New evidence for the role of calcium in the glycosidase
reaction of GH43 arabinanases
Daniele de Sanctis
1,2,
*, Jose
´
M. Ina
´
cio
1,
*
,
, Peter F. Lindley, Isabel de Sa
´
-Nogueira
1,3
and
Isabel Bento
1
1 Instituto de Tecnologia Quı
´
mica e Biolo
´
gica, Universidade Nova de Lisboa, Oeiras, Portugal
2 Structural Biology Group, European Synchrotron Radiation Facility, Grenoble, France
3 Departamento de Cie
ˆ
ncias da Vida, Faculdade de Cie
ˆ
ncias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal
Keywords

Fax: +351 21 441 1277
Tel: +351 21 446 9100
E-mail:
Present address
Instituto de Biotecnologia e Bioengenharia-
Centro de Biomedicina Molecular e Estrutural,
Universidade do Algarve, Campus de
Gambelas, Faro, Portugal
*These authors contributed equally to this work
Database
Structural data for the native BsArb43B, the
BsArb43B H318A mutant and the BsArb43B
D171A mutant in complex with
arabinohexose have been submitted to the
Protein Data Bank under the accession num-
bers 2X8F, 2X8T and 2X8S, respectively
(Received 13 May 2010, revised 27 July
2010, accepted 6 September 2010)
doi:10.1111/j.1742-4658.2010.07870.x
Endo-1,5-a-l-arabinanases are glycosyl hydrolases that are able to cleave
the glycosidic bonds of a-1,5-l-arabinan, releasing arabino-oligosaccharides
and l-arabinose. Two extracellular endo-1,5-a-l-arabinanases have been
isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named
AbnA and Abn2, respectively). BsArb43B shows low sequence identity with
previously characterized 1,5-a-l-arabinanases and is a much larger enzyme.
Here we describe the 3D structure of native BsArb43B, biochemical and
structure characterization of two BsArb43B mutant proteins (H318A and
D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in
complex with arabinohexose. The 3D structure of BsArb43B is different
from that of other structurally characterized endo-1,5-a-l-arabinanases, as

saccharides that includes xylans, arabinans, galactans,
mannans and glucans. l-arabinose, the second most
abundant pentose in nature, is found in significant
amounts in homopolysaccharides, branched and
de-branched arabinans, and heteropolysaccharides
such as arabinoxylans and arabinogalactans. Arabinan
is composed of a-1,5-linked l-arabinofuranosyl units,
some of which are substituted with a-1,3- and a-1,2-
linked chains of l-arabinofuranosyl residues [4,5]. Two
major enzymes hydrolyse arabinan: a-l-arabinofurano-
sidases (AFNs; EC 3.2.1.55) and endo-1,5-a-l-arab-
inanases (ABNs; EC 3.2.1.99). AFNs catalyze the
hydrolysis of terminal non-reducing a-l-1,2-, a-l-1,3-
and a-l-1,5-arabinosyl residues from various oligosac-
charides and polysaccharides, including arabinan, ara-
binoxylan and arabinogalactan [6,7]. ABNs attack the
glycosidic bonds of the a-1,5-l-arabinan backbone,
releasing a mixture of arabinooligosaccharides and l-
arabinose [4]. These types of enzyme have attracted
much attention due to their application in various
fields such as food technology, nutritional medical
research, plant biochemistry and organic synthesis
[4,5,8].
Bacillus subtilis, a saprophytic Gram-positive
endospore-forming bacterium, which is a commonly
used micro-organism in the antibiotic and enzyme pro-
duction industries, synthesizes two AFNs, encoded by
the genes abfA and abf2, and two endo-ABNs,
BsArb43A and BsArb43B, which are the products of
abnA and abn2 genes, respectively. Recently, the four

