Positional specificity of Flavobacterium johnsoniae acetylxylan esterase and acetyl group migration on xylan main chain

Article abstract of DOI:10.1016/j.carbpol.2019.115783

A new Flavovacterium johnsoniae isolate encodes an enzyme that is essentially identical with a recently discovered novel acetylxylan esterase, capable of liberating 3-O-acetyl group from 4-O-methyl-D-glucuronic acid-substituted xylopyranosyl (Xylp) residues (Razeq et al., 2018). In addition to deesterification of the 2-O-MeGlcA-substituted Xylp residues in acetylglucuronoxylan, the enzyme acts equally well on doubly acetylated Xylp residues from which it liberates only the 3-O-acetyl groups, leaving the 2-O-acetyl groups untouched. 3-O-Monoacetylated Xylp residues are attacked with a significantly reduced affinity. The resulting 2-O-acetylated xylan was used to investigate for the first time the migration of the 2-O-acetyl group to position 3 within the polysaccharide. In contrast to easy acetyl group migration along the monomeric xylopyranosides or non-reducing-end terminal Xylp residues of xylooligosaccharides, such a migration in the polymer required much longer heating at 100 °C. The specificity of the xylan 3-O-deacetylase was, however, no so strict on acetylated methyl and 4-nitrophenyl xylopyranosides.

Full text of DOI:10.1016/j.carbpol.2019.115783

Journal Pre-proof
Positional specificity of Flavobacterium johnsoniae acetylxylan esterase
and acetyl group migration on xylan main chain
´
´
Vladim´ır Puchart, Morten Gjermansen, Maria Mastihubova, Kristian
B.R. Mørkeberg Krogh, Peter Biely
PII:
S0144-8617(19)31451-1
DOI:
https://doi.org/10.1016/j.carbpol.2019.115783
CARP 115783
Reference:
To appear in:
Carbohydrate Polymers
Received Date:
Revised Date:
Accepted Date:
17 October 2019
11 December 2019
24 December 2019
´
Please cite this article as: Puchart V, Gjermansen M, Mastihubova M, Mørkeberg Krogh KBR,
Biely P, Positional specificity of Flavobacterium johnsoniae acetylxylan esterase and acetyl
group migration on xylan main chain, Carbohydrate Polymers (2019),
doi: https://doi.org/10.1016/j.carbpol.2019.115783
This is a PDF file of an article that has undergone enhancements after acceptance, such as
the addition of a cover page and metadata, and formatting for readability, but it is not yet the
definitive version of record. This version will undergo additional copyediting, typesetting and
review before it is published in its final form, but we are providing this version to give early
visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal
pertain.
© 2019 Published by Elsevier.

Positional specificity of Flavobacterium johnsoniae acetylxylan esterase and acetyl group
migration on xylan main chain
Vladimír Pucharta *, Morten Gjermansenb, Mária Mastihubováa, Kristian B. R. Mørkeberg
Kroghb, Peter Bielya
a Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9,
SK-845 38 Bratislava, Slovak Republic
b Novozymes A/S, Kogshoejvej 36, DK-2880 Bagsvaerd, Denmark
E mail: addresses:
vladimir.puchart@savba.sk
mgjm@novozymes.com
maria.mastihubova@savba.sk
KBK@novozymes.com
peter.biely@savba.sk
* Correspondence author:
Phone: +421-2-59410229
Fax: +421-2-59410222
E-mail: vladimir.puchart@savba.sk
Highlights
 This is the second study of a novel unclassified acetylxylan 3-O-deacetylase
 The acetylxylan esterase is a product of a new Flavobacterium johnsoniae isolate
 The xylan 3-O-deacetylation proceeds most rapidly on 2-O-substituted Xylp residues
 The enzyme converts acetylglucuronoxylan to 2-O-acetylated polysaccharide
 The latter served for the first time to study acetyl group migration in the polymer
1

Abstract
A new Flavovacterium johnsoniae isolate encodes an enzyme that is essentially identical with
a recently discovered novel acetylxylan esterase, capable of liberating 3-O-acetyl group from
4-O-methyl-D-glucuronic acid-substituted xylopyranosyl residues (Xylp) (Razeq et al., 2018).
In addition to deesterification of the 2-O-MeGlcA-substituted Xylp residues in
acetylglucuronoxylan, the enzyme acts equally well on doubly acetylated Xylp residues from
which it liberates only the 3-O-acetyl groups, leaving the 2-O-acetyl groups untouched. 3-
O-Monoacetylated Xylp residues are attacked with a significantly reduced affinity. The
resulting 2-O-acetylated xylan was used to investigate for the first time the migration of the 2-
O-acetyl group to position 3 within the polysaccharide. In contrast to easy acetyl group
migration along the monomeric xylopyranosides or non-reducing-end terminal Xylp residues
of xylooligosaccharides, such a migration in the polymer required much longer heating at
100 °C. The specificity of the xylan 3-O-deacetylase was, however, no so strict on acetylated
methyl xylopyranosides.
Keywords: hardwood acetylglucuronoxylan; novel acetylxylan esterase; novel
regiospecificity; 3-O-deacetylase; 2-O-acetyglucuronoxylan; acetyl group migration
Abbreviations
CE, carbohydrate esterase; MeGlcA; 4-O-methyl--D-glucopyranosyluronic acid; Xylp,
-D-xylopyranosyl;
2

