de Sa´-Nogueira, Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Avenida de Repu´blica-EAN, 2780-157 Oeiras, Portugal Fax: +351 21 441 1277 Tel: +351 21 446 9
Trang 1reaction of GH43 arabinanases
Daniele de Sanctis1,2,*, Jose´ M Ina´cio1,*,, Peter F Lindley, Isabel de Sa´-Nogueira1,3and
Isabel Bento1
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 Cieˆncias da Vida, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal
Keywords
Bacillus subtilis; catalytic mechanism;
crystallography; endo-a -L- arabinananase
GH43; mutagenesis
Correspondence
I Bento, Instituto de Tecnologia Quı´mica e
Biolo´gica, Universidade Nova de Lisboa,
Avenida de Repu´blica-EAN, 2780-157
Oeiras, Portugal
Fax: +351 21 441 1277
Tel: +351 21 446 9100
E-mail: bento@itqb.unl.pt
I de Sa´-Nogueira, Instituto de Tecnologia
Quı´mica e Biolo´gica, Universidade Nova de
Lisboa, Avenida de Repu´blica-EAN,
2780-157 Oeiras, Portugal
Fax: +351 21 441 1277
Tel: +351 21 446 9100
E-mail: sanoguei@itqb.unl.pt
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
it comprises two domains, an N-terminal catalytic domain, with a 3D fold similar to that observed for other endo-1,5-a-l-arabinanases, and an additional C-terminal domain Moreover, this work also provides experi-mental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzy-matic mechanism of BsArb43B
Abbreviations
ABN, arabinanase; AFN, arabinofuranosidase; APBS, adaptive Poisson-Boltzmann solver; CBM, carbohydrate- binding module; GH, glycoside hydrolase; MPD, 2-methyl-2,4-pentadiol; SAD, single wavelength anomalous dispersion; Se-Met, Se- Methionine; TLS, translation/libration/ screw.
Trang 2The plant cell wall is structurally complex and
bio-logically recalcitrant Micro-organisms, in particular
saprotrophs, play a fundamental role in the
decompo-sition processes of plant biomass, secreting numerous
polysaccharide-degrading enzymes that attack
cellu-lose, hemicellulose and pectin Mobilization of plant
biomass for chemical and fuel production is a major
biotechnological challenge of the 21st century, and the
use of polysaccharide-hydrolysing enzymes in biomass
saccharification is promising [1,2] Although recent
years have seen significant advances in interpretation
of new structures of (hemi)cellulose hydrolytic
enzymes, a full understanding of the details of
sub-strate recognition and catalysis by these varied and
highly specific enzymes remains an important goal [3]
Hemicellulose is the second most abundant
renew-able biomass polymer after cellulose This fraction of
plant cell walls comprises a complex mixture of
poly-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 polysacoligosac-charides, 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
Bacillus subtilis arabinases were independently
charac-terized at the genetic and biochemical level [9–12]
Both the endo-ABNs, BsArb43A and BsArb43B,
belong to glycoside hydrolase (GH) family 43, a heter-ogeneous group of enzymes comprising endo- and exo-a-l-arabinanases (EC 3.2.1.99), b-xylosidases (EC 3.2.1.37), a-l-arabinofuranosidases (EC 3.2.1.55), xylanases (EC 3.2.1.8) and galactan
1,3-b-galactosidas-es (EC 3.2.1.145) (http://www.cazy.org/) [13] The crys-tal structures of an exo-ABN, CjArb43A from Cellvibrio japonicus [14] and three endo-ABNs, BsArb43A from B subtilis [15], ABN-TS from Bacil-lus thermodenitrificansTS-30 [16] and AbnB from Geo-bacillus stearothermophilus [17], have been determined, and showed a catalytic domain consisting of a five-bladed b-propeller fold However, BsArb43B is a much larger enzyme, and displays less than 23% amino acid identity with previously characterized ABNs We have previously reported the crystallization and preliminary X-ray analysis of BsArb42B [18] Here we present the 3D structure of the wild-type enzyme and describe mutant proteins, providing new evidence for the roles
of the calcium cluster observed in the active cleft and particular amino acids in enzymatic activity
Results and Discussion
BsArb43B (Abn2) structure The three dimensional structure of BsArb43B comprises all 443 amino acids of the mature protein BsArb43B consists of two domains, an N-terminal catalytic 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
