Carbohydrate esterases of family 2 are 6-O-deacetylases

Article abstract of DOI:10.1016/j.febslet.2009.11.095

Three acetyl esterases (AcEs) from the saprophytic bacteria Cellvibrio japonicus and Clostridium thermocellum, members of the carbohydrate esterase (CE) family 2, were tested for their activity against a series of model substrates including partially acet

Full text of DOI:10.1016/j.febslet.2009.11.095

FEBS Letters 584 (2010) 543–548
journal homepage: www.FEBSLetters.org
Carbohydrate esterases of family 2 are 6-O-deacetylases
a,*
a
b
b
Evangelos Topakas , Sarantos Kyriakopoulos , Peter Biely , Ján Hirsch ,
a
a
Christina Vafiadi , Paul Christakopoulos
a
BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str, Zografou Campus,
5700 Athens, Greece
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovak Republic
1
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Three acetyl esterases (AcEs) from the saprophytic bacteria Cellvibrio japonicus and Clostridium
thermocellum, members of the carbohydrate esterase (CE) family 2, were tested for their activity
against a series of model substrates including partially acetylated gluco-, manno- and xylopyrano-
sides. All three enzymes showed a strong preference for deacetylation of the 6-position in aldohex-
oses. This regioselectivity is different from that of typical acetylxylan esterases (AcXEs). In aqueous
medium saturated with vinyl acetate, the CE-2 enzymes catalyzed transacetylation to the same posi-
tion, i.e., to the primary hydroxyl group of mono- and disaccharides. Xylose and xylooligosaccha-
rides did not serve as acetyl group acceptors, therefore the CE-2 enzymes appear to be 6-O-
deacetylases.
Received 2 October 2009
Revised 29 November 2009
Accepted 30 November 2009
Available online 4 December 2009
Edited by Stuart Ferguson
Keywords:
Acetyl esterase
Transesterification
CE-2 family
Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
6
-O-Deacetylase
Cellvibrio japonicus
Clostridium thermocellum
1
. Introduction
Acetylxylan esterases (AcXEs; EC 3.1.1.72) are common compo-
fold [4]. The esterases CjCE2B and CtCE2 provide a rare example
of a catalytic dyad (Ser & His) with stabilization of the His residue,
mediated by main chain carbonyl groups. Surprisingly, the CtCE2
module, which is linked to the cellulase CtCel5C, displays divergent
catalytic esterase and non-catalytic carbohydrate binding func-
tions, illustrating a rare example of the ‘‘gene sharing” hypothesis,
where the introduction of a second functionality into the active site
of an enzyme does not compromise the original activity of the bio-
catalyst [4].
nents of the hemicellulolytic and cellulolytic enzyme systems of
microorganisms proliferating on plant biomass residues [1]. Their
function is to deesterify partially acetylated hardwood 4-O-
methyl-D-glucuronoxylan and xylans of annual plants. These
esterases are found in 8 of 16 known carbohydrate esterase (CE)
families (http://www.cazy.org/fam/acc_CE.html, [2]).
Members of the CE-2 family are especially intriguing with re-
spect to the multiple functions required of plant degrading enzyme
systems. CE-2 family esterases were first described in Neocallimas-
tix patriciarum and were shown to be acetyl esterases (AcEs), which
are active on synthetic aryl-esters, with low or no activity against
acetylated birchwood xylan [3]. Recently, the crystal structure and
biochemical properties of several CE-2 members, both single-mod-
ule CE-2 enzymes from Cellvibrio japonicus (CjCE2A, B), and a com-
Members of the CE-2 family act as AcEs, releasing acetate from
activated artificial substrates, such as 4-nitrophenyl acetate, while
some show high deacetylating activity against acetylated konjac
glucomannan, compared to acetylated birchwood xylan [4]. It is
interesting that in the elegant work of Montanier et al. [4], the
positional specificity of the enzymes as potential 6-O-deacetylases
was not investigated, despite some hints that konjac glucomannan
is a linear random copolymer of (1?4) linked b-D-mannose and b-
ponent of
a
modular cellulase CtCel5C from Clostridium
/b hydrolase
D-glucose with a low degree of acetylation at the C-6 position [5,6].
