Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-β-1,3-glucanase from Thermotoga petrophila

Article abstract of DOI:10.1016/j.bbrc.2011.02.098

1,3-β-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal β-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90. °C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules.

Full text of DOI:10.1016/j.bbrc.2011.02.098

Biochemical and Biophysical Research Communications 406 (2011) 590–594
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Mode of operation and low-resolution structure of a multi-domain
and hyperthermophilic endo-b-1,3-glucanase from Thermotoga petrophila
Junio Cota a,d, Thabata M. Alvarez a, Ana P. Citadini a, Camila Ramos Santos b, Mario de Oliveira Neto c,
Renata R. Oliveira b, Glaucia M. Pastore d, Roberto Ruller a, Rolf A. Prade e, Mario T. Murakami b,
Fabio M. Squina a,
⇑
a Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), do Centro Nacional de Pesquisa em Energia e Materiais, Campinas SP, Brazil
b e Laboratório Nacional de Biociências (LNBio), do Centro Nacional de Pesquisa em Energia e Materiais, Campinas SP, Brazil
c Instituto de Física de São Carlos (IFSC), Universidade de São Paulo, São Carlos SP, Brazil
d Faculdade de Engenharia de Alimentos (FEA), Universidade Estadual de Campinas, Campinas SP, Brazil
e Department of Microbiology and Molecular Genetics, Oklahoma State University (OSU), Stillwater, OK, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 February 2011
Available online 23 February 2011
1,3-b-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel
production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional
characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga pet-
rophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal
b-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue
from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products.
Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and
the secondary structure is maintained after incubation at 90 °C. The structure resolved by small angle
X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic
domain flanked by two carbohydrate-binding modules.
Keywords:
Endo-b-1,3-glucanase
Glycoside hydrolase family 16
Hyperthermostable enzyme
Capillary zone electrophoresis
Small angle X-ray scattering
Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved.
1. Introduction
glycoside linkages, respectively [28]. These enzymes are further
classified as family members (GH) 16, 17, 55, 64, and 81 of the
Renewable structural polysaccharides are abundant resources in
the biosphere and represent a valuable industrial substrate for
many industrial processes, such as biofuels production, feedstock-
chemicals and pharmaceuticals [1]. b-1,3-Glucans are important
polymers found mainly in yeast and filamentous fungi and are pro-
duced by plants (as callose) in response to tissue damage. This poly-
mer is a major structural storage polysaccharide (laminarin) of the
marine brown macro-algae of the genus Laminaria. b-1,3-glucan is
also produced as insoluble exopolysaccharide by some fungal and
bacterial species [2–5].
CaZy system, which is based on structural similarities of the amino
acid sequence [28]. However, CaZy GH families display a dispersed
range of enzymatic activity, and updating biochemical and bio-
physical information strengthens the Cazy pupose.
To date, crystal structures from family GH 16 endo-b-1,3-glu-
canases (3.2.1.39) have been determined for Pyrococcus furiosus
[6], Rhodothermus marinus [6], Streptomyces sioyaensis [7], and
Nocardiopsis sp [8]. The active site region of family GH16 consists
of three acidic residues, two glutamic and one aspartic acid in
the active site, and two conserved tryptophan residues, which pro-
mote substrate binding and transition-state stabilization [9]. The
overall fold of these enzymes consists of a classical sandwich-like
b-jelly-roll motif formed by the face-to-face packing of two ant
parallel sheets containing seven and eight strands with a deep cav-
ity [6–8,10].
The b-1,3-glucanases are widely distributed among bacteria,
fungi, and plants and are generally classified into two types,
exo-b-1,3-glucanases (EC 3.2.1.58) and endo-b-1,3-glucanases (EC
3.2.1.6 and 3.2.1.39), which hydrolyze terminal or internal
In this work is reported a comprehensive functional character-
ization of an endo-b-1,3-glucanase from Thermotoga petrophila
(TpLam), including a detailed description of the hydrolytic mode
of operation, along with structural insights of this hyperthermosta-
ble enzyme.
⇑
Corresponding author. Address: Rua Giuseppe Máximo Scolfaro, 10.000 Polo II
de Alta Tecnologia, Caixa Postal 6170, Campinas, SP 13083-970, Brazil. Fax: +55 19
35183104.
E-mail address: fabio.squina@bioetanol.org.br (F.M. Squina).
0006-291X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2011.02.098