1. INTRODUCTION
Major hemicellulose in hardwoods is acetylglucuronoxylan. It is a polysaccharide with
main chain of -1,4-linked xylopyranosyl (Xylp) residues, 10 % of which carry 4-O-methyl-
-D-glucopyranuronosyl side residues attached to position 2. About 60 % of Xylp residues
are acetylated. Major portion is monoacetylated at position 2 and 3 and minor portion is 2,3-
di-O-acetylated. Recent observations suggested that in various hardwood xylans 3-O-acetyl
group occurs on almost all MeGlcA-substituted Xylp residues (Evtuguin, Tomás, Silva, &
Neto, 2003; Naran, Black, Decker, & Azadi, 2009; Puchart, Mørkeberg Krogh, & Biely,
2019; Teleman, Lundqvist, Tjerneld, Stålbrand, & Dahlman, 2000). Thus, there are four types
of acetylated Xylp residues within the xylan chain: 2-O-monoacetylated,
3-O-monoacetylated, 2,3-di-O-acetylated and 3-O-acetylated-2-MeGlcA-glycosylated
(Fig. 1).
Fig. 1 Hypothetical hardwood acetylglucuronoxylan containing four types of acetylated Xylp
residues (the middle formula) and their deacetylation by AcXEs belonging to CE1. CE4, CE5
and CE6 families. AcXEs of all four CE families are unable to release the 3-O-acetyl on
MeGlcA-subtituted Xylp residues, and AcXEs of family CE4 attack neither doubly acetylated
Xylp residues. The upper polysaccharide is acetylated exclusively at the 3-position of
MeGlcA-substituted Xylp residues.
3

Microbial decomposition of such a complex polysaccharide by glycoside hydrolases,
namely endo--1,4-xylanases (EC 3.2.1.8, 3.2.1.136), -xylosidases (EC 3.2.1.37) and
-glucuronidases (EC 3.2.1.131, 3.2.1.139), requires also polysaccharide deesterification
catalyzed by acetylxylan esterases. They are a quite diverse group of enzymes differing in
primary and tertiary structures as well as in regiospecificity. In Carbohydrate Active enZymes
(CAZy) database they are classified in carbohydrate esterase (CE) families 1, 4, 5, 6 and 16
(Lombard, Golaconda Ramulu, Drula, Coutinho, & Henrissat, 2014). The members of these
families differ slightly in the mode of action on singly and doubly acetylated Xylp residues,
however, enzymes of neither mentioned families were found capable of liberating the acetyl
group from position 3 on Xylp residue MeGlcA-glycosylated at position 2 (Fig. 1) (Biely et
al., 2012). These heterogeneously doubly substituted Xylp residues represent the most
recalcitrant structural arrangement in the acetylated polysaccharide. MeGlcA is a steric barrier
for AcXEs and the 3-O-acetyl interferes with the action of α-glucuronidase. In view of the
yields of hardwood xylan saccharification such a new deacetylase could play an important
role, since its absence in enzyme cocktails may leave almost one third of the polysaccharide
in the form of enzyme resistant tetrasaccharide (Biely, Singh, & Puchart, 2016).
Recently a new AcXE was identified in a xylan utilization locus of Flavobacterium
johnsoniae UW101 which showed capability of attacking the 3-O-acetyl group vicinal to the
MeGlcA side residue (Razeq et al., 2018). A clear experimental evidence for this action was
obtained by 1H-NMR spectroscopy. Attracted by this new development in the area of
glucuronoxylan deacetylation, but also due to an incomplete interpretation of the changes in
the acetyl group signals in the NMR spectra caused by the enzyme (Razeq et al., 2018), we
have prepared highly similar recombinant enzyme (98.6% identity) from a different
F. johnsoniae strain. The enzyme was applied to glucuronoxylan acetylated solely at
MeGlcA-substituted Xylp residues. Moreover, the regiospecificity of the enzyme was
reexamined on aspen acetylglucuronoxylan extracted by steam explosion. The results
obtained were similar as those reported by Razeq et al. (2018) on a mixture of acetylated
aldouronic acids, however, our NMR signal assignments also expand current knowledge on
the mode of action of these novel type AcXEs on singly and doubly acetylated Xylp residues.
The action of the enzyme resulted in a 2-O-acetylated polysaccharide which was exploited to
study for the first time the acetyl group migration between position 2 and 3 on internal Xylp
residues. The positional specificity of the enzyme on the polymeric substrate was also
4

compared with its action on synthetic substrates, partially and fully acetylated β-
xylopyranosides.
2. METHODS
2.1. Substrates
All acetates derived from methyl -D-xylopyranoside (Puchart et al., 2016, and
references therein) and its deoxy- and deoxy-fluoro- analogues (Mastihubová & Biely, 2004b)
as well as monoacetates of 4-nitrophenyl -D-xylopyranoside (Mastihubová & Biely, 2004a)
have been synthesized previously. Isolation of steam explosion extracted aspen
acetylglucuronoxylan has also been reported (Biely et al., 2013; Horn, Nguyen, Westereng,
Nilsen, & Eijsink, 2011). This hemicellulose was used as a substrate for Orpinomyces sp. CE6
AcXE (Megazyme, Bray, Ireland)-catalyzed deacetylation according to published procedure
(Puchart, Mørkeberg Krogh, & Biely, 2019) that resulted in a polysaccharide selectively
3-O-acetylated at MeGlcA-substituted Xylp residues. Monosaccharide composition as well as
acetic acid and MeGlcA content of the polysaccharide have been published (Puchart,
Mørkeberg Krogh, & Biely, 2019). The degree of polymerization was estimated to be ca. 35
based on the reducing sugar determination.
2.2. Cloning and expression of an acetylxylan esterase from Flavobacterium johnsoniae
1WA
The gene encoding the acetylxylan esterase (GenBank accession number: MN306533)
from the bacterium Flavobacterium johnsoniae 1WA which was originally isolated from a
soil sample collected in the USA, was codon optimized for Bacillus subtilis codon usage via a
publicly available codon optimizer tool (Raab, Graf, Notka, Schödl, & Wagner, 2010). The
resulting optimized gene was further modified by exchanging the coding region for the
natural signal peptide with the coding region from the Bacillus clausii AprE signal peptide
and by addition of a C-terminal 6×His affinity purification tag. The gene was then ordered as
a synthetic gene from the commercial provider GeneArt, Regensburg, Germany.
5

