acetate–benzene–isopropanol (2:1:0.1, v/v). Sugars were detected
with N-(1-naphthyl)ethylenediamine dihydrochloride reagent [22].
The positional specificity of the enzymes on birch acetylglucuronoxylan
was examined by 1H-NMR. 10 mg of the polysaccharide, twice evapo-
rated from D2O, was dissolved in 0.65 ml D2O, the pH of the solution
was adjusted to 6.0 with 0.2 M solution of deuterized sodium acetate
(Aldrich Chemicals, USA) in D2O. After recording the 1H-NMR spectrum
of the starting polysaccharide, AcXEs desalted and lyophilized twice
from D2O were added (SlCE4 at 0.045 mg/ml and OCE6 at 0.3 mg/ml)
and the changes in signal intensity of the acetyl groups were monitored
in time course.
0.35 s, the data size of 2048 in t1×1024 in t2), NOESY spectrum (τmix
=
0.09 s, relaxation delay 1.5 s, acquisition time 0.45 s, the data size 1024
in both, t1 and t2), and 1H–13C heteronuclear HSQC spectrum (relaxation
delay 0.5 s, acquisition time 0.19 s, the data size 2048 in t1×1024 in t2,
1
with optimization on JCH =146 Hz) and HMBC spectrum (relaxation
n
delay 0.5 s, acquisition time 0.15 s, optimization on JCH =8 Hz long
range coupling constant, the data size 2048 in t1×2048 in t2). The
selective HMBC spectrum was measured on 600 MHz spectrometer
n
(relaxation delay 0.5 s, acquisition time 0.15 s, optimization on JCH
=
8 Hz long range coupling constant, the data size 2048 in t1×2048 in t2).
The spectral widths employed in all 2D NMR experiments were typically
1600 Hz for proton and 14,000 Hz (HSQC spectrum) or 20,000 Hz
(HMBC spectrum) for carbon.
3. Results
3.1. Action of Orpinomyces AcXE on acetylated polysaccharide and glycosides
2.4. NMR measurements
The Orpinomyces CE6 AcXE exhibited similarly as other typical
AcXEs ability to precipitate beechwood acetylglucuronoxylan from
solution as a result of liberation of the acetyl groups. Examination of po-
sitional specificity of the enzyme on monoacetates of 4-nitrophenyl
NMR spectra were measured at 25 °C in D2O on VNMRS 400 MHz
Varian spectrometer equipped with 5 mm 1H–19 F/15 N–31P PFG AutoX
Table 1
1H and 13C NMR data (δ, ppm) of acetylglucuronoxylan at 25 °C in D2O. The following designations are used: Xyl-(Xyl), non-acetylated Xylp; Xyl-(Xyl-Ac), Xylp linked to acetylated
Xylp; Xyl-3Ac, 3-O-acetylated Xylp; Xyl-2Ac, 2-O-acetylated Xylp; Xyl-2,3Ac, 2,3-di-O-acetylated Xylp; Xyl-3Ac-2MeGlcA, 3-O-acetylated Xylp 2-O-linked with MeGlcA.