thermocellum (CtCE2) were resolved, revealing an
a
In the present work, we describe the substrate and positional spec-
ificity of three recombinant CE-2 esterases from C. japonicus and C.
thermocellum against a series of model substrates, including par-
tially acetylated gluco-, manno- and xylopyranosides. The enzymes
were also successfully tested for their synthetic activity and
regioselectivity of acetylation of different biomass-derived
Abbreviations: CE, carbohydrate esterases; AcE, acetyl esterase; AcXE, acetylxy-
lan esterase; 4-NPh–OH, 4-nitrophenol
*
Corresponding author. Fax: +30 210 7723163.
E-mail address: vtopakas@central.ntua.gr (E. Topakas).
0
014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2009.11.095

5
44
E. Topakas et al. / FEBS Letters 584 (2010) 543–548
carbohydrates. This is the first report that members of CE-2 family
show, in both hydrolytic and synthetic functions, a regioselectivity
distinct from that of AcXEs and appear to be specialized for the 6-
O-deacetylation of glucopyranosyl and mannopyranosyl residues.
from the 4-nitrophenyl b-
ble rate within 15 min. Kinetic constants (kcat, K
using a non-linear regression model (GraFit) that also gives an esti-
mate of the standard error of each parameter [16].
D
-xyloside monoacetates at an apprecia-
m
) were estimated,
In order to probe the substrate specificity of CE-2 esterases
against acetylated
xylopyranosides (compounds 4–8, Fig. 1), respectively, 10 mM of
each substrate was tested at 40 °C in a total volume 105 l in
.1 M sodium phosphate buffer, pH 6.0. The reaction was started
by the addition of the enzyme solution (5 l). The b- -xylopyrano-
D-mannopyranose, methyl b-D-gluco- and b-D-
2
. Materials and methods
l
2.1. Enzyme source
0
l
D
Recombinant C. japonicus CjCE2B (ACE85322.1), CjCE2C
sides diacetate solutions were freshly prepared in order to avoid
acetyl group migration [8]. Deesterification of compounds was fol-
lowed qualitatively by TLC and quantitatively by determination of
the remaining ester, according to Hestrin [17]. This method is
based on the conversion of the ester into hydroxamic acid by a
reaction with hydroxylamine under alkaline conditions. The
hydroxamic acid is in turn complexed with ferric ion, giving rise
to a brownish chromophore measured spectrophotometrically at
(
ACE85140.1) and C. thermocellum CtCE2 domain (ABN52032.1)
were heterologously expressed in Escherichia coli BL21 (DE3) and
purified by immobilized metal affinity chromatography, as de-
scribed previously [4]. b-Xylosidase used in the enzyme-coupled
assay of AcEs, was a product of Aspergillus niger gene xlnD, ex-
pressed in recombinant Saccharomyces cerevisiae Y294 [7].
2
.2. Model compounds and reagents
5
40 nm.
The protein concentration (mg ml ) of the purified CE-2 ester-
À1
The three monoacetylated 4-nitrophenyl b-
D-xylopyranosides
ases were estimated spectrophotometrically at 280 nm, using mo-
lar extinction coefficient values calculated from their protein
sequence.
1
–3 were synthesized previously [8]. Methyl 6-O-acetyl-b-D-gluco-
pyranoside 4 was prepared according to Lindberg [9]. Compound 5
was prepared by enzymatic acetylation in this work (see below).
The three methyl di-O-acetyl-b-
described by Kovac and Alföldi [10–12]. 6-O-Methyl-
nose 15 and its methyl -glycoside 14 were synthesized according
to Bourne et al. [13] and Evtushenko [14], respectively. The model
disaccharides related to glucoxylans and xyloglucans 17, 18 and 20
were prepared according to Petráková et al. [15].