The synthetic gene was fused via overlap extension PCR to a strong triple promoter
consisting of the PamyL4199, PamyQsc and Pcry3A promoters in series (Jørgensen & Pedersen, 2015)
and flanking homology regions matching the B. subtilis pectate lyase gene. The resulting
expression cassette was inserted into the genomic pectate lyase locus of a B. subtilis host by
homologous recombination. The B. subtilis host strain used was protease deficient derivative
(Zhao et al., 2019) of Bacillus subtilis ATCC 6051, which had the following protease genes
deleted: nprE, aprE, wprA, bpr, vpr, mpr, epr and ispA. The recombinant B. subtilis clone
containing the integrated expression construct was selected on chloramphenicol and
subsequently cultivated for 48 hours in LB medium at 30 °C. The enzyme-containing
supernatant was harvested by centrifuging of the culture broth for 30 minutes at 15 000×g.
2.3. Purification of the enzyme
Harvested culture broth was separated from cellular material by passage through an
0.22 m PES filter (Nalgene), before addition of 3M TRIS to a final concentration of 85 mM.
The resulting solution was filtered through an 0.22 µm PES filter, and applied to a 5 ml
HisTrap Excel column (GE Healthcare) pre-equilibrated with 50 mM TRIS/HCl, pH 8.0. The
loaded column was washed with 2 column volumes of 50 mM HEPES, pH 7.0, to wash out
unbound proteins. The bound esterase was eluted with 50 mM HEPES containing 0.75 M
imidazole, pH 7.0. The collected fractions were analyzed by measuring A280 and A280/A260.
The esterase containing fractions were pooled and desalted by gel-filtration into 50 mM
HEPES, pH 7.0, containing 100 mM NaCl. The fractions collected in this step contained an
electrophoretically homogeneous protein according to SDS PAGE. The identity of the
isolated esterase to the expected acetylxylan esterase was confirmed by in-gel tryptic digest
followed by MS analysis.
2.4. Monitoring of polysaccharide deacetylation by NMR spectroscopy
Deacetylation of polymeric substrates was followed by 1H NMR spectroscopy.
Prior to use, the substrates, enzyme and phosphate buffer were three times freeze dried from
heavy water to ensure deuterium exchange of all ionisable proton atoms. The enzyme
reactions were carried out at 35 °C directly in narrow NMR tubes (outer diameter 2.5 mm).
The polysaccharide (2 mg) selectively acetylated at position 3 of MeGlcA-
glycosylated Xylp residues (Puchart, Mørkeberg Krogh, & Biely, 2019) was dissolved in
6

160 l of 15 mM deuterium-exchanged sodium phosphate buffer, pH 6.0, and after thermal
equilibration to 35 °C proton NMR spectrum was recorded. Then 40 g of enzyme was added
and the reaction was monitored by NMR spectroscopy.
Aspen acetylglucuronoxylan (2 mg) was dissolved in 150 l of 100 mM deuterium-
exchanged sodium phosphate buffer, pH 6.0, and initial proton NMR spectrum was recorded
at 35 °C. Then 6.3 g of the enzyme was added and the reaction course was followed by
proton NMR spectroscopy at 2-min intervals. After 80 minutes ten times higher amount of the
enzyme (63 g) was applied and the reaction was continued for 21 h.
2.5. Acetyl group migration on xylan main chain
The solution of 2-O-acetylated xylan prepared as described above, which did not show
any sign of acetyl group migration at 35 °C, was heated at 100 °C for several 5-10 min
intervals, and after cooling to 35 °C subjected to NMR spectroscopy at this temperature.
2.6. NMR spectroscopy
1H NMR spectra were recorded using automatic chemical shift calibration on
AVANCE III HD 400 MHz spectroscope equipped with a broad band BB-(H-F)-D-05-Z
liquid N2 Prodigy probe (both from Bruker BioSpin, Rheinstetten, Germany). The following
parameters were used: pre-saturation zgpr sequence, pre-saturation delay 2 s, r.f. 90° pulse,
and acquisition time 2.5 s.
2.7. Deacetylation of acetylated methyl -D-xylopyranosides
Stock solutions (50 mM) in dimethyl sulfoxide were prepared from the following
multiply acetylated derivatives of methyl -D-xylopyranoside: 2,3,4-tri-O-acetyl, 2,3-di-
O-acetyl, 2,4-di-O-acetyl, 3,4-di-O-acetyl, 2-deoxy-3,4-di-O-acetyl, 3-deoxy-2,4-di-O-acetyl,
2-deoxy-2-fluoro-3,4-di-O-acetyl, and 3-deoxy-3-fluoro-2,4-di-O-acetyl. Working 5 mM
solution of each was prepared by 10-fold dilution with 50 mM sodium phosphate buffer,
pH 6.0. After thermal equilibration the reaction was initiated by addition of the enzyme
(0.187 mg/ml, final concentration) and the reaction was performed at 35 °C and monitored by
7