domain (Ala28–Tyr367) and a C-terminal domain
(Ala368–Ala470). The catalytic domain displays a char-
acteristic b-propeller fold [19,20], with five b-sheets,
called blades, arranged radially around a pseudo five-
fold axis (Fig. 1). Each blade comprises four anti-
parallel b-strands, and the catalytic domain comprises
20 b-strands and three a-helices. Two a-helices are
located after blade I, while the third is observed in a coil
region between the third and the fourth b-strands of
blade IV (Fig. 1). In BsArb43B, a connection between
the N- and C-terminal domains is made from the last
blade through a long linker, making the last b-strand of
this blade much shorter than the other strands. The
extra C-terminal domain comprises eight anti-parallel
b-strands and a small a-helix, arranged in a b-barrel-like
fold (Fig. 1).
The catalytic domain
The BsArb43B catalytic domain has the b-propeller
fold that is characteristic of this type of enzymes.
Superposition of the Ca trace of the BsArb43B
D. de Sanctis et al. Role of calcium in the glycosidase reaction
FEBS Journal 277 (2010) 4562–4574 ª 2010 The Authors Journal compilation ª 2010 FEBS 4563
catalytic domain with the Ca trace of BsArb43A from
B. subtilis (Protein Data Bank code 1UV4 [15]), a-l-
arabinanase (CjArb43A) from C. japonicus (Protein
Data Bank code 1GYD [14]), endo-1,5-a-l-arabinanase
(ABN-TS) from B. thermodenitrificans TS-30 (Protein
Data Bank code 1WL7 [16]) and endo-1,5-a-l-arab-
inanase (AbnB) from Geobacillus stearothermolhilus
(Protein Data Bank code 3CU9 [17]) using the

observed either between residues located in blades IV
and V and the C-terminal domain or between the hair-
pin that joins blades IV and V and the C-terminal
domain (Fig. 1 and Table S1). The apolar interactions
in the interface between the two domains include the
following residues: His37, His355, His345, Val36,
Pro39, Ile41, Phe48, Val50, Leu63, Trp66, Tyr322,
Tyr331, Ile333, Val347 and Val 349, while the polar
interactions are mainly hydrogen bonds and are listed
in Table S1.
In a similar manner to the other members of the
GH43 family, the BsArb43B catalytic domain contains
a large cavity that extends across the protein. During
refinement of the structural model, additional electron
density was observed in this cavity close to the catalytic
site, which could not be accounted for by protein
atoms. This density was modelled as a metal ion, here
refined as a calcium ion, hepta-coordinated by six water
molecules and a histidine residue (His318), giving a
cluster with a pentagonal bi-pyramid shape (Fig. 3A).
BsArb43B active site
The active site of BsArb43B is located in the deep cav-
ity at the centre of the b-propeller and comprises three
Fig. 1. Three-dimensional structure of BsArb43B created using
PyMol [21]. The N-terminal catalytic domain comprises a five-blade
b-propeller [blade I (residues 40–44, 47–51, 57–60, 67–70) shown in
orange; blade II (residues 103–106, 112–119, 126–133, 141–149)
shown in magenta; blade III (residues 173–176, 182–186, 193–197,
213–216) shown in blue; blade IV (residues 223–231, 236–243,
253–259, 295–298) shown in dark red; blade V (residues 312–323,

substrate [14,27]. In the case of BsArb43B, the general
base and the general acid (OD1 Asp38 and OE2
Glu224, respectively) are located approximately 5.8 A
˚
apart, and the third catalytic carboxylic acid (Asp171)
is located 4.1 A
˚
from the general acid. To probe the
function of the three residues, each of them was inde-
pendently substituted by an alanine. The arabinanase
activity of the mutants D38A, D171A and E224A was
assayed in an Escherichia coli periplasmic fraction and
compared to that of the wild-type (WT). Under these
conditions, the mutants displayed no measurable activ-
ity (data not shown), confirming the key roles of each
member of the triad of carboxylates in the catalytic
activity of BsArb43B.
As described above, a metal ion was observed fur-
ther down in the catalytic cavity, approximately 5 A
˚
below the catalytic carboxylates, which was hepta-
coordinated to six water molecules and a histidine
ligand. The presence of ions in an equivalent location
has been previously reported for other arabinanases
structures. In the ABN-TS model, a chloride ion was
assigned to this site [16]. A chloride was also indicated
for CjArb43A, but the authors did not exclude the
possibility that a calcium ion was present [14], and two
Ca
2+