Trang 3catalytic 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
LSQKAB program [22] gave the following rmsd
val-ues: 1.50 A˚ for 192 Ca pairs, 1.66 A˚ for 189 Ca pairs,
1.68 A˚ for 211 Ca pairs and 1.66 A˚ for 189 Ca pairs,
respectively (Fig 2) These values show that the
sec-ondary structure of the b-propeller is well conserved,
with the differences located mainly in the coil regions
that connect the five blades, and in the region that
connects the N- and C-terminal domains within the
fifth blade (Fig 2) As described above, BsArb43B has
two a-helices after blade I, where usually only one is
observed, and a third a-helix in blade IV that is not
observed in the other endo-arabinanases
The BsArb43B catalytic domain does not show the
‘Velcro’ closure [19,23,24] that is characteristically
observed in other proteins with the b-propeller fold In
the ‘Velcro’ closure, the N- and C- termini are joined
in the same sheet to ‘seal’ the circular array of the b-propeller [20] In BsArb43B, closure of the b-propel-ler is achieved in a different way, by a set of polar and hydrophobic interactions established within the N-ter-minal catalytic domain and between the N- and C-ter-minal domains (Fig 1) These types of interactions are 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,
330–337, 346–354, 360–362) shown in cyan] and three short
a-heli-ces Regions connecting the blades are shown in green The
C-ter-minal domain comprises eight b-strands arranged in a distorted
b-barrel-like configuration (shown in yellow) The hairpin that joins
blades IV and V in the N-terminal domain and interacts with the
C-terminal domain is shown in red.
Fig 2 Structural overlay with other arabinanases (EC 3.2.1.99) belonging to the GH43 family BsArb43B is shown in blue, Cellvib-rio japanicus exo-arabinanase (Protein Data Bank code 1GYD) in red, Bacillus subtilis arabinanase (Protein Data Bank code 1UV4) in green, Bacillus thermodenitrificans arabinanase (Protein Data Bank code 1WL7) in orange, and arabinanase from Geobacillus ste-arothermolhilus (Protein Data Bank code 3CU9) in yellow.
Trang 4carboxylate residues: Asp38, Asp171 and Glu224
(Fig 3A) These three acidic residues, conserved in all
members of GH families 32, 43, 62 and 68 [25], are
responsible for the general acid catalysis that leads to
hydrolysis of the glycosidic bond Within GH family
43, the enzymes work by an inversion mechanism, in
which one carboxylate acts as a general base catalyst, deprotonating the nucleophilic water molecule that attacks the bond, and the second acidic residue acts as
a general acid catalyst, protonating the departing agly-cone [26] Putative roles of the third residue include acting as a pKamodulator and maintaining the correct alignment of the general acid residue relative to the 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
Ca2+ ions were modelled with in the axial cavity of BsArb4A [15] Recently, a calcium ion was also modelled at this position in the structure of the endo-arabinanase from G stearothermophilus [17] In the BsArb43B structure, the coordination distances and geometry at this site strongly suggest the presence of a
Ca2+ ion in the axial cavity (Table 1) This was first confirmed by an X-ray fluorescence spectrum on a native crystal, for which a peak corresponding to cal-cium was observed (Fig S1), even when no calcal-cium salt was added to the protein solution during purifica-tion or crystallizapurifica-tion In addipurifica-tion, a 12r peak was observed at that position on an anomalous difference Fourier map, calculated from data collected at 1.067 A˚ wavelength, and was refined perfectly as a calcium ion (Fig 3B) However, a chloride peak was also observed
in the X-ray fluorescence spectrum, and chloride is present in the protein buffer (NaCl + Tris⁄ HCl) and the crystallization solution [18] However, chloride ions
do not normally adopt such a coordination, but it is typical of calcium ions (Table 1) [28], and would not result in such a high peak of density in the anomalous
A
B
Fig 3 (A) Detail of the BsArb43B active site showing the three
catalytic carboxylates (D38, D171 and E224), the Ca2+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 Ca2+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.