Structural unit
H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5,5′/C-5
CH3-CO\
OCH3
Xyl-(Xyl)
Xyl-(Xyl-Ac)
Xyl-3Ac
4.44/101.76
4.38/102.74
4.54/101.44
4.66/99.85
4.79/99.47
4.68/101.03
5.26/97.13
3.25/72.67
3.18/72.25
3.45/70.95
4.66/73.44
4.79/71.42
3.66/74.82
3.53/72.53
3.52/73.58
3.49/71.21
4.96/75.27
3.76/71.46
5.14/72.96
5.04/73.87
3.81/72.51
3.76/76.35
3.72/76.50
3.91/75.55
3.83/76.10
4.02/75.23
3.96/75.56
3.16/82.23
3.35, 4.07/62.99
3.34, 4.03/63.07
3.44, 4.11/63.02
3.40, 4.13/62.76
3.50, 4.17/62.52
3.46, 4.10/62.95
4.32, −/72.16
–
–
–
–
2.13/20.55
2.14/20.45
2.07,2.08/20.29
2.19/21.06
–
–
Xyl-2Ac
–
Xyl-2,3Ac
Xyl-3Ac-2MeGlcA
MeGlcA
–
–
3,42/60,09

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region of the 1H-NMR spectrum the following signals of Xylp residues
were present: 2-O-acetyl Xylp residue H-1 and H-2, overlapped at
4.66 ppm; 3-O-acetyl-Xylp residue H-1 and H-3 at 4.54 ppm and
4.96 ppm, respectively; 3-O-acetyl-Xylp residue with 2-O-linked
MeGlcA H-1 and H-3 at 4.68 ppm and 5.04 ppm, respectively;
2,3-di-O-acetylated Xylp residue H-1 and H-2, overlapped at
4,79 ppm, and H-3 at 5.14 ppm. The skeletal proton assignment of
Xylp units agrees with literature data [4–6,24,25], however, the anal-
ysis of HMBC and NOESY spectra resulted in a different assignment of
the acetyl methyl groups signals. On the basis of H/C_O (proton of
the backbone/carbon of the acetyl carbonyl group) and C_O/CH3
(carbon of the acetyl carbonyl group/methyl of the acetyl group)
cross peaks observed in the selective HMBC spectrum (Fig. 1), proton
and carbon chemical shifts of differently acetylated Xylp residues and
the corresponding acetyl groups could be assigned (Table 2). NOESY spec-
trum (Fig. 3) confirmed this assignment with the H-2/CH3 cross-peak
of 2-O-acetyl-Xylp residue which appeared at 4.66/2.14 ppm, and
the H-3/CH3 cross-peak due to 3-O-acetyl-Xylp residue at 4.96/
2.13 ppm. On the basis of these data, signals of the methyl protons
of the acetyl groups at 2.07 and 2.08 ppm were attributed to
2,3-di-O-acetyl-Xylp residue. Methyl group signal at 2.13 ppm was
assigned to 3-O-acetyl-Xylp residue, while those at 2.14 and 2.19 ppm
belong to 2-O-acetyl-Xylp and to 3-O-acetyl-Xylp residue 2-O-substituted
with MeGlcA, respectively. The data are summarized in Table 2.
Table 2
The assignment of proton chemical shifts (δ, ppm) of signals of acetyl groups in acetylated
glucuronoxylan. The following designations are used: Xyl-3Ac, 3-O-acetylated Xylp;
Xyl-2Ac, 2-O-acetylated Xylp; Xyl-2,3Ac, 2, 3-di-O-acetylated Xylp; Xyl-3Ac-2MeGlcA,
3-O-acetylated Xylp 2-O-linked with MeGlcA.
Structural unit
HMBC
NOESY
H/CH3
H/C_O
C_O/CH3
Xyl-3Ac
Xyl-2Ac
Xyl-2,3Ac
H-3 4.96/173.79
H-2 4.66/173.66
H-2 4.79/173.57
H-3 5.14/173.70
H-3 5.04/173.82
173.79/2.13
173.66/2.14
173.57/2.07
173.70/2.08
173.82/2.19
H-3 4.96/2.13
H-2 4.66/2.14
n.d.
Xyl-3Ac-2MeGlcA
n.d.
β-D-xylopyranoside gave quite surprising results. In contrast to other
AcXEs capable to precipitate acetylated beechwood xylan from solu-
tion and showing strong preference for deacetylation of 2-O-acetyl
β-D-xylopyranoside [12], the ratio of specific activities of the studied
enzyme towards 4 mM 2-O-, 3-O- and 4-O-acetyl derivative was
1.0:0.51:0.048. The action of the enzyme was also examined on di-O-
and tri-O-acetyl derivatives of methyl β-D-xylopyranoside. A visual
evaluation of the rate of hydrolysis of the derivatives followed by TLC
indicated only a slight enzyme preference for attacking position 2 in
2,4-di-O-Ac-MeXylp and 2,3-di-O-Ac-MeXylp (Fig. 2). Fully acetylated
MeXylp was deacetylated somewhat faster than the above diacetates.
The Orpinomyces CE6 AcXE obviously does not have any requirement
for free vicinal hydroxyl group as the CE4 AcXEs do [16,17,23].