TLC. On the TLC plate 8-l aliquots were spotted for deoxy-analogues and 4-l aliquots for
other substrates.
2.8. Deacetylation of isomeric monoacetates of 4-nitrophenyl -D-xylopyranoside
Stock solutions (200 mM) in dimethyl sulfoxide were prepared from the all the
monoacetates of 4-nitrophenyl -D-xylopyranoside, namely 2-O-acetyl, 3-O-acetyl and
4-O-acetyl. Working 4 mM solutions in 50 mM sodium phosphate buffer, pH 6.0, were
thermal equilibrated and supplied with the enzyme (final concentration: 212 ng/ml for TLC
experiment, 51.8 ng/ml for HPLC experiment). The reaction was performed at 35 °C and its
course analyzed by either TLC or HPLC.
2.9. HPLC
HPLC analysis was carried out at 35 °C using an MZ-AquaPerfect® C18 5 m
column (150×4 mm; MZ-Analysentechnik, Meinz, Germany) equipped with a
spectrophotometric detector operating at 275 nm. The column was eluted isocratically with a
solvent system of 60% acetonitrile (v/v) at a flow rate of 1 ml/min. Aliquots (10 l) of the
reaction mixtures were directly loaded at defined time intervals. Each chromatogram was
automatically peak-analyzed and area of the remaining substrate (acetylated 4-nitrophenyl
-D-xylopyranoside) and deacetylated product was integrated. Spontaneous deesterification
(control) was taken into consideration and the relative proportion of enzyme-catalyzed
product formation was used for specific activity calculation.
2.10. TLC
Deacetylation of low molecular weight synthetic compounds was analyzed by TLC
which was performed on aluminum-coated silica plates (Merck, Darmstadt, Germany). The
plates were developed twice in a solvent system of either ethyl acetate/toluene/isopropanol
(for methyl xyloside derivatives) or ethyl acetate/benzene/isopropanol (for 4-nitrophenyl
xyloside acetates) 2:1:0.1 (by vol.). The carbohydrates were visualized by orcinol reagent
(0.5% orcinol, 5% sulfuric acid in ethanol) after heating.
8

3. RESULTS
3.1. Amino acid sequence comparison
In this study 414 amino acid long protein encoded by F. johnsoniae strain 1WA was
recombinantly produced. Its amino acid sequence, not deposited yet, was compared with the
previously studied esterase (Razeq et al., 2018), originating from a strain F. johnsoniae
UW101. Both ORFs are of the same length and code for native proteins that are highly similar
(Supplementary Fig. 1S). Signal sequences (21 residues) are identical, but in the secreted
proteins there are 6 substitutions, 3 of which are conservative. All the substitutions are distant
from catalytically important amino acids, therefore they are expected hardly to affect
significantly catalytic properties. Both proteins, showing homology to none CEs classified in
CAZy database, contain a putative catalytic triad Ser217, Asp392 and His395 (native enzyme
numbering), and belong to a SGNH/GDSL hydrolase superfamily of proteins (CDD 238868).
In addition to the residues of serine esterase catalytic triad, other amino acids conserved in the
superfamily are Gly258 and Asn294. It is interesting that in the two proteins deposited in
GenBank database, having the same number of substitutions and originating from different
F. johnsoniae strains, some of the amino acid changes occur at the same loci as in our
enzyme.
3.2. Action on 3-O-acetylglucuronoxylan
The ability of the enzyme to deesterify 3-O-acetyl group in a vicinity of 2-O-linked
MeGlcA side chain was studied using a polymeric substrate, 3-O-acetylxylan specifically
acetylated only at position 3 on MeGlcA-substituted Xylp residues (Fig. 1, upper formula). In
the 1H-NMR spectrum of this polysaccharide there is a single ester peak in the region of
acetyl groups (Fig. 2). Its chemical shift corresponds to the 3-O-acetyl group in the vicinity of
2-O-linked MeGlcA. After addition of the enzyme this singlet completely disappeared, thus
confirming the crucial result of the previous report (Razeq et al., 2018) on the ability of F.
johnosniae UW101 AcXE to deesterify 3-O-acetyl group in the vicinity of MeGlcA residue.
The disappearance of the acetate signal was accompanied with changes in the skeletal proton
signals. The most obvious was a lack of a triplet at 5.04 ppm which was present in the starting
9

acetylated polymer and assigned to H-3 atom linked to C-3 carbon atom of Xylp residue
whose oxygen O-3 was acetylated. It is noteworthy that the intensity of H-5 doublet of
MeGlcA residue increased upon deacetylation.
Fig. 2. Deacetylation of 3-O-acetylglucuronoxylan by F. johnsoniae AcXE monitored by
1H-NMR spectroscopy. The reaction was performed at 35 °C in NMR tube containing 160 l
comprising 15 mM sodium phosphate, pH 6.0, 2 mg of the polysaccharide and 40 g of the
enzyme. The spectra on the right side is extension of the region with signals of the acetyl
methyl groups of acetate ester and acetic acid. Note. the starting polysaccharide contained
some acetic acid.
3.3. Action of the enzyme on polymeric acetylglucuronoxylan
10

To learn more about positional preferences for deesterification, the enzyme was
applied onto a steam explosion extracted hardwood acetylglucuronoxylan that had been used
for the preparation of the 3-O-acetylglucuronoxylan. In the native polysaccharide there are
four types of acetylated Xylp residues and the signals of these distinct acetyl groups have
been assigned previously (Fig. 3, bottom spectrum) (Uhliariková, Vršanská, McCleary, &
Biely, 2013). Upon enzyme addition, there was a rapid decrease in the intensity of the peaks
corresponding to doubly substituted Xylp residues, namely to 3-O-acetylated-2-O-(4-
O-methyl)glucuronylated (2.19 ppm) and 2,3-di-O-acetylated (2.07-2.08 ppm) (Fig. 3, the
second spectrum from bottom). The decrease in the intensity of the diacetate was
accompanied with an increase in the signal of 2-O-monoacetate (2.14 ppm), suggesting that
the doubly acetylated epitope is, at least preferentially, if not exclusively, deacetylated at
position 3.
AcOH
2-O-Ac
After the heat treatment,
+63 mg E, 200 min
3-O-Ac
Treated at 100 °C
for 45 min
+6.3 g E, 81 min,
then +63 g E, 180 min
+6.3 g E, 61 min
2,3-O-Ac2
3-O-Ac-2-MeGlcA
0 min
Fig. 3. 1H-NMR spectroscopy monitoring of the deacetylation of aspen acetylglucuronoxylan
by F. johnsoniae AcXE (the bottom three spectra), and of acetyl group migration upon a
treatment at 100 °C (the second spectrum from the top) from position 2 to position 3 in
glucuronoxylan exclusively monoacetylated at position 2. The top spectrum shows again the
11