D
-xylopyranosides 6–8 have been
2
.4. Enzymatic transacetylation reactions
D
-glucopyra-
a
Enzymatic acetylation of monosaccharides (D-glucose (Glc), D-
À1
mannose (Man), D-galactose (Gal) and D-xylose (Xyl); 40 mg ml ),
their methyl glycosides and their mono-methyl ethers
À1
(
40 mg ml ), was carried out in 100 mM 3-(N-morpholino)pro-
panesulfonic acid–NaOH buffer pH 6.0 saturated with vinyl ace-
tate, as reported previously [18]. The reactions were initiated by
2.3. Substrate specificity reactions
the addition of the enzyme (16
0 °C in an Eppendorf Thermomixer Comfort (Eppendorf, Ger-
many), operating at 1400 rpm for 24 h and analyzed by HPLC
monosaccharides) or TLC. All reaction mixtures contained carbo-
hydrate acceptors (100 l), purified recombinant CE-2 esterases
and 20 l of vinyl acetate. Control samples lacking enzyme prepa-
ration or acyl donor were run simultaneously.
lg), followed by agitation at
The b-xylosidase-coupled assay, using 2-O-, 3-O-, or 4-O-acetyl
3
4
-nitrophenyl b-
D-xylopyranoside (compounds 1–3, Fig. 1), was
conducted at 40 °C in a total volume of 105
ll in 0.1 M sodium
(
phosphate buffer, pH 5.5. Stable 200 mM stock solutions of sub-
strates were prepared in dimethyl sulfoxide and diluted to working
concentrations with the preheated phosphate buffer just before the
assay. To measure deacetylase activity, the reaction was started by
l
l
the addition of the CE-2 enzyme solution (2.5
ll) to a mixture of
2.5. Analytical procedures
the substrate and b-xylosidase (102.5 l). The absorbance of the
l
liberated 4-nitrophenol (4-NPh–OH) was monitored at 405 nm
against substrate and enzyme blanks, using a SPECTRAmax 250
Microplate Spectrophotometer (Molecular Devices, Sunnyvale,
CA). The recombinant b-xylosidase, did not liberate 4-NPh–OH
Reaction mixtures using compounds 9–22 (Fig. 3) as acetyl
acceptors, were analyzed by TLC on aluminium sheets coated with
Silica gel 60 (Merck, Germany), in the acetonitrile/water 9:1 (v/v)
Fig. 1. Chemical structures of model substrates used for probing the hydrolytic specificity of CE-2 esterases, including 4-nitrophenyl b-
partially acetylated -mannopyranose and methyl b- -gluco- and b- -xylopyranosides.
D-xylopyranoside monoacetates,
D
D
D

E. Topakas et al. / FEBS Letters 584 (2010) 543–548
545
Table 1
Kinetic constants for hydrolysis of 4-nitrophenyl b-D-xylopyranoside monoacetates by CjCE2B (A) and CjCE2C (B) from C. japonicus and CtCE2 (C) from C. thermocellum. Numbers in
parentheses are the estimates of the standard errors. n.d., activity not determined.
À1
Substrates
Name
K
m
(mM)
kcat (min
)
kcat/K
m
A. CjCE2B
1
2
3
2-O-Acetyl 4-nitrophenyl b-
3-O-Acetyl 4-nitrophenyl b-
4-O-Acetyl 4-nitrophenyl b-
D-xylopyranoside
D-xylopyranoside
D-xylopyranoside
4.42 (0.82)
1.55 (0.38)
1.24 (0.15)
2496 (332)
126 028 (24 674)
192 614 (12 122)
565 (129)
81 361 (25 467)
155 912 (21 345)
B. CjCE2C
1
2
3
2-O-Acetyl 4-nitrophenyl b-
3-O-Acetyl 4-nitrophenyl b-
4-O-Acetyl 4-nitrophenyl b-
D-xylopyranoside
D-xylopyranoside
D-xylopyranoside
n.d.
n.d.
4.31 (1.06)
n.d.
n.d.
14 493 (2637)
n.d.
n.d.
3365 (1030)
C. CtCE2
1
2
3
2-O-Acetyl 4-nitrophenyl b-
3-O-Acetyl 4-nitrophenyl b-
4-O-Acetyl 4-nitrophenyl b-
D-xylopyranoside
D-xylopyranoside
D-xylopyranoside
n.d.
n.d.
2.46 (0.42)
n.d.
n.d.