cluster and
the Tris molecule observed in the binding pocket. The water mole-
cules are represented by red spheres. The anomalous Fourier map
that corresponds to the Ca
2+
ion is shown by a green mesh, and is
contoured at the 5r level. (B) Detail of the active site of the
BsArb43B H318A mutant (shown in dark grey) superposed on
native Abn2 (coloured according to the atom type). The cluster
undergoes a small reorganization, and a more planar conformation
of the five water molecules and the metal ion is observed.
D. de Sanctis et al. Role of calcium in the glycosidase reaction
FEBS Journal 277 (2010) 4562–4574 ª 2010 The Authors Journal compilation ª 2010 FEBS 4565
Fourier map at this wavelength. Together, these results
confirm that the atom in the cluster is calcium.
To determine whether the calcium ion has a specific
role in the activity of BsArb43B, assays were per-
formed in the presence of EGTA, a chelating agent
that binds Ca
2+
with a significantly larger affinity than
EDTA does. The results revealed a drastic decrease in
the activity of the enzyme in the presence of 1 mm
EGTA (14.94 ± 2.93 UÆmg
)1
), compared with the
activity values in the presence of EDTA (86.18 ±
13.78 UÆmg
)1
) or in the absence of chelators (90.94 ±

main chain (Fig. 3B). It is therefore not surprising
that the calcium cluster contributes to overall stabi-
lization of the b-propeller fold.
To further investigate the importance of the calcium
cluster, two mutants were produced in which the histi-
dine residue that coordinates the calcium was mutated
into an alanine (H318A) and a glutamine (H318Q).
These mutations aimed to disrupt the calcium cluster in
order to determine its importance for this type of pro-
tein. In enzymatic assays performed with both mutants,
there was a drastic decrease in enzymatic activity is
observed for the H318A mutant, and enzymatic activity
was completely lost for the H318Q mutant (Table 2).
Structure determination of the H318A mutant showed
essentially the same structure as that for the native
enzyme, with a rmsd of 0.20 A
˚
for 442 Ca pairs. How-
ever, in this mutant, a major difference was observed in
coordination of the calcium cluster. Surprisingly, muta-
tion of His318 to Ala does not unduly disrupt the
cluster, and hepta-coordination of the calcium ion is
maintained by an extra water molecule that is posi-
tioned where the NE2 of the histidine imidazole ring
would be located (Fig. 4C). Removal of the histidine
residue has the effect of relaxing the geometry of
the cluster, resulting in a more planar arrangement of
the Ca
2+
ion with five of the water molecules.

)
Ca 5.52 9.28 11.84
NE2 His318 2.54 7.26 2.53 9.55
w
1
2.52 7.28 2.49 7.53 2.45 11.17
w
2
2.47 8.27 2.60 11.51 2.44 9.37
w
3
2.48 7.22 2.48 11.11 2.50 9.58
w
4
2.46 6.05 2.54 8.10 2.52 10.58
w
5
2.44 5.61 2.57 6.28 2.50 10.72
w
6
2.43 6.97 2.49 6.49 2.42 10.03
w
7
2.49 10.96
Table 2. Catalytic activity of wild-type and mutants of BsArb43B.
Kinetic parameters were determined using linear arabinan as the
substrate. NA, no detectable activity.
Enzyme k
cat
(s