selective removal of the 3-O-acetyl groups from thus obtained singly 2- and 3-O-acetylated
Xylp residues. The enzyme reactions were performed at 35 °C in NMR tube containing
160 l of 100 mM sodium phosphate buffer (pH 6.0) containing 2 mg of the polysaccharide
and the enzyme amount indicated.
The rates of the disappearance of the signals of 2,3-diacetate and 3-O-Ac-2-MeGlcA-
Xylp residue were similar and both peaks disappeared after about 80 minutes at the used
enzyme concentration. At this time the decrease of the 3-monoacetate signal was negligible,
while the signal of the 2-monoacetate clearly increased on account of the 2,3-diacetate 3-O-
deacetylation. Ten times higher enzyme load of the enzyme and 3 h long incubation were
required for complete deesterification of the 3-monoacetate (a peak at 2.13 ppm) (Fig. 3,
middle spectrum). However, no change in the signal intensity of the 2-monoacetate was
observed during further incubation for 24 h. These results serve as a clear evidence that: i) the
enzyme deacetylates 3-monoacetylated Xylp residues extremely slowly when compared with
its action on the 3-acetyl group on Xylp residues with the vicinal OH group either substituted
with MeGlcA or acetylated; ii) the 2-monoacetate is resistant to the enzyme attack; iii) the
acetyl group at position 2 does not migrate to position 3 at 35 °C. The enzyme treatment can
lead to two different patterns of glucuronoxylan acetylation (Fig. 4).
Fig. 4. Scheme of hardwood acetylglucuronoxylan composed of four types of acetylated Xylp
residues (middle formula) and its conversion to 2- and 3-monoacetylated polysaccharide
12

(upper formula) and to exclusively 2-O-acetylated polysaccharide (bottom formula) by F.
johnsoniae AcXE depending on the robustness of the enzymatic treatment.
3.4. Acetyl group migration studied on polysaccharide
The 2-O-monoacetylated glucuronoxylan, prepared by the action of F. johnsosniae
AcXE on steam explosion-extracted polymer, was for the first time used to study acetyl group
migration in the polysaccharide. It has previously been shown that such a migration is
common at ambient temperature in xylopyranosides (Mastihubová & Biely, 2004a) and quite
rapid at increased temperature at the non-reducing-end Xylp residue of methyl xylotrioside
(Puchart & Biely, 2015). In the latter compound the equilibrium of all three monoacetates (2-,
3- and 4-monoacetate) was established during a 5 min treatment at 100 °C regardless the
position of the acetyl group in the starting monoacetylated isomer. In the case of the
2-O-monoacetylated glucuronoxylan, after a 5-min heating at 100 °C less than 10 % of the 2-
O-acetyl group migrated to position 3. To reach equilibrium between 2-monoacetate and
slightly predominating 3-monoacetate, the heating at 100 °C had to be extended for 45-60
minutes (Fig. 3, the second spectrum from the top). It is also noteworthy that during the
equilibrium experiments no signal appeared corresponding to 2,3-diacetate or 3-O-acetylated-
2-O-glucuronylated Xylp residue. This serves as an evidence that the acetyl group migration
is strictly intracircular, i.e. it does not occur between two xylopyranoside rings.
After reaching the equilibrium between 2-monoacetate and 3-monoacetate (Fig. 3, the
second spectrum from the top), the polysaccharide was treated with fresh F. johnsoniae AcXE
in the same NMR tube. NMR spectroscopy confirmed again the selective 3-O-deacetylation.
The 3-acetyl group was completely removed after 3 hours, while the 2-monoacetate remained
intact even after 24 hours (Fig. 3, top spectrum).
3.5. Action of the enzyme on acetylated methyl xylopyranosides
The enzyme was also tested on fully and doubly acetylated methyl
-D-xylopyranosides. Two of them, 2,4- and 3,4-di-O-acetates, were also available in the
form of deoxy and deoxy-fluoro-analogues which were earlier used to characterize the mode
of action of AcXEs of families CE1, CE4 and CE5 (Biely & Mastihubová, 2004b; Biely,
Mastihubová, Côté, & Greene, 2003; Biely, Mastihubová, & Puchart, 2007). The
semiquantitative data on the rate and pattern of deacetylation were obtained by TLC analysis
13

of enzyme-substrate reaction mixtures run in parallel under the same substrate (5 mM) and
enzyme concentration (187 g/ml) (Figs. 5 and 6). Table 1 summarizes the results. In
consonance with the behavior of the enzyme on acetylated xylan, the best substrates among
the tri- and diacetates with vicinal hydroxyl group were 2,3,4- triacetate and 2,3-diacetate.
Both compounds were rapidly deacetylated at position 3. 2,4- and 3,4-diacetates served as
much worse substrates, their first deacetylation was very slow and in the case of the 3,4-
diacetate deacetylation was not selective. The resulting monoacetates, in general, served as
poor substrates resembling the slow deacetylation of 3-monoacetylated Xylp residues in the
polymer. The 2-acetate, resistant to the enzyme in the polymer, was, however, also a subject
of slow deacetylation, possibly due to acetyl group migration to position 3 or formation of a
productive enzyme-substrate complex in a different substrate orientation.
2,3-Ac2-Xylp-OMe
2,4-Ac2-Xylp-OMe
3,4-Ac2-Xylp-OMe
2,3,4-Ac3-Xylp-OMe
Ac3
Ac2-s
2,4-Ac2
3-Ac
4-Ac
2-Ac
2-Ac and
Xyl-OMe
Xyl-OMe
1
1
3
1
1
3
1
1
3
1
1
3
S
0
1
4 20 C
0
1
4 20 C
0
1
4 20 C
0
1 4 20 C S
12
12
12
12
Time (h)
Fig. 5. Time course of deacetylation of unmodified acetylated methyl -D-xylopyranosides by
F. johnsoniae AcXE followed by TLC. The reaction was performed at 35 °C using 5 mM
substrate and 187 g/ml enzyme concentration.
14