79 873 (8662)
n.d.
n.d.
32 493 (6567)
solvent system. Sugars were detected on dried chromatograms,
using the N-(1-naphthyl)ethylenediamine dihydrochloride re-
agent, according to Bounias [19]. The chromatograms treated with
the detection reagent were heated at 110 °C in an oven and cooled
prior to analysis.
mass spectra were obtained in positive mode, using the electro-
spray ionization (ESI) technique. Various parameters, such as cap-
illary voltage, drying gas temperature, drying and nebulizer gas
pressure, were adjusted in order to optimize the signal.
The acetylation of monosaccharides (Glc, Man, Gal, Xyl) was
quantified, using an HPLC (Waters 600E), equipped with a refrac-
3
. Results and discussion
2
tometer (Waters 410), on a NH -Macherey Nagel 250/4.6 100–5
3
.1. Substrate specificity of CE-2 family AcEs
column. Elution was conducted with a mixture of acetonitrile/
À1
water (9:1, v/v) at a flow rate of 1 ml min . Yields for the synthe-
To probe the function and role of the diverse members of the
sis of the acetylated monosaccharides were calculated from the
amount of each individual monosaccharide reacted compared to
its initial unreacted concentration. The monosaccharide concentra-
tions were calculated from standard curves, obtained under the
same analytical conditions.
CE-2 esterase family, which currently contains 38 members
http://www.cazy.org/fam/CE2.html), the biochemical properties
(
of three CE-2 enzymes were assessed. In addition to CtCE2, which
corresponds to the C-terminal region of the C. thermocellum bifunc-
tional protein CtCel5C-CE2, we also characterized two C. japonicus
CE-2 members, CjCE2B and CjCE2C, which were cloned and ex-
pressed in E. coli, as described previously [4].
2.6. Structure elucidation of acetylated monosaccharides
In order to probe the positional specificity of CE-2 esterases on
the xylopyranose ring, we used the enzyme-coupled assay with
Proton NMR spectroscopy was performed in CD OD with a Bru-
ker DRX 400 spectrometer, equipped with a 5 mm H/ C dual in-
verse broad probe at 400.13 MHz.
3
1
13
three different monoacetylated 4-nitrophenyl b-
D-xylopyranosides
(Fig. 1; compounds 1–3). As shown in Table 1, all CE-2 tested
Mass spectrometry was done by direct infusion of the reaction
products dissolved in 50:50 methanol/water (v/v) with 0.2% formic
acid solution, in a Varian 500 MS Quadrupole Iontrap LC/MS. The
showed strong preference for the deacetylation of positions 4
and 3. This finding is in contrast to the preference of AcXEs for po-
sition 2 [20], but similar to the positional specificity of the AcE
Table 2
Hydrolysis of synthetic substrates by CE-2 AcEs from C. japonicus and C. thermocellum. Specific activities for partially acetylated D-mannopyranose and methyl b-D-gluco- and b-D-
xylopyranosides. Compound 5 was synthesized by CjCE2B.
Substrates
Name
Specific activity (U/mg)
CjCE2B
CjCE2A
CtCE2
4
5
6
7
8
Methyl 6-O-acetyl-b-
6-O-Acetyl-D-mannopyranose
Methyl 3,4-O-diacetyl-b-
Methyl 2,3-O-diacetyl-b-
Methyl 2,4-O-diacetyl-b-
D
-glucopyranoside
328
395
4
0
5
60
3
3
0
2
183
35
8
0
2
D-xylopyranoside
D-xylopyranoside
D-xylopyranoside
Fig. 2. Transacetylation reaction of monosaccharides with vinyl acetate catalyzed by CE-2 AcEs.

5
46
E. Topakas et al. / FEBS Letters 584 (2010) 543–548
Fig. 3. Chemical structures of model substrates used for probing the synthetic specificity of CE-2 esterases, including various methyl ethers of D-glucose and methyl
glycosides of D-glucopyranose and D-xylopyranose.