Trang 5Fourier 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 Ca2+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 ±
7.85 UÆmg)1) These results suggest that the presence
of calcium is important for the optimal activity of
BsArb43B
Furthermore, to determine whether the role of the
calcium is structural, thermal shift assays were
per-formed to determine the Tmof the protein in the
pres-ence and abspres-ence of the calcium ion, using EDTA
and EGTA as chelators, as described above The
sam-ples were incubated with both chelators for 24 h prior
to the thermal shift assays being performed The Tm
values obtained for the native enzyme and for the
samples incubated with EGTA and EDTA differ by
2C and 1 C, respectively In addition, whereas the
native and EDTA samples show the same type of
sharp transition, the transition in the sample
incu-bated with EGTA is much smaller, with a broader
minimum (Fig S3) These results indicate that the
protein is less stable when incubated with EGTA As
EGTA is a strong calcium chelator, it can be
postu-lated that the loss of stability is associated with loss
of the calcium cluster As described above, the
calcium atom is coordinated by six water molecules
and a histidine residue (His318) (Fig 3A) Moreover,
the water molecules all lie within hydrogen bonding
distance of oxygen carbonyl atoms from the protein
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 Ca2+ion with five of the water molecules
As stated above, both mutations affect the enzy-matic activity of the mutant proteins, with a complete
Table 1 Coodination distances of the calcium cluster.
Distance to
Ca atom (A ˚ ) Bfactor(A˚2 )
Distance to
Ca atom (A ˚ ) Bfactor(A˚2 )
Distance to
Ca atom (A ˚ ) Bfactor(A˚2 )
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.
k cat ⁄ K m (s)1l M )1)
BsArb43B (WT) 191.6 ± 5.9 111.0 ± 3.0 1.72
Trang 6loss of activity in the H318Q mutant As the calcium
cluster is present in the H318A mutant, it appears that
the decrease in activity observed in this mutant may be
due to absence of the histidine residue This histidine
residue is conserved in the majority of arabinanases
(Fig S2), and a histidine residue was also found in an
equivalent position in the structure of a b-xylosidase
from the GH43 family For this b-xylosidase from
G stearothermophilus, it was suggested that the
histi-dine residue was involved in substrate recognition by
establishing a hydrogen bond with a unit of xylose in
a substrate–enzyme complex [27] Likewise, in the
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 pKa 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
characterized endo-arabinanases [14–17] The
sugar-binding cleft traverses the catalytic domain, and is
located between blades II and III on one side and
blades V and I on the other Structural comparison of
the BsArb43B binding cleft with the binding cleft of
the other four arabinanases with known structure
(BsArb43A, ABN-TS, AbnB and CjArb43A; see above
for details) shows that differences are mainly found in
three loop regions (Fig 5) Loop region I is located in
one side of the binding cleft, comprises residues 53–55,
and aligns with the loop that includes residues 30–35
A
B
B
Fig 4 (A) Surface charge distribution of BsArb43B in the proximity
of the active site Arabinotriose in the binding cleft is represented using sticks (B) Detailed view of the binding cleft with arabinotri-ose (C) Details of the residues defining the binding cleft Hydrogen bond interactions between arabinotriose and the polar residues of the cleft are shown as dashed lines.