3-H-2,4-Ac2-Xylp-OMe 3-F-2,4-Ac2-Xylp-OMe 2-H-3,4-Ac2-Xylp-OMe 2-F-3,4-Ac2-Xylp-OMe
2-F-3,4-Ac2
2-deoxy-3,4-Ac2
3-F-2,4-Ac2
3-deoxy-2,4-Ac2
3-F-2-Ac
2-F-4-Ac
2-deoxy-4-Ac
3-deoxy-2-Ac
3-F-Xylp-OMe
2-F-Xylp-OMe
2-deoxy-Xylp-OMe
Xyl-OMe
3-deoxy-Xylp-OMe
Xyl-OMe
1
1
3
1
1
3
1
1
3
1
1
3
S 0
1
4 20 C
0
1
4 20 C
0
1
4 20 C
0
1 4 20 C S
12
12
12
12
Time (h)
Fig. 6. Deacetylation of deoxy and deoxy-fluoro analogues of methyl -D-xylopyranoside
diacetates by F. johnsoniae AcXE followed by TLC. The reaction was performed at 35 °C
using 5 mM substrate and 187 g/ml enzyme concentration.
Table 1. Acetylated methyl -D-xylopyranosides and their deoxy and deoxy-fluoro analogues
used to study regiospecificity of F. johnsoniae AcXE. The compounds are listed descendently
according the rate of their first deacetylation.
Substrate
Formula and the position
of the first deacetylation
Rate of the first Rate of the second
deacetylation/
Approximate
time required
for 50%
deacetylation /
Approximate time
required for 50%
conversion
conversion
Rapid/
<5 min
2,3,4-Ac3-Xylp-OMe
Slow (position 4)
~30 min
2,3-Ac2-Xylp-OMe
Rapid/
<5 min
Very slow
~12 h
2F-3,4-Ac2-Xylp-OMe
Rapid/
Rapid
<5 min
~10 min
Position 3
faster than
position 4
Rapid/
2H-3,4-Ac2-Xylp-OMe
3F-2,4-Ac2-Xylp-OMe
Slow
~2 h
<5 min
Rapid/
~5 min
Slow
~1 h
3H-2,4-Ac2-Xylp-OMe
Slower/
~30 min
Slow
~2 h
15

2,4-Ac2-Xylp-OMe
3,4-Ac2-Xylp-OMe
Slow/
~20 min
Slow
~12 h
Slow
~60 min
Slow
~6 h
The replacement of the free hydroxyl group in methyl β-D-xylopyranoside 2,4- and
3,4-diacetate by either hydrogen or fluorine resulted in a considerable increase in the rate of
the first and the second deacetylation. In the analogues lacking the 3-hydroxyl group, the
acetyl group migration to adjacent position is excluded, so the second deacetylation of
positions 2 is not related to this phenomenon. In general, the regioselectivity of deacetylation
of deoxy and deoxy-fluoro methyl xylopyranosides is similar to that of the derivatives with
the vicinal hydroxyl group.
3.6. Regiospecificity of the enzyme on monoacetylated 4-nitrophenyl
-D-xylopyranosides
Regiospecificity of the enzyme was also studied using all three monoacetylated 4-
nitrophenyl -D-xylopyranosides as glycosides having a bulkier aromatic aglycone. These
compounds have previously been shown to restrict regiospecificity of common AcXEs more
than acetylated methyl xylosides (reviewed in Biely, 2012).The best substrate for AcXEs of
CE families 1, 4, 5 and 6 was the 2-O-acetate. On the basis of deacetylation of
acetylglucuronoxylan and methyl xylosides it was expected that F. johnsoniae enzyme would
deesterify 4- and 3-monoacetylated aromatic xyloside. Both these substances actually served
as a substrate, however, surprisingly, 2-monoacetate was deacetylated as well. Moreover,
TLC did not reveal significant differences in the reaction efficiency (Supplementary Figure
2S). This was confirmed by HPLC allowing determination of specific activities towards 4-, 3-
and 2-monoacetylated -D-xylopyranoside to be 0.285, 0.188 and 0.128 U/mg, respectively.
4. Discussion
AcXEs play an important role in enzymatic conversion of natural partially acetylated
form of hardwood xylan. Such xylan is extracted from wood applying non-alkaline
biorefinery processes that avoid saponification of ester linkages. The removal of the acetyl
16

groups from the polysaccharide main chain is extremely important in view of the extent of its
enzymatic saccharification since the deacetylation creates new sites on the polymeric
substrate for productive binding to the enzymes hydrolyzing the glycosidic linkages. Almost
all so far recognized AcXEs were found to deacetylate singly and doubly acetylated Xylp
residues, but they did not recognize as a substrate the 3-O-acetyl group on MeGlcA-
substituted Xylp residue (Fig. 1) (Koutaniemi et al., 2013; Uhliariková, Vršanská, McCleary,
& Biely, 2013). The existence of an esterase liberating the acetyl group shielded by vicinal
MeGlcA residue in nature had been predicted for a long time, but has been demonstrated only
recently (Razeq et al., 2018). A gene coding for such an enzyme was found in a
polysaccharide utilizing locus of the bacterium F. johnsoniae UW 101 (Razeq et al., 2018).
The positional specificity of the enzyme, however, has not been sufficiently elucidated.
Moreover, due to a misinterpretation of the NMR spectra in the above quoted paper (the
signals of doubly acetylated Xylp residues have been considered to be the signals of
monoacetylated Xylp residues and the signals of monoacetylated residues have been ignored),
we have decided to confirm the 3-O-deacetylase activity of an enzyme having almost identical
amino acid sequence (98.6% identity) from a different F. johnsoniae strain. The capacity of
the enzyme to remove efficiently the 3-O-acetyl group from MeGlcA-glycosylated Xylp
residues, the most recalcitrant epitope of hardwood xylan, was confirmed not only on the
corresponding 3-O-acetylated polysaccharide, but also on native steam explosion extracted
hemicellulose. The native polysaccharide also allowed us to estimate relative deesterification
rates of the four distinct types of acetyl groups. It should be noted that the changes in the
pattern of NMR spectrum observed upon incubation with the esterase studied in this work
were essentially the same as those reported earlier (Razeq et al., 2018). The NMR spectra
provided information that the new esterase is specific xylan 3-O-deacetylase able to liberate
all three types of acetyl groups attached to position 3 (Fig. 1), but unable to deesterify position
2. However, the deacetylation of position 3 is affected by a modification of the vicinal
position 2. The deacetylation appears to be easier for the enzyme in case the hydroxyl group
at the neighboring position 2 is substituted, either MeGlcA-glycosylated or acetylated,
because in addition to the unique ability of the enzyme to liberate 3-O-acetyl group vicinal to
MeGlcA side residue, the esterase equally well deesterifies 3-O-acetyl group from doubly
acetylated Xylp residues. 3-O-Acetylated Xylp residues having free 2-OH group are attacked
by at least one order of magnitude slower. Only application of a larger enzyme amounts or
longer incubations resulted in glucuronoxylan acetylated exclusively at position 2. This
enzyme specificity in combination with a control of the extent of deacetylation provides a tool
17

