From the Laboratorium voor Eiwitbiochemie en
Eiwitengineering, Ghent University, Ledeganckstraat 35, B-9000
Gent, Belgium and the
Laboratoire de Biochimie, University of
Liège, Institute of Chemistry, Sart-Tilman B-4000
Liège, Belgium
Received for publication, July 10, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Enzymes from psychrophilic organisms differ from
their mesophilic counterparts in having a lower thermostability and a
higher specific activity at low and moderate temperatures. The current consensus is that they have an increased flexibility, enhancing accommodation and transformation of the substrates at low energy costs.
Here we describe the structure of the xylanase from the Antarctic
bacterium Pseudoalteromonas haloplanktis at 1.3 Å resolution. Xylanases are usually grouped into glycosyl hydrolase
families 10 and 11, but this enzyme belongs to family 8. The fold
differs from that of other known xylanases and can be described as an ( It has been stated that life can be successful almost
everywhere on our planet and micro-organisms have indeed been isolated from some of the most extreme environments on earth, including the
extremes of pH, pressure, and temperature. Of these extremophilic micro-organisms, the thermophiles and hyperthermophiles have been extensively studied for several years, while cold-dwelling organisms have only recently attracted attention. Cold-adapted, or psychrophilic, organisms have developed adaptation mechanisms to overcome the low
temperature challenge, the most important of which is the production of
cold-active enzymes. Various adaptational strategies have been proposed
for these enzymes. The current accepted consensus is that they have an
increased flexibility, thus enabling the conformational changes
necessary for activity at low temperature (see Refs. 1 and 2 for recent
reviews). They are characterized by an increased turnover number and
physiological efficiency
(kcat/Km) at low and moderate
temperatures, as well as by a reduced stability. To date, only four
crystal structures of cold-adapted enzymes originating from
psychrophilic micro-organisms have been reported (3-6) and no general
rules as to how these proteins maintain sufficient flexibility have
been deduced. The structures of psychrophilic enzymes can provide clues
to how their stability at higher temperatures can be improved while
their flexibility in a colder environment is maintained, a trait that
makes them useful for many biotechnological applications (7).
Here we describe the structure of a xylanase from the Antarctic
bacterium Pseudoalteromonas haloplanktis. Xylanases (EC
3.2.1.8) catalyze the hydrolysis of the
The psychrophilic xylanase hydrolyzes xylan to principally xylotriose
(X3) and xylotetraose (X4) and, in contrast to
other currently identified xylanases, it operates with inversion of anomeric configuration. The activity of the enzyme on X5 is
extremely low, while the catalytic efficiency on X6 is much
higher, indicating that the enzyme has a large substrate binding cleft,
containing at least six xylose binding subsites (12). The structure of the xylanase is analyzed in terms of cold adaptation, and features that
may be important for substrate binding and selectivity are described.
In addition, the native enzyme is compared with an enzyme/xylobiose
complex and a partly inactivated D144N mutant.
Wild Type Psychrophilic Xylanase--
The psychrophilic xylanase
(pXyl)2 was expressed in
Escherichia coli and purified as previously described
(12).
D144N Mutant--
The psychrophilic xylanase gene,
including its signal sequence, was introduced into the Ndel
and Xhol sites of the cloning vector pSP 73® (Novagen). The
mutation was introduced by PCR with Pwo polymerase,
using the sense primer (5'-GCCCCGCTCCCAATGGCGAAGAGTAC-3') containing the Asp to Asn mutation D144N (underlined), and the antisense primer (5'-CTCATCCACTTTATAAACAAAGCCGTTTTGA-3'). The PCR
product was purified and circularized, used to transform to E. coli DINO RR1®, and double-strand sequenced using an ALF DNA sequencer (Amersham Biosciences). The mutated xylanase gene was excised
with NdeI and XhoI, ligated into the pET22b(+)
cloning vector (Novagen) and used to transform to E. coli
BL21 (DE3) cells (Stratagene). Production and purification was carried
out as described for the wild type recombinant xylanase (12). The
kcat/Km ratio was determined
from initial rates at substrate concentrations of 2.0, 2.2, and 2.5 mg/ml soluble birchwood xylan, using the following relation:
kcat/Km = v0/S0E0, which is valid at
S0 Crystallization and Data Collection--
Crystallization, data
collection, and SAD phasing of the native enzyme and the
selenomethionine-labeled mutant were performed as described previously
(13). Crystals of the D144N mutant were obtained under the same
conditions as the wild type enzyme. A complex of the native enzyme with
xylobiose was obtained by soaking the native crystals in a solution
containing 70% 2-methyl-2,4-pentanediol, 0.1 M sodium
phosphate, pH 7.0, and 10 mg/ml xylobiose for 48 h. Data
collection statistics for the complexed xylanase and the D144N mutant
are shown in Table I. These data were
collected at the protein crystallography beamline of the ELETTRA
synchrotron (Trieste, Italy). Statistics for the native xylanase and
the selenomethionine-labeled mutant used in SAD phasing have already
been described (13).
Refinement and Structural Analysis--
A
FOM of 0.93 was obtained for the
selenomethionine mutant after density modification, resulting in an
interpretable electron density map for the native enzyme and the
autotracing of 400 residues using ARP-wARP (14). The structure was
further refined against the native 1.3 Å data using the graphics
program TURBO-FRODO (15) and Refmac5 (16). B-factors were refined
anisotropically in the latter refinement rounds. The stereochemistry
was checked using PROCHECK (17), which showed that all the residues are in allowed regions of the Ramachandran plot. The structures of the
xylobiose complex and mutant D144N were refined using the coordinates
of the native enzyme as a starting model. Refinement was as for the
native enzyme except that the B-factors were refined isotropically for
mutant D144N. Refinement statistics are shown in Table
II. Side chains with missing
electron density were not modeled. For the xylobiose complex, final
electron density was good enough to place only one xylosyl residue. The
hydroxyl group on the anomeric carbon of this residue was refined with
a double conformation corresponding to 65%
Hydrogen bonds and ligand interactions were calculated with the
programs HBPLUS (19) and LIGPLOT (20), respectively. Superpositions were performed using PROSUP (21) and LSQKAB (16) while surface features
were analyzed with GRASP (22). Unless otherwise stated, figures were
prepared using MOLSCRIPT (23) and BOBSCRIPT (24).