Table 3
AcE, which might explain the high deacetylating activity reported
for this enzyme against konjac glucomannan [4]. The resistance
of methyl 2,3-di-O-acetyl-b-D-xylopyranoside to hydrolysis is more
or less in consonance with the behaviour of the enzymes on
xylopyranoside monoacetates (Table 1). The conclusion from the
above results is that all three CE-2 enzymes show clear preference
for deacetylation of the primary hydroxyl group in gluco- and man-
nopyranosyl residues.
Transacetylation of various monosaccharides with vinyl acetate catalyzed by CE-2
AcEs at 30 °C after 24 h.
Name
Transacetylation conversion (%)
CjCE2B
CjCE2A
CtCE2
D-Glucose
D-Mannose
D-Galactose
D-Xylose
31
46
22
0
25
26
48
0
86
71
89
0
3.2. Synthetic potential and specificity of CE-2 AcEs
from Hypocrea jecorina, which is a member of the recently estab-
lished CE-16 family [21]. Thus, the mode of action of CE-2 and
CE-16 AcEs on xylopyranosyl residues could be complementary
to the action of AcXEs for the complete removal of acetyl groups
during hemicellulose biodegradation.
Recently, CE-2 AcEs and in particular CjCE2B and CtCE2, have
been reported to display a significant preference for acetylated
konjac glucomannan [4] which, in view of the scarce information
on 6-O-acetylation of the polysaccharide [5,6], might indicate
activity for the deacetylation of gluco- and mannopyranoside resi-
In order to evaluate the synthetic potential of CE-2 AcEs, we
carried out transacetylation reactions in two-phase system, com-
posed of an aqueous solution of the acceptor and vinyl acetate
as the acetyl group donor (Fig. 2; [18,22]). As shown in Table
3, all CE2 tested were able to catalyze the acetylation of Glu,
Man and Gal with the CtCE2 esterase showing the highest con-
version yields. On the other hand, none of the esterases were
able to acetylate Xyl, a reaction that was carried out successfully
by the CE-16 AcE from H. jecorina in the same reaction system
and conditions [18,22], indicating a divergence with respect to
the synthetic mode of action between AcEs belonging to differ-
ent CE families. The products of Glc and Man acetylation by
CjCE2B were isolated from the reaction mixtures by chromatog-
raphy on Silica gel column. Monoacetylation of both hexoses
was demonstrated by ESI MS operating in the positive ion mode,
dues at 6-position. A series of partially acetylated
nose and methyl b- -gluco- and b- -xylopyranosides (compounds
–8) were tested as substrates for probing the catalytic specificity
D-mannopyra-
D
D
4
of the CE-2 esterases. As shown in Table 2, all CE-2 esterases tested
showed strong preference for deacetylation of methyl 6-O-acetyl-
+
b-
D
-glucopyranoside and 6-O-acetyl-
D
-mannose, compared to their
resulting in a singly charged adduct ion [M+Na] at m/z 245. The
activity against diacetylated xylopyranosides. The highest specific
activity for 6-O-acetylated substrates was exhibited by CjCE2B
presence of the acetyl group at position 6 was confirmed by 1
NMR (Table 4).
H

E. Topakas et al. / FEBS Letters 584 (2010) 543–548
547
Table 4
1
H NMR data of acetylated Glc and Man. Chemical shifts (d) in ppm.
H1a
H2–H5
H6
CH
3
COOÀ
6
-O-Acetyl-
-O-Acetyl-
D
D
-glucopyranose
5.10 (d, J = 3.56 Hz, 0.1H), 4.50
d, J = 7.74 Hz, 0.9H)
3.52–3.14 (m, 4H)
4.42 (bs, 0.4H), 4.39 (bs, 0.6H), 4.20
(d, J = 6.0 Hz, 0.6H), 4.17 (d, J = 6.0 Hz, 0.4H)
2.07 (s, 3H)
(
6
-mannopyranose
5.07 (bs, 0.8H), 4.78 (bs, 0.2H)
3.62–3.96 (m, 4H)
4.40 (d, J = 1.9 Hz 0.4H), 4.37 (d, J = 1.9 Hz 0.6H),
2.07 (s, 3H)
4.25 (d, J = 6.2 Hz, 0.6H), 4.22 (d, J = 6.2 Hz, 0.4H)
a
Two peaks due to mixture of anomers.