The fact that 1H-NMR spectroscopy enables to distinguish posi-
tions and environments of acetyl groups in acetylglucuronoxylan
prompted us to examine in this way the positional specificity and
mode of action of AcXEs on natural substrate dissolved in D2O. This
was done by monitoring changes in intensity of signals observed in
the region 5.3–4.35 ppm (H-1 signals and skeletal protons of acety-
lated Xylp residues) and signals of acetyl group methyl protons un-
ambiguously assigned in this work. Because, H-1 and H-2 signals of
2,3-di-O-acetylated Xylp residues (4.79 ppm) were hidden by residual
3.2. Assignment of resonances related to the acetyl groups
For a structural analysis of acetylglucuronoxylan used as a sub-
strate of AcXEs, 1D and 2D NMR experiments were used. Analysis of
the spectra resulted in a complete assignment of proton and carbon
chemical shifts. The data are reported in Table 1. In the anomeric
F1
(ppm)
Xyl-3Ac, Xyl-2Ac,
H-3/CH3 H-2/CH3
2.5
3.0
3.5
4.0
4.5
5.0
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
F2 (ppm)
Fig. 3. NOESY spectrum of hardwood acetylglucuronoxylan showing the H-2/CH3 cross-peak of 2-O-acetyl-Xylp residue and the H-3/CH3 cross-peak of 3-O-acetyl-Xylp residue. The
correlations are marked by vertical and horizontal lines in the upper left corner. The corresponding resonances, derived from a considerably magnified spectrum, are shown in
Table 2.

I. Uhliariková et al. / Biochimica et Biophysica Acta 1830 (2013) 3365–3372
3369
A
B
Chemically
deacetylated
Chemically
deacetylated
Free acetate
*
*
X
5h
5h
X
X
X
1h
0h
1h
0h
H D O
5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3
2.30 2.25 2.20 2.15 2.10 2.05 2.00 1.95 1.90
f1 (ppm)
f1 (ppm)
Fig. 4. Monitoring of the action of Streptomyces lividans CE4 AcXE on birch acetylglucuronoxylan by 1H-NMR spectroscopy. The changes of signal intensity of acetylated Xylp res-
idues in the anomeric region of the spectrum are shown in Part A, the changes of intensity of the methyl protons of the acetyl groups are shown in Part B. The time of incubation in
hours is indicated. The most rapidly decreasing signals are marked with thick arrows, slower decreasing signals with thinner arrows. Signals of protons not influenced by the en-
zyme are marked with crossed arrows. The spectrum of the chemically deacetylated polysaccharide is shown on the top of Part A, B. The following designations are used: Xyl,
non-acetylated Xylp; Xyl-3Ac, 3-O-acetylated Xylp; Xyl-2Ac, 2-O-acetylated Xylp; Xyl-2,3Ac, 2,3-di-O-acetylated Xylp; Xyl-3Ac-2MeGlcA, 3-O-acetylated Xylp 2-O-substituted
with MeGlcA. The H-1 signals of the reducing end Xyl residues, αXylred at 5.15 ppm and βXylred at 4.55 ppm, are marked by asterisk.
water signal, its H-3 signal (5.14 ppm) was used for monitoring of the
deacetylation reaction. The anomeric region of the spectrum also con-
tains signals of non-acetylated Xyl at the reducing end (αXylred at
5.15 ppm and βXylred at 4.55 ppm) which are visible in the 1H-NMR
spectrum of the alkali deacetylated glucuronoxylan (Fig. 4).
3.3. Positional specificity of S. lividans AcXE
Fig. 4 shows that S. lividans AcXE StCE4 releases acetyl groups from
position 2 and position 3, but only from singly acetylated Xylp residues.
The intensity of the resonance corresponding to 2-O-acetylated Xylp
25
120
B
A
100
20
80
60
40
20
0
15
10
5
0
0
1
2
3
4
0
1
2
3
4
Time (h)
Time (h)
Fig. 5. Time course of the changes in intensity of 1H-NMR signals of acetylated and non-acetylated Xylp residues in the anomeric region of the spectrum (Part A) and signals of the
methyl protons of acetyl group (Part B) during incubation of birch acetylglucuronoxylan with S. lividans CE4 AcXE. Symbols of xylopyranosyl residues: 5, internal non-acetylated;
●, 3-O-acetyl; o, 2-O-acetyl; ▲, 2,3-di-O-acetyl, △ 3-O-acetyl-2-O-MeGlcA. The values of the signal of non-acetylated Xylp residues in part A (5) were divided by 2 to make the
graph more illustrative.