J. Cota et al. / Biochemical and Biophysical Research Communications 406 (2011) 590–594
591
2.3. Small angle X-ray scattering
2. Material and methods
Small Angle X-ray Scattering (SAXS) data for TpLam at the con-
centrations 4 and 8 mg/mL were collected on the SAXS2 beam line
at the Brazilian Synchrotron Light Laboratory. The radiation wave-
length was set to 1.48 Å and a 165 mm MarCCD detector was used
to record the scattering patterns. The sample-to-detector distance
was set to 1022.5 mm to give a scattering vector-range from 0.013
to 0.33 nmꢀ1, where q is the magnitude of the q-vector defined by
2.1. Cloning and purification of TpLam
The full-length coding TpLam gene (Tpet_0899) derived from
genomic DNA of T. petrophila was amplified by standart PCR
method for cloning the mature enzyme without signal peptide.
The primer set used for amplification was 50-GCTAGCCAAAACATC
CTTGGCAACGC-30 and 50-GGATCCTCATTGAGGACTCACCGAAA-30,
and the gene segment was cloned into the pET28a (Novagen)
vector using NheI and BamHI restriction sites. The protein expres-
sion and purification steps, included a Ni2+-chelating affinity and
size-exclusion chromatography, were performed following Squina
et al., 2010 [11]. The purified TpLam was further analyzed by
SDS–PAGE. Protein concentration was determined by Bradford
method.
q = 4p/k sinh (2h is the scattering angle). Protein samples were pre-
pared in 20 mM phosphate 50 mM NaCl buffer pH 6.0. The integra-
tion of SAXS patterns were performed using Fit2D software [17]
and the curves were scaled by protein concentration. The radius
of gyration (Rg) was approximated using two independent proce-
dures, by Guinier equation [18] and by indirect Fourier transform
method using GNOM program [18]. The distance distribution func-
tions p(r) also was evaluated by GNOM and the maximum diame-
ter (Dmax) was obtained. Molecular weight was calculated using the
procedure implemented on web tool SAXSmoW [19]. Dummy
atom model (DAMs) was calculated from the experimental SAXS
data using ab initio procedure implemented in Dammin program
[20]. Several runs of ab initio shape determination with different
starting conditions led to consistent results as judged by the struc-
tural similarity of the output models, yielding nearly identical scat-
tering patterns and fitting statistics in a stable and self-consistent
process. CRYSOL program calculated the simulated scattering curve
from DAM and homology model [21]. The evaluation of Rg and Dmax
were performed with the same program.
2.2. Enzyme characterization
Standard enzymatic assays for TpLam evaluation were per-
formed following previous work [29]. The determination of the
optimum pH and temperature profiles, thermo stability evaluation
and 8-aminopyreno-1,3,6-trisulfonic acid (APTS) labeling was per-
formed as described previously [28]. The mixture was incubated at
90 °C for 10 min in standard assays with laminarin or 60 min aim-
ing at to determine the specific activity in a set of polysaccharides
(purchased from the best source possible, Sigma Aldrich and Mega-
zyme). The enzymatic activity and substrate specificity were deter-
mined from the amount of reducing sugar liberated from different
polysaccharide substrates by the DNS method [12]. One unit of en-
zyme was defined as the quantity of enzyme that released reducing
2.4. Homology molecular modeling
sugars at rate of 1 lmol/min. The kinetic parameters for TpLam
Homology models for each domain of TpLam were constructed
using the HHPred server [22]. These models were renumbered
according to the wild protein and spatially disposed in this order:
1, 2, and 3, in a manner to avoid clashes, with MASSHA [23]. CRY-
SOL program calculated the of the scattering amplitudes for atoms,
excluded volume and border for each model and it was input for
the next approach [24]. BUNCH software, which employs rigid
body model (RBM) and simulated annealing routines, was used
to find the best relative positions of the high-resolution models
[25]. This solution was superposed in the DAM with SUPCOMB
[25].
activity were estimated from initial rates at twelve substrates con-
centration in the 1.6–65 mg/mL range in standard assay.
A Response Surface Methodology was performed to optimize
the reaction conditions for TpLAM. The variables analyzed here
were the pH together with temperature, whereas a Central Com-
posite Design (k = 2) with four central points totalizing 12 experi-
ments (Table 1) was considered for optimization of these
variables. Details concerning the statistical approaches for these
experiments can be found in Myers and Montgomery [13] and all
b-1,3-glucanase activity assays were carried out following our pre-
vious work using laminarin as substrate (stock 0,5% in water)
[14,15].
3. Results and discussion
Capillary electrophoresis of oligosaccharides and Far-UV circu-
lar dichroism (CD) measurements (190–260 nm) and prediction
of secondary structure from the dichroism circular spectrum was
conducted as described as previously [14–16].
3.1. TpLam is a specific endo b-1–3 acting glucanase with remarkable
thermostable properties
TpLam activity was investigated at different pH values and tem-
peratures. The enzyme exhibited at least 80% of its optimal activity
over a pH range, from 5 to 7 (data not shown), which resemble
other hyperthermophylic laminarinases [2,26]. Purified TpLam
showed a relative activity ranging from 82% to 86% at temperatures
of 78 °C and 95 °C and pH 6, respectively (data not shown). A
Central Composite Design with two variables was performed to
optimize reaction conditions. The model (Fig. 1A and B) was vali-
dated by an analysis of variance (ANOVA) showing that the model
was significant at high confidence level (95%), with R2 = 0.96. The
best condition for enzyme activity was reached at pH 6.2 and
91 °C. To validate the model, at pH 6 and temperature of 91 °C
the predicted laminarinase activity of 250.0 IU/mg protein,
whereas experimentally determined was 260.8 IU/mg protein.
TpLam was heat-stable at 70 °C and maintained 60% of residual
activity after 16 h at 80 °C. The enzyme showed a half-life time
of 577 and 126 min at 90 and 95 °C, respectively (Fig. 1C). The
Table 1
Central composite design matrix (22) and the response of TpLam activity after 10 min
of incubation.
Run no.
Coded levels
(X1 = pH; X2 = T)
Actual levels
(X1 = pH; X2 = T)
Laminarinase activity
(IU/mg protein)
X1
X2
X1
X2
1
2
3
4
5
6
7
8
9
ꢀ1
ꢀ1
4.6
7.4
4.6
7.4
4.0
8.0
6.0
6.0
6.0
6.0
6.0
6.0
74.2
74.2
94.8
94.8
84.5
84.5
70.0
99.0
84.5
84.5
84.5
84.5
139,8
170,9
175,2
204,2
68,6
154,6
159,7
238,9
234,6
241,3
247,8
247,1
1
ꢀ1
ꢀ1
1
1
0
0
1
ꢀ1.414
1.414
0
0
0
0
0
0
ꢀ1.414
1.414
0
0
0
0
10
11
12