Trang 7(amino acid sequence LTEER) in BsArb43A In this
latter enzyme, the loop was suggested to induce
endo-activity in the exo-arabinanase CjArb43A from
C japonicus[15] However, in BsArb43B and the other
two endo-arabinanases, this loop is more similar in size
to that found in the exo-ABN, and does not show the
same sequence motif These observations indicate that
this loop is not in itself responsible for this type of
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
of the cleft is defined by residues Trp100, Cys119,
Pro124, Tyr189, His220, Leu246, Phe284 and Phe285,
and by the main chain of residues Asp122 and Ser123
The trisaccharide molecule was found to interact
directly with the enzyme by establishing hydrogen
bonds with residues Ser190, His220 and Glu224, and
indirectly, through water molecules, with residues
Asn166, Ser188, Tyr189 and Leu246 These
interac-tions are established by the two more deeply buried
arabinose residues (AHR2 and AHR3) Stacking
inter-actions were also observed between these two
arabi-nose rings and Tyr189 and Pro124, respectively No
interaction was observed between the most exposed
arabinose residue of the trisaccharide (AHR1) and the
protein surface This arabinose residue is oriented
towards the solvent region, and presumably the other residues of the arabinohexose are disordered and do not interact directly with the protein (Fig 4B)
The location of the trissacharide residue in the BsArb43B D171A mutant is similar to that observed for AbnB of G stearothermophilus in complex with arabinotriose (Protein Data Bank code 3D5Z [17]) However, in the latter structure, the arabinotriose is more buried in the binding pocket, occupying subsites )1 to +2, and the first arabinose residue interacts directly with the catalytic residues Asp27 and Asp147, adopting an equivalent position to that observed for a Tris molecule in the native BsArb43B structure Indeed, in BsArb43B D171A complex structure, the most internal arabinose ring (AHR3) is located 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 difdif-ferent colours: blue for BsArb43B, magenta for BsArb43A, green for
ABN-TS, dark grey for AbnB, red for exo-Abn.
Trang 8mutant 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
model Nevertheless, this electron density could result
from a partially occupied alternative binding of the
polysaccharide chain or from a saccharide product that
resulted from residual activity of the mutant
The C-terminal domain
The most obvious structural difference between
BsArb43B and other arabinanases is the presence of
the additional C-terminal domain This domain
com-prises 103 amino acids (residues 367–470) organized in
a short piece of a-helix and eight b-strands, arranged
in a distorted b-barrel-like configuration (Fig 1)
Although the reported structures of arabinanases do
not show such domains, other members of GH43
fam-ily contain an extra domain, the carbohydrate- binding
module (CBM), namely b-xylosidase from G
staero-thermophilus(XynB3, Protein Data Bank code 2EXH)
[27] and arabinoxylan arabinofuranohydrolase
AXH-m2,3 from B subtilis (BsAXH-AXH-m2,3; Protein Data
Bank code 3C7E) [29] Structural comparison of the
BsArb43B C-terminal domain with these two
struc-tures reveals that its orientation is different In
BsArb43B, the extra C-terminal domain is close to
bla-de V of the catalytic domain, but the CBM domains in
XynB3 and BsAXH-m2,3 are located close to blade I
In addition, the CBMs of XynB3 and BsAXH-m2,3
appear to be completely independent from the catalytic
domain, interacting mainly through polar contacts In
BsArb43B, the putative CBM appears to interact more
tightly with the N-terminal domain, and hydrogen
bonds are observed between these two entities [residues
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 particular those of the Bacillus⁄ Clostridium group CBMs are commonly found by glycoside hydrolases, which utilize an insoluble substrate in order to attack this polysaccharide more efficiently For this reason, CBMs retain the ability to concentrate enzymes onto the polysaccharide substrate, leading to more rapid degradation of the polysaccharide [33] Detailed analy-sis of the putative CBM of BsArb43B does not show any evidence of extra sugar binding sites However, determination of the surface charge distribution using adaptive Poisson-Boltzmann solver (APBS) [34] (Fig 6) showed that the C-terminal domain presents a
Fig 6 Surface charge distribution of Abn2, viewed from the oppo-site side of the active oppo-site It is possible to identify the entrance of the funnel between the blades of the propeller, which is partially negatively charged The putative CBM shows a mostly positive charge.