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I. Uhliariková et al. / Biochimica et Biophysica Acta 1830 (2013) 3365–3372
A
B
Free acetate
Chemically
Chemically
deacetylated
deacetylated
5h
5h
1h
0h
X
X
*
*
1h
0h
5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4
2.30 2.25 2.20 2.15 2.10 2.05 2.00 1.95 1.90
f1 (ppm)
f1 (ppm)
Fig. 6. Monitoring of the action of Orpinomyces CE6 AcXE on birch acetylglucuronoxylan by 1H-NMR spectroscopy. The changes of signal intensity of acetylated Xylp residues in the
anomeric region of the spectrum are shown in Part A, the changes of intensity of the methyl protons of the acetyl groups are shown in Part B. The time of incubation in hours is
indicated. The most rapidly decreasing signals are marked with thick arrows, slower decreasing signals with thinner arrows. Signals of protons not influenced by the enzyme
are marked with crossed arrows. The spectrum of the chemically deacetylated polysaccharide is shown on the top of Part A, B. The following designations are used: Xyl,
non-acetylated Xylp; Xyl-3Ac, 3-O-acetylated Xylp; Xyl-2Ac, 2-O-acetylated Xylp; Xyl-2,3Ac, 2,3-di-O-acetylated Xylp; Xyl-3Ac-2MeGlcA, 3-O-acetylated Xylp 2-O-linked with
MeGlcA. The H-1 signals of the reducing end Xyl residues, αXylred at 5.15 ppm and βXylred at 4.55 ppm, are marked by asterisk.
residues decreased faster than the resonances of 3-O-acetylated resi-
dues (Fig. 5). A clear sign of the enzyme performance is the remarkable
increase of the H-1 signal of completely deacetylated internal Xylp res-
idues at 4.44 ppm. The changes in the intensity of signals in the
anomeric region were in accordance with changes of the intensity of
signals of methyl protons in the acetyl groups (Fig. 4 and Fig. 5). The en-
zyme did not attack 2,3-di-O-acetylated xylopyranosyl residues and the
3-O-acetyl group on xylopyranosyl residues α-1,2-substituted with
4-O-methyl-D-glucuronic acid.
3.4. Positional specificity of Orpinomyces AcXE
Action of the CE6 AcXE on acetylglucuronoxylan is shown in Fig. 6
and Fig. 7. The favorite targets of the enzyme are 2,3-di-O-acetylated
Xylp residues and 2-O-acetyl-Xylp residues. A somewhat slower de-
crease of the signal of 3-O-acetyl Xylp residue could be explained by
generation of this type of acetylated Xylp from 2,3-di-O-acetylated res-
idue after removal of the acetyl group from position 2. However, the en-
zyme similarly as SlCE4 does not recognize the 3-O-acetyl group on Xylp
8
35
A
B
7
30
6
5
4
3
2
1
0
25
20
15
10
5
0
0
1
2
3
4
5
0
1
2
3
4
5
Time (h)
Time (h)
Fig. 7. Time course of the decrease in intensity of 1H-NMR signals of acetylated and non-acetylated Xylp residues in the anomeric region of the spectrum (Part A) and signals of the
methyl protons of acetyl group (Part B) during incubation of birch acetylglucuronoxylan with Orpinomyces sp. AcXE. Symbols of xylopyranosyl residues: 5, internal
non-acetylated;●, 3-O-acetyl; o, 2-O-acetyl;▲, 2,3-di-O-acetyl, △, 3-O-acetyl-2-O-MeGlcA. The values of the signal of non-acetylated Xylp residues in part A (5) were divided
by 2 to make the graph more illustrative.