for a preparation of acetylglucuronoxylan either 3- and 2-O-monoacetylated, or just 2-O-
monoacetylated (Fig. 3). We should note here that the resistance of the 2-acetyl group to
F. johnsoniae AcXE can also be seen in the NMR spectra shown by Razeq et al. (2018). Due
to an incomplete assignment of the signals by the authors this result is mentioned for the first
time in this work.
In exclusively 2-O-acetylated xylan, the 2-O-acetylation appeared to be very stable,
since it did not change due to acetyl group migration during relatively long incubations with
the enzyme during NMR measurements. This is in contrast to common acetyl group migration
observed at ambient temperature in synthetic monomeric xylopyranosides (Mastihubová and
Biely, 2004a). Quite surprisingly, the acetyl group transfer from position 2 to position 3 in the
polymer required a significant input of energy, such as a long heating at high temperature.
These findings pointed out that xylopyranoid rings in the polysaccharide main chain are not
flexible enough to enable conformation changes required for spontaneous acetyl group
migration via orthoester intermediates observed on non-reducing-end xylopyranoid rings
(Puchart & Biely, 2015). If there is an acetyl group migration on the polymer, it is
undoubtedly extremely slow at physiological temperatures. Different situation may, however,
occur during non-alkaline wood cooking. The finding of limited acetyl group migration
within a polymer might also be relevant to biosynthesis of the acetylated polysaccharide(s).
At least some plant xylan acetyl transferases could be specialized for esterification of
hydroxyls at position 2 and position 3 (Zhong, Cui, & Ye, 2018).
In contrast to polymeric substrate that was deesterified exclusively at position 3, a
lower regiospecificity of the F. johnsoniae AcXE was observed on multiply acetylated methyl
xylopyranosides. The first deesterification proceeded exclusively at position 3 of 2,3-
diacetate as well as 2,3,4-triacetate. However, 2,4-diacetate itself as well as that generated
quickly from the triacetate was slowly deesterified at position 4 and yielded 2-monoacetate
persisting in the reaction mixtures for a long time. Thus the preference of the enzyme for
deacetylation of position 3, eventually 4, may be exploited in preparative synthesis of 2-
monoacetylated xylopyranosides from multiply acetylated precursors. The replacement of the
2- or 3-hydroxyl group by hydrogen or fluorine in 2,4- and 3,4-diacetates accelerated the
initial deesterification and also facilitated the second deacetylation. The modification of the
2,4-diacetate at position 3 excludes the role of the acetyl group migration to vicinal group in
the second slow 2-O-deacetylation, which indicates that the enzyme may form productive
complexes with xylopyranosides in different orientations. In conclusion we may conclude that
the partially acetylated methyl xylopyranosides confirmed the preference of F. johnsoniae
18

AcXE for the 3-O-acetyl group, however, the enzyme did not show the same regioselectivity
on monoacetylated 4-nitrophenyl xylopyranosides. The substitution of the hydroxyl group at
position 2 facilitates greatly the 3-O-deacetylation in both polymeric substrate and
xylopyranosides. These observations are difficult to be understood and their explanation will
await elucidation of the 3D structure of the esterase.
Finally, we should stress the significance of this type of deacetylases in
saccharification yields of acetylated plant xylans. In the case of hardwood glucuronoxylan,
the 3-O-deacetylation of MeGlcA-substituted Xylp residues unblocks 20-30 % of acetylated
hardwood xylan to saccharification with glycoside hydrolases. The new deacetylase will also
be important for a breakdown of herbaceous arabinoglucuronoxylans since they also contain
3-O-acetylated MeGlcA-substituted Xylp residues (Naran, Black, Decker, & Azadi, 2009).
The novel F. johnsoniae 3-O-deacetylase is complementary to other common AcXEs of
families CE1, CE5 and CE6 deacetylating mono- and diacetylated Xylp residues, and also to
CE4 AcXEs ignoring not only the 3-O-acetyl group on MeGlcA-substituted Xylp residues but
also diacetylated Xylp residues (Uhliariková, Vršanská, McCleary, & Biely, 2013). Similarly
to other AcXEs, the action of the 3-O-deacetylase decreases hydrophilicity and solubility of
native, partially acetylated polysaccharide. The enzyme allows to obtain selectively 2-O-
acetylated polysaccharide. This may serve as a source of 2-O-acetylated xylooligosaccharides,
however, if the acetyl group in the fragments occurs on the non-reducing-end Xylp residues,
the acetyl group will migrate to other two positions.
Funding
This work was supported by the Slovak Research and Development Agency under the
contract No. APVV-0602-12, by Scientific Grant Agency under the contract No. 2/0016/18,
and by Research Agency under the contract No. ITMS 26230120008.
Declaration of interest: none
Acknowledgements
The authors are thankful to Dr. Bjørge Westereng (University of Aas, Norway) for supplying
aspenwood acetyglucuronoxylan. The author are also grateful to Dr. Iveta Uhliariková
(Institute of Chemistry, Slovak Academy of Sciences) for NMR spectra measurement, and to
Trine Berg (Novozymes) for protein purification.
References
Biely, P. (2012). Microbial carbohydrate esterases deacetylating plant polysaccharides.
Biotechnology Advances, 30, 1575-1588.
Biely, P., Mastihubová, M., Côté, G. L., & Greene, R. V. (2003). Mode of action of
acetylxylan esterase from Streptomyces lividans: a study with deoxy and deoxy-fluoro
19