structure of native BsArb43B, the histidine residue
(His318) also has the ND1 atom within hydrogen
bonding distance of a Tris molecule which has been
modelled in the active site. Superposition of the struc-
tural models for BsArb43B and 2EXJ shows that the
Tris molecule is located where the xylose molecule is
observed in the b-xylosidase. These observations sug-
gest that the histidine residue is also involved in sub-
strate recognition and stabilization in BsArb43B. In
the absence of the histidine residue, recognition and
stabilization of the substrate are not as efficient as for
the native enzyme, and the efficiency of the enzyme
therefore decreases. On the other hand, when the histi-
dine residue is mutated into a glutamine, not only are
recognition and stabilization of the substrate compro-
mised, but there may also be disruption of the calcium
cluster, with a concomitant complete loss of activity,
as observed. It is probable that the calcium ion con-
tributes to modulation of the pK
a
values of the cata-
lytic carboxylates, thus ensuring the protonation
equilibrium necessary for enzyme activity.
Substrate-binding cleft
Analysis of the molecular surface of BsArb43B shows
an elongated surface groove across the face of the pro-
peller, which acts as the substrate-binding cleft (Figs 4
and 5). This type of cleft, open on both sides, can
accommodate several sugar units of a polymeric sub-
strate, and has been observed in other structurally

enzymatic activity. The loop II region comprises resi-
dues 227–233 in BsArb43B, and is similar in the four
arabinanases BsArb43B, ABN-TS, CjArb43A and
AbnB, but not BsArb43A. BsArb43A has a longer
loop located in a different position (Fig. 5). Loop III
is located in the other side of the binding cleft and
comprises residues 279–286 in BsArb43B (Fig. 5). This
loop is of similar size in all four endo-arabinanases
(BsArb43B, ABN-TS, BsArb43A and AbnB), but is
much longer in the exo-ABN (CjArb43A) and blocks
one of the ends of the binding cleft (Fig. 5). In fact,
in the structure of the exo-ABN complex with
arabinohexose [15], this loop makes the reducing end
of the carbohydrate chain bend towards the solvent,
probably optimizing binding of the carbohydrate chain
in the cleft, and resulting in trioses as products. These
observations suggest that, by blocking one of the ends
of the binding cleft, loop III in exo-ABN may be asso-
ciated with the exo activity observed in this enzyme,
whereas a much shorter loop, as observed in the
endo-arabinanases BsArb43B, BsArb43A, AbnB and
ABN-TS), which leaves this side of the binding cleft
open, is more suitable to accommodate a long poly-
meric carbohydrate chain [16].
In order to identify the residues involved in sub-
strate recognition, the structure of the D171A
BsArb43B mutant in complex with arabinohexaose
was determined. Some electron density was found in
the proximity of the catalytic site that could be mod-
elled as an arabino-trisaccharide (Fig. 4). The surface

approximately 3.5 A
˚
closer to one of the catalytic resi-
dues (Glu224), positioning the arabinose rings AHR3
and AHR2 in positions equivalent to subsites +1 and
+2 of the arabinotriose saccharide observed in the
G. stearothermophilus AbnB complex. The fact that a
fully occupied arabinose residue was not observed in a
position equivalent to the )1 subsite in the D171A
mutant structure was interpreted as a consequence of a
strongly reduced affinity of the mutant enzyme for
binding arabinose due to mutation of the catalytic resi-
due Asp171 into an alanine. This is further supported
by the fact that, even in the structure of the D171A
Fig. 5. Molecular surface of BsArb43B showing the loops that dif-
fer between the endo- and exo-arabinanases highlighted in different
colours: blue for BsArb43B, magenta for BsArb43A, green for ABN-
TS, dark grey for AbnB, red for exo-Abn.
Role of calcium in the glycosidase reaction D. de Sanctis et al.
4568 FEBS Journal 277 (2010) 4562–4574 ª 2010 The Authors Journal compilation ª 2010 FEBS
mutant crystallized without arabinohexose, no Tris
molecule is present in the catalytic site (data not
shown).
In the D171A BsArb43B mutant, additional electron
density was observed on the other side of the binding
cleft, opposite to where the arabinotriose molecule was
modelled (Fig. 4B). This residual electron density
could not be accounted for protein atoms or water
molecules, but its paucity did not enable any addi-
tional saccharide molecule to be inserted into the