I. Uhliariková et al. / Biochimica et Biophysica Acta 1830 (2013) 3365–3372
3371
residues 2-O-substituted with MeGlcA. The intensity of the correspond-
ing signal at 2.19 and 5.04 ppm did not change with time. Thus MeGlcA
appears to be a serious steric barrier for OCE6 because the enzyme does
not require a free vicinal hydroxyl group as CE4 AcXEs. The enzyme tol-
erates well another acetyl group at the vicinal position.
residues α-1,2-substituted with MeGlcA. The 1H-NMR spectroscopy ap-
proach to study positional and substrate specificity of AcXEs outlined in
this work appears to be a simple way to characterize catalytic properties
of enzymes belonging to various CE families. Particularly clear data are
generated by following the changes in the signals of the acetyl methyl
groups. A detailed knowledge of the mode of action of polysaccharide
deacetylating enzymes is important in view of the development of effi-
cient bioconversion of plant materials that did not undergo alkaline
pretreatment leading to hydrolysis of ester linkages.
4. Discussion
In accord with the requirement for a free vicinal hydroxyl group of
the CE4 enzymes for deacetylation of position 2 or 3 [16,17,23], Sl CE4
does not recognize as its targets the doubly acetylated xylose residues
and also the 3-O-acetylated residues substituted by MeGlcA. The in-
tensity of the corresponding resonances in the anomeric region of
the spectrum and in the region of signals of the acetyl methyl group
did not change during several hours (Fig. 5). The fact that the SlCE4
deacetylates both positions 2 and 3 in monoacetylated Xylp residues
is in contrast to the high selectivity of the enzyme for position 2 in
monoacetates of 4-nitrophenyl xylopyranoside [12]. However, SlCE4
was shown to be capable of deacetylating positions 2 and 3 in 2,4-
and 3,4-di-O-acetyl-methyl β-D-xylopyranoside [16]. A question re-
mains therefore to be answered whether the deacetylation of both
positions is a consequence of acetyl group migration from position 3
to position 2 prior the nucleophile attack on the ester carbonyl
group [11,8], or whether the polysaccharide can form with the en-
zymes productive complex also in orientation by 180° reversed to
that in which the position 2 is deacetylated [8]. The formation of
such two productive complexes was suggested for acetylated MeXylp
when the 3D structure of Trichoderma reesei CE5 AcXE was
established [11]. The stereochemistry of the position 2 and 3 on
Xylp residue in both orientations is the same [8]. Since the formation
of the reverse productive complex can be excluded with CE4 chitin
deacetylases hydrolyzing the amide of GlcNH2 exclusively at the posi-
tion 2 [26–28], we hypothesize that the deacetylation of both posi-
tions 2 and 3 by SlCE4 takes place at the same orientation of the
substrate in the enzyme–substrate complex. In the case the enzyme
facilitated acetyl group migration would not be involved in the mech-
anism of deacetylation of the two positions, the enzyme should be
able to form productive complexes with both 2-O- and 3-O-acetylated
Xylp residues at the same orientation of the xylan main chain.
Similar considerations concern the mode of action of OCE6. In
acetylglucuronoxylan the enzyme attacks preferentially position 2 in
both mono- and di-O-acetylated Xylp residues. The rate of deacetylation
of position 3 stays only slightly behind, perhaps due to generation of the
3-O-acetylated Xylp by preferential removal of the acetate from the
2-position of the 2,3-di-O-acetylated residues. This mode of action is
in accord with substrate specificity of the enzyme on acetylated deriva-
tives of MeXylp and monoacetyl derivatives of 4-nitrophenyl Xylp.
Anyway, the ability of the CE6 enzyme, belonging to the serine-type
esterases [15,29], to deacetylate both position 2 and 3 remains
difficult to be explained similarly as in the case of SlCE4, an aspartate
metalloenzyme [14,30]. Thus, the formation of two different spatially
distorted enzyme-substrate complexes with the same xylan main
chain orientation cannot be excluded also in the case of the CE6 AcXE.
More detailed studies of the reaction mechanisms and particularly
three-dimensional structures of the enzymes with appropriate ligands
are required to answer these questions.
Acknowledgements
The authors are grateful to Mrs. Mária Cziszárová for excellent
technical assistance. This work was supported by the grants from
the Slovak Academy of Sciences grant agency VEGA 2/0001/10 and
VEGA 2/0007/13, and also by the Research & Development Operation-
al Programme ITMS 26220120054 funded by the ERDF.
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