analogues of acetylated methyl β-D-xylopyranoside. Biochimica et Biphysica Acta,
1622, 82-88.
Biely, P., Mastihubová, M., & Puchart V. (2007). The vicinal hydroxyl group is prerequisite
for metal activation of Clostridium thermocellum acetylxylan esterase. Biochimica et
Biophysica Acta, 1770, 565-570.
Biely, P., Cziszárová, M., Uhliariková, I., Agger, J. W., Li, X.-L., Eijsink, V. G. H., &
Westereng, B. (2013). Mode of action of acetylxylan esterases on acetyl glucuronoxylan
and acetylated oligosaccharides generated by a GH10 endoxylanase. Biochimica et
Biophysica Acta, 1830, 5075-5086.
Biely, P., Singh, S., & Puchart, V. (2016). Towards enzymatic breakdown of complex plant
xylan structures: State of the art. Biotechnology Advances, 34, 1260-1274.
Evtuguin, D. V., Tomás, J. L., Silva, A. M. S., & Neto, C. P. (2003). Characterization of an
acetylated heteroxylan from Eucalyptus globulus Labill. Carbohydrate Research, 338,
597-604.
Horn, S. J., Nguyen, Q. D., Westereng, B., Nilsen, P. J., & Eijsink, V. G. H. (2011).
Screening of steam explosion conditions for glucose production from non-impregnated
wheat straw. Biomass & Bioenergy, 35, 4879–4886.
Jørgensen, S, T., & Pedersen, P. E. (2015). Stably maintained multiple copies of at least two
ORF in the same orientation. Patent No. WO 2005/123915.
Koutaniemi, S., van Gool, M. P., Juvonen, M., Jokela, J., Hinz, S. W., Schols, H. A., &
Tenkanen, M. (2013). Distinct roles of carbohydrate esterase family CE16 acetyl
esterases and polymer-acting acetyl xylan esterases in xylan degradation. Journal of
Biotechnology, 168, 684–692.
Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M., & Henrissat, B. (2014). The
Carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Research, 42,
D490-D495.
Mastihubová, M., & Biely, P. (2004a). Lipase-catalyzed preparation of acetates of
4-nitrophenyl β-D-xylopyranoside and their use in kinetic studies of acetyl migration,
Carbohydrate Research, 339, 1353-1360.
Mastihubová, M., & Biely, P. (2004b). Deoxy and deoxyfluoro analogues of acetylated
methyl β-D-xylopyranoside – substrates for acetylxylan esterases. Carbohydrate
Research, 339, 2101-2110.
Naran, R., Black, S., Decker, S. R., & Azadi, P. (2009). Extraction and characterization of
native heteroxylans from delignified corn stover and aspen. Cellulose, 16, 661-675.
Puchart, V., & Biely, P. (2015). Redistribution of acetyl groups on the non-reducing end
xylopyranosyl residues and their removal by xylan deacetylases. Applied Microbiology
and Biotechnology, 99, 3865-3873.
Puchart, V., Agger, J. W., Berrin, J.-G., Várnai, A., Westereng, B., & Biely, P. (2016).
Comparison of fungal carbohydrate esterases of family CE16 on artificial and natural
substrates. Journal of Biotechnology, 233, 228-236.
Puchart, V., Mørkeberg Krogh, K. B. R., & Biely, P. (2019). Glucuronoxylan 3-O-acetylated
on uronic acid-substituted xylopyranosyl residues and its hydrolysis by GH10, GH11
and GH30 endoxylanases. Carbohydrate Polymers, 205: 217-224.
Raab, D., Graf, M., Notka, F., Schödl, T., & Wagner, R. (2010). The GeneOptimizer
algorithm: using a sliding window approach to cope with the vast sequence space in
multiparameter DNA sequence optimization. Systems and Synthetic Biology,
4,:215-225.
Razeq, F. M., Jurak, E., Stogios, P. J., Yan, R., Tenkanen, M., Kabel, M. A., Wang, W., &
Master, E. R. (2018). A novel acetyl esterase enabling complete deacetylation of
20

substituted xylans. Biotechnology for Biofuels, 11, art. 74.
https://doi.org/10.1186/s13068-018-1074-3.
Teleman, A., Lundqvist, J., Tjerneld, F., Stålbrand, H., & Dahlman, O. (2000).
Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen
employing 1H and 13C NMR spectroscopy. Carbohydrate Research, 329, 807-815.
Uhliariková, I., Vršanská, M., McCleary, B. V., & Biely, P. (2013). Positional specifity of
acetylxylan esterases on natural polysaccharide: An NMR study. Biochimica et
Biophysica Acta, 1830, 3365-3372.
Zhao, L., Ye, B., Zhang, Q., Cheng, D., Zhou, C., Cheng, S:, & Yan, X. (2019). Construction
of second generation protease-deficient hosts of Bacillus subtilis for secretion of foreign
proteins. Biotechnology and Bioengineering, 116, 2052-2060.
Zhong, R., Cui, D., & Ye, Z.-H. (2018). A group of Populus trichocarpa DUF231 proteins
exhibit differential O-acetyltransferase activities toward xylan. PLoS One, 13, art.
e0194532; https://doi.org/10.1371/journal.pone.0194532.
21