Table 5
Semiquantitative determination of the rate of acetylation of various methyl ethers of D-glucose and methyl glycosides of D-glucopyranose and D-xylopyranose by CjCE2B and
CjCE2A from C. japonicus and CtCE2 from C. thermocellum in aqueous solution saturated with vinyl acetate. Rate of acetylation: excellent acetyl group acceptors, +++; weaker acetyl
group acceptors, ++ and +; no acceptor, À.
Substrates
Name
Rate of acetylation
CjCE2B
CjCE2A
CtCE2
9
Methyl b-
D
-glucopyranoside
-glucopyranoside
Methyl 2-O-methyl-b- -glucopyranoside
3-O-Methyl -glucopyranose
+++
+++
+++
+++
+++
À
++
+
++
++
+
+
10
11
12
13
14
15
16
17
18
19
20
21
22
Methyl
a
-
D
+++
+++
++
+++
À
D
D
Methyl 4-O-methyl-
Methyl 6-O-methyl-
6-O-Methyl
Methyl b- -xylopyranoside
Xylopyranosyl b-(1,4)-glucopyranose
Glucopyranosyl b-(1,3)-xylopyranose
Glucopyranosyl b-(1,4)-xylopyranose
a
a
-
-
D
-glucopyranoside
-glucopyranoside
D
À
D-glucopyranose
À
À
À
D
À
À
À
++
À
+
+++
+++
+
+++
À
+++
+
+++
++
À
a-
D-Glucopyranosyl
a-
D-glucopyranoside (
a
,a-trehalose)
À
Cellobiose
Xylobiose
+++
À
+++
À
The synthetic reaction of CE-2 recombinant AcEs was also
examined, using various mono-methyl ethers of -glucose and
methyl -glucopyranoside and also methyl b- -xylopyranoside
compounds 9–16; Fig. 3) as acetylation acceptors. As shown in Ta-
ble 5, the three CE-2 esterases acetylated both anomeric forms of
methyl -glucopyranoside (compounds 9 and 10) with CtCE2 ester-
ase showing a slight preference for the acetylation of the -ano-
mer. The results in Table 5 show that CE-2 esterases acetylated
all compounds, with exception of 6-O-protected -glucose or
Acknowledgements
D
D
D
The authors thank Professor Harry J. Gilbert from Complex Car-
bohydrate Research Center, University of Georgia and Professor
Carlos M.G.A. Fontes from Faculty of Veterinary Medicine, Techni-
cal University of Lisbon, who supplied the plasmids for the produc-
tion of the recombinant CE-2 esterases.
(
D
a
D
D
-
References
glucopyranoside, indicating a strong regioselectivity for acetyla-
tion of the primary hydroxyl group. This finding is in accordance
with their hydrolytic specificity profile. The 6-O-acetylation also
appears to be the predominant reaction with disaccharides as acet-
yl group acceptors (compounds 17–22, Fig. 3, Table 5). Xylobiose
did not serve as an acceptor. Of three mixed-type disaccharides
containing glucose and xylose (compounds 17–19), a higher degree
of acetylation was observed with dimmers having a glucopyrano-
syl residue at the non-reducing end. These results indicate that
the enzymes can differentiate between reducing and non-reducing
aldohexose residues in oligosaccharides as their target. This is fur-
ther justified by the monoacetylation of cellobiose compared to
trehalose. An explanation of this phenomenon, which might be
associated with sugar binding subsites in the substrate binding site
of the CE-2 enzymes, would require a study that would include lar-
ger oligosaccharides and eventually polysaccharides as acetyl
group acceptors.
A unique property of the esterase enzymes studied in the pres-
ent paper is catalysis of acetyl transfer to alcohols in water, in par-
ticular the efficient regioselective 6-O-acetylation of aldohexoses
in aqueous medium saturated with vinyl acetate. This reaction
may be considered to achieve another hitherto unattainable goal
of biocatalysis with important future applications in carbohydrate
and material chemistry. Elucidation of the physiological role of CE2
esterases requires further studies that include identification of the
natural substrate for the enzymes.
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