Overall Fold--
The structure of the psychrophilic xylanase
consists of 13 Comparison with Clostridium thermocellum Endoglucanase--
The
psychrophilic xylanase belongs to glycosyl hydrolase family 8, which
mainly comprises endoglucanases (9). The structure of the catalytic
domain of one of these endoglucanases, CelA from the thermophilic
bacterium C. thermocellum, has been reported earlier (25).
The ( General Adaptation to Low Temperature--
It is generally
accepted that cold-adapted proteins are more flexible than their
mesophilic counterparts, with a reduced number of weak interactions.
This flexibility often coincides with a reduced stability of the
psychrophilic protein. In order to identify the cold-adapted properties
of pXyl, the structure was analyzed and compared in detail with that of
the C. thermocellum CelA, a summary of which is given in
Table III. It must be taken into account,
however, that the amount of sequence identity between both enzymes is
quite low (23%), and differences may therefore be due to this distant
relationship. In relation to their adaptation to temperature, it has
been found that CelA has an optimum temperature for activity near
80 °C (30), while pXyl has maximum activity near 35 °C (12).
Differential scanning calorimetry studies have also demonstrated the
lower stability of the psychrophilic enzyme, with an estimated melting
temperature (Tmm) of 52.6 °C (12) and
83.4 °C 3 for pXyl and
CelA (minus the C-terminal dockerin domain), respectively.
It can be seen from Table III that the most striking difference between
the two proteins is in the number of salt bridges, with clearly more
stabilizing ionic pairs in CelA. This latter protein also has two extra
arginine residues, and as arginines can form 5 different hydrogen
bonds, they are therefore expected to be important for stabilization.
In addition, pXyl has a higher accessible surface area than CelA and it
exposes a larger percentage of hydrophobic residues, a destabilizing
factor due to the ordering of water molecules. An increased exposure of
hydrophobic residues has also been reported for the structures of
Previous studies have indicated that, in addition to the features
already mentioned, some psychrophilic enzymes may be characterized by a
decreased number of hydrogen and/or disulfide bonds, a decreased number
of proline residues, an increased number of glycine residues and/or by
insertions in loops, as compared with their mesophilic and thermophilic
counterparts (32, 33). From Table III, however, it can be seen that
this is not the case for pXyl, indicating that this enzyme does not use
these strategies for cold-adaptation.
In addition to the destabilizing adjustments noted above, many
psychrophilic enzymes are characterized by alterations in their active
site with, in particular, an increased size and accessibility (3, 32)
as well as an optimization of the electrostatic potential (6, 32). Fig.
3 gives a surface representation of CelA and pXyl, and it is clear that
the substrate binding groove is larger and contains more acidic
residues in the case of CelA. The xylanase is known to cleave xylan,
which can be substituted with negatively charged
D-glucuronic acid or
4-O-methyl-D-glucuronic acid at the OH-2
position (12, 34, 35). At low temperatures, both a decreased
flexibility of the enzyme and a decreased diffusion rate can influence
the overall enzymatic reaction rate. Thus, the decreased negativity of
pXyl compared with CelA may be an adaptation of the enzyme to
circumvent repulsive interactions between negatively charged residues
and substrate, and may enhance the rate at which pXyl binds substituted
xylan. Nonetheless, it is necessary to compare this situation with the
structure of mesophilic family 8 xylanases in order to determine
whether or not this is a true factor of cold adaptation. Indeed, the
importance of redistribution of charges has also been described for a
cold-adapted citrate synthase (3) and a psychrophilic malate
dehydrogenase (6) where decreased negative potentials or increased
positive potentials are found at the protein surface surrounding the
binding site for negatively charged substrate.
A greater accessibility of the active site may enhance the binding of
substrate to enzyme and may be an adaptive strategy of cold-adapted
enzymes (3). Mutagenesis studies have, however, questioned this
proposition (36). In pXyl, the accessibility of the substrate-binding
region is substantially lower than in CelA (Fig. 3), which is mainly
the result of loop 263-276 folding over the groove in pXyl. The pXyl
structure therefore also questions the importance of substrate
accessibility in the cold adaptation of this enzyme.
The flexibility of specific residues in the substrate binding site is
another factor suggested to be important for adaptation to low
temperatures and, indeed, in pXyl the aromatic residues that line
subsites +1 and +2, Tyr-381 and Phe-280 respectively, were refined with
a double conformation. Both double conformations are dependent on one
another: conformation B from Tyr-381 sterically clashes with
conformation A from Phe-280 (Fig. 5). In the case of a cold-adapted
protease, it has been observed that an active site tyrosine adopts a
double conformation as well, corresponding to both a substrate-bound
and a substrate-free form (37). The flexibility of residues in the
active site was therefore proposed to be a cold-adapted feature. In
pXyl it is not clear why extra flexibility of these two residues is
necessary because, in CelA, a similar conformation is found in both the
native and the substrate-bound enzyme (25, 38). However, small
movements of these side chains may be necessary for the accommodation
of substrate, and an increased flexibility of these residues may
therefore assist in binding the substrate rapidly.
In the