592
J. Cota et al. / Biochemical and Biophysical Research Communications 406 (2011) 590–594
Fig. 1. Response surface (A) and contour plot (B) for the influence of temperature and pH on TpLam activity. (C) Curve of decay of time of half life (t1/2) using four different
temperatures (70, 80, 90, and 95 °C). (D) Thermal stability of laminarinase analyzed by circular dichroism. CD spectra were taken at 20 °C (solid line), at 90 °C (dashed line)
and after 18 h at 90 °C.
Fig. 2. Capillary zone electrophoresis of APTS-labeled oligosaccharides. (A) Incomplete and complete hydrolysis of APTS-reducing-end-labeled-laminarihexaose. (B)
Incomplete and complete hydrolysis of laminarin (APTS-labeled after the enzymatic reaction). G1,G2, G3, G4, G5, G6, G7, G8, and G9 indicate the degree of polymerization of
glucose oligomers.
optimum temperature reported for the TpLam is comparable to
other thermophilic bacterial endo-b-1,3-glucanases from T. neapol-
itana and P. furiosus, which display temperatures of 95 °C and
100 °C, respectively [2,26].
The recombinant enzyme was highly specific on b 1–3-linked
polysaccharides, such as laminarin (48.07 IU/mg) and b-glucan
from barley (41.55 IU/mg) and lichenan (21.37 IU/mg). No b 1–4
glucanase activity was detected, based on enzymatic assays using
avicel, carboxymethylcellulose and cellopentaose, as well as xylan,
arabinan and manan containing polysaccharides. Kinetic values
were determined experimentally from the initial rates of laminarin
hydrolysis at each substrate concentration and standard assay con-
ditions. A Vmax of 558.2
lmol/min/mg and a Km of 3.3 mg/mL were
found for purified TpLam. Two other kinetic constants were