Arg366(NH1)–Glu33(OE2), Arg366(NH2)–Glu33(OE1),
Asp392(OD1)–Lys296(NZ), Asp392(O)–Arg255(NH2),
Asp392(N)–Glu291(OE2), Lys398(O)–Thr302(N), Gln
443(OE1)–Tyr365(N)], together with a hydrophobic
core nestled between these entities (residues Val256,
Ala269, Val295, Met298, Tyr301, Trp359, Pro364,
Ile387, Leu458 and Trp466).
Structural alignment using DALI [30] or SSM from
EBI [31] does not show any relevant matches between
this domain and other structures in the databases. The
highest hit obtained with both servers is the exclusion
domain of dipeptidyl peptidase I or cathepsin C
(Protein Data Bank code 1K3B [32]). The role of the
C-terminal domain was further investigated by con-
struction of two truncated versions of the enzyme.
Based on structural and sequence alignments, two
truncated proteins were engineered that lack 119
residues (trunc1) or 106 residues (trunc2) at the C-
terminus (Fig. S2). The genes encoding the two mutant
proteins were individually expressed in E. coli, but the
proteins were not detected. The lack of accumulation
in vivo indicates poor stability, and suggests that the
presence of the C-terminal domain is crucial for
the acquisition of the correct enzyme fold. Analysis of
the interactions between the two domains led us to
presume that expression of the truncated version of
BsArb43B may expose the hydrophobic core described
above and reduce the protein stability.
An extra domain is found in putative arabinanases
that are present in the genomes of other bacteria, in

Concluding remarks
The work presented here shows that BsArb43B has a
3D fold that is different from those of other arabinan-
ases with a known structure. In addition to the
catalytic domain that is common to the other arab-
inanases, the BsArb43B 3D fold comprises an extra
C-terminal domain. Whether this extra domain is a
CBM or has a different function is still under investi-
gation. Detailed analysis of the binding cleft of
BsArb43B and the other structurally determined arab-
inanases showed that the exo-ABN from C. japonicus
has a long loop that occludes one of the sides of the
cleft, whereas all the endo-ABNs have loops of smaller
and similar size that leave the binding cleft open at
both sides, allowing it to act in endo mode. The pres-
ent work also enabled precise identification of the
metal in the active cleft as calcium, and suggested the
nature of its role in the enzymatic mechanism. Based
on data reported here, calcium appears to be impor-
tant for the enzymatic mechanism of the enzyme,
probably by directly influencing the protonation state
of the catalytic carboxylate. In addition, these data
also show that the histidine residue (His318) that coor-
dinates with the calcium also plays a role in the
enzyme mechanism by binding and stabilizing the
substrate in the active site.
Experimental procedures
Substrates
Debranched arabinan (linear a-1,5-l-arabinan, purity 95%)
and a-1,5-l-arabinooligosaccharides (arabinohexose, purity

Wavelength (A
˚
) 1.06725 0.93100 1.0332
Detector ADSC Quantum Q315r ADSC Quantum 4 ADSC Quantum Q315r
Distance 172.37 172.36 265.56
Resolution (A
˚
) 1.50 1.79 1.90
Space group P1 P1 P1
Cell parameters
a, b, c (A
˚
) 51.9, 57.9, 85.6 51.8, 57.4, 85.5 51.9, 57.6, 86.2
a, b, c (°) 96.2, 91.8, 117.3 82.1, 88.2, 63.6 82.3, 87.9, 63.6
Number of unique hkl
a
133 640 76 283 67 116
Completeness (%)
a
94.9 (92.7) 93.3 (72.2) 95.7 (87.1)
Mean I, r(I)
a
13.5, 2.2 13.7, 3.3 14.5, 6.4
R
symm
a
0.038 (0.377) 0.077 (0.385) 0.047 (0.136)
Multiplicity
a
2.0 (2.0) 3.9 (3.4) 2.0 (1.9)