Trang 9largely positively charged surface, due to the residues
Lys376, Lys378, Lys421, Lys424, Arg442, Arg448 and
Lys469, and this feature could be related to its
hypo-thetical function Whether this extra domain
consti-tutes a CBM or is an evolutionary relic of a longer
ancestoral enzyme is currently under investigation It is
worth noting that no CBM with a similar b-barrel-like
shape has yet been reported The b-trefoil folding of a
protein of the CBM family 13 (http://www.cazy.org/)
[13] resembles a b-barrel fold, but structural
compari-son between the putative CBM of BsArb43B and
CBM13 does not suggest any relevant similarity
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 idpres-entification 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 95%) were purchased from Megazyme International (Bray, Ireland)
Bacterial strains and growth conditions Escherichia coli DH5a (Gibco BRL Richmond, CA, USA) was used for routine molecular cloning, and E coli BL21 (DE3) pLysS [35] was used as the host for expression of the recombinant protein BsArb43B from Bacillus subtilis 168T+ and mutant proteins All strains were grown on Luria–Bertani medium [36], with kanamycin (30 lgÆmL)1), chloramphenicol (20 lgÆmL)1) and isopropyl-b-d-thiogalac-topyranoside) being added as appropriate
DNA manipulation and mutagenesis DNA manipulations were performed as described previ-ously [37] PCR amplifications were performed in a MyCy-cler thermal cycler (Bio-Rad, Hercules, CA, USA) A QIAprep Spin Miniprep kit (Qiagen, Valencia, CA, USA) was used to purify the plasmids DNA was sequenced using
an ABI PRIS BigDye Terminator Ready Reaction Cycle
Table 3 Data collection statistics.
Beam line at European
Synchrotron Radiation Facility
Cell parameters
a For the highest-resolution shell: 1.58–1.50 A ˚ for D171A, 1.89–1.79 A˚ for H318A b Data adapted from de Sanctis et al 2008 [18].
Trang 10Sequencing kit (Applied Biosystems, Foster City, CA,
USA) Amino acid substitutions in BsArb43B were created
using the QuikChange site-directed method (Strategene, La
Jolla, CA, USA) using the respective mutagenic
oligonu-cleotide pairs (Table S2) and plasmid pZI39 as template
Truncation of BsArb43B was performed based on sequence
(first 363 residues) and structure (first 350 residues)
align-ments Briefly, the truncated C-terminus of the protein was
amplified by PCR using primers ARA246 and ARA404
(sequence alignment) or primers ARA246 and ARA384
(structure alignment), with pZI39 as template (Table S2)
The resulting 623 or 581bp DNA fragments, respectively,
were digested using SalI and XhoI, and cloned into the
same sites of pZI39 The presence of mutations and correct
truncation of BsArb43B were verified by sequencing of the
resulting plasmids
Protein expression and purification
For protein over-production, E coli BL21 (DE3) pLysS
cells carrying the desired plasmid were grown on LB
med-ium, and the extracellular BsArb43B and derived mutants
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 Kmand Vmaxvalues) were determined from the Lineweaver–Burk plot at optimum pH and tem-perature using linear a-1,5-l-arabinan as the substrate at concentrations ranging from 1 to 10 mgÆmL)1
Thermal shift assays Samples were prepared by adding 5· Sypro Orange (Molecular Probes, Carlsbad, CA, USA) to a mixture con-taining the protein solution in a 96-well thin-wall PCR plate sealed with optical-quality sealing tape (Bio-Rad) and
Table 4 Refinement statistics.
Mean B values (A ˚ 2 )
Distance deviations
Ramachandran analysis (%) [44]
a Calculated with 5% of reflections excluded from refinement.