J. Cota et al. / Biochemical and Biophysical Research Communications 406 (2011) 590–594
593
determined from the experimental data and the values for catalytic
constant (Kcat) and Kcat/Km were 680.3 sꢀ1 and 206.2, respectively.
The CD spectrum of native laminarinase presents a negative
peak at 217 nm and a positive peak at 232 nm (Fig. 1D). The
217 nm peak is characteristic of b-structures and the 232 nm peak
is explained by the aromatic residues contribution. All domains
mode of action for TpLam. During the course of hydrolysis of
APTS-labeled laminarihexaose (non-reducing end available) the
enzyme most likely attacks the internal glycosidic linkages releas-
ing fluorescent dimers, trimers and tetramers (Fig. 2A). Final
hydrolysis products were dimers and trimers, suggesting that they
cannot be degraded further (Fig. 2A). Nevertheless through APTS-
labeling (after the enzymatic reaction) of hydrolysis products from
laminarihexaose showed formation of glucose (data not shown).
Using natural laminarin the degradation pattern was characteristic
for endoglucanases (APTS-labeled after the reaction) with produc-
tion of intermediates with different degree of polymerization
(Fig. 2B), yielding as final products; glucose, laminaribiose and
laminaritriose (Fig. 2B).
have a high content of beta-sheet, with few
a-helical structures.
Glycoside hydrolases are rich in aromatic residues, which are nota-
bly important to form the active site pocket and to interact with
the substrate. Those side chains contribute to the CD spectrum,
especially in proteins of low
a-helix content, such as the lamina-
rinase, giving positive bands in the 220–230 region [27]. At 90 °C
little change is observed in the CD spectrum of laminarinase, show-
ing its stability in terms of secondary structure (Fig. 1D). After
incubation at 90 °C for 18 h, CD spectrum was changed with the
loss of the 232 nm peak indicating protein unfolding (Fig. 1D).
Enzymatic assays demonstrated its inactivation under such condi-
tions, which confirmed CD analysis.
3.2. Low-resolution structure of TpLam reveals a multi-domain
V-shape conformation
The structural parameters derived from experimental curve,
DAM and RBM are shown in Table 2. The values obtained from
these different calculations are similar, indicating an agreement
among the SAXS data, the SAXS-derived envelope and the RBM.
Moreover, these data indicate that the maximum intra particle
distance (Dmax) and Rg of TpLam are 130 and 42 Å, respectively,
meaning that the Dmax is three times greater than the Rg, and the
particle should have an elongated form.
The scattering curve, the distance distribution function and the
low-resolution structure are shown in Fig. 3A. As expected, the
DAM is elongated, exhibiting a V-shape with two asymmetric arms.
The three-domain high-resolution model was well fitted into the
DAM with the catalytic core (CC2) occupying the central region
and two carbohydrate-binding modules (CBM) forming the arms.
The asymmetry of the molecule is due to the fact that the linker
Capillary zone electrophoresis analysis of the corresponding
hydrolysis products was performed in an attempt to define the
Table 2
Structural parameters derived from SAXS data for TpLam.
Parameters/Sample Expa
DAMb
130.8
41.99
19.0
–
RBMc
135.3
42.20
–
0
130.0 5.0
Dmax (AÅ)
0
40.10 (Guinier) 42.0 0.1 (GNOM)
Rg (AÅ)
0
19.0
67.9
Resolution (AÅ)
MWSAXS (kDa)
–
Resolution: 2p/q.
a
Calculated from the experimental data at 4 mg/mL.
Calculated from dummy atom model.
Calculated from rigid body model (RBM), BUNCH program.
b
c
Fig. 3. Laminarinase analysis by SAXS. (A) Experimental scattering curve (open circles) and fit produced by GNOM (solid line). (B) Distance distribution function computed
from the experimental data. (C) Laminarinase model with CBM1 (magenta), CD2 (green) and CBM3 (blue) domains fitted into the envelope obtained from SAXS data is shown
in different orientations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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J. Cota et al. / Biochemical and Biophysical Research Communications 406 (2011) 590–594
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