were extracted from the periplasmic protein fraction by
cold osmotic shock, as previously described [18]. Bio-
chemical analyses revealed that all of the mutants were suc-
cessfully expressed and had a migration pattern on
SDS ⁄ PAGE identical to that of wild-type BsArb43B,
except for the truncated versions, which were not detected.
For purification of recombinant BsArb43B and the
BsArb43B H318A and H318Q mutants, the periplasmic
protein fraction was filtered and loaded onto a 1 mL
Histrap column (Amersham Pharmacia Biotech, Piscataway,
NJ, USA). The bound proteins were eluted by discontinu-
ous imidazole gradient, and fractions containing more than
95% pure protein were dialysed overnight against a dialysis
buffer (1 · phosphate buffer, 10% glycerol), and then
frozen in liquid nitrogen and kept at )80 °C until further
use.
Enzyme assays
The source of the enzyme was the periplasmic protein
fraction of E. coli cultures or purified arabinanases. The
enzyme activity was determined as previously described
[10]. The reducing sugar content after hydrolysis of the
polysaccharides was determined by the Nelson–Somogyi
method, with l-arabinose as standard [11]. One unit of
activity was defined as the amount of enzyme that produces
1 lmol of arabinose equivalents per minute. The kinetic
parameters (apparent K
m
and V
max
values) were determined

2+
) 21.3 (TRIS) 12.9 (Ca
2+
) 14.1 (TRIS)
Maximal estimated error (A
˚
) 0.044 0.071 0.071
Distance deviations
Bond distances (A
˚
) 0.017 0.010 0.015
Bond angles (A
˚
) 1.403 1.185 1.471
Planar groups (A
˚
) 0.006 0.005 0.005
Chiral volume deviation (A
˚
3
) 0.126 0.089 0.089
Ramachandran analysis (%) [44]
Favourable 98.1 (924 ⁄ 942) 98.1 (925 ⁄ 943) 98.1 (909 ⁄ 927)
Allowed 100 (942 ⁄ 942) 100.0 (943 ⁄ 943) 100.0 (927 ⁄ 927)
a
Calculated with 5% of reflections excluded from refinement.
D. de Sanctis et al. Role of calcium in the glycosidase reaction
FEBS Journal 277 (2010) 4562–4574 ª 2010 The Authors Journal compilation ª 2010 FEBS 4571
heated in an iCycler iQ5 Real Time PCR detection system
(Bio-Rad) from 20 to 90 °C, using increments of 1 °CÆ

crystal in the orthorhombic form. An initial structural model
was obtained as previously reported by de Sanctis et al. [18],
and was used as a search model for molecular replacement
using a 1.7 A
˚
resolution data set obtained from the native tri-
clinic (P1) crystal. The program PHASER [41] successfully
placed two molecules in the asymmetric unit, and model
building and refinement proceeded using the COOT [42] and
PHENIX [43] programs, iteratively. The PHENIX program
was used to refine atomic coordinates together with indivi-
dual isotropic atomic displacement parameters for all the
structures presented. TLS thermal anisotropic parameteri-
zation was included in the final stages of refinement. Each
molecule was divided into two TLS groups, corresponding to
the N- and C-terminal domains.
The structures of the mutant proteins H318A and
D171A were solved by the molecular replacement method,
using the program PHASER [41], with the model of the
native protein as refined from the triclinic crystal form as
the search model, without the solvent molecules. The struc-
tures were refined according to the procedure used for the
native BsArb43B in the triclinic form; refinement statistics
for all three structures (native BsArb43B, H318A and
D171A mutant proteins) are reported in Table 4.
Acknowledgements
Cla
´
udio M. Soares Andrea Spallarossa (Department of
Pharmaceutical Science, University of Genova) and

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Table S1. Residues involved in the interface between
the N- and C-terminal domains.
Table S2. Plasmids and oligonucleotides used in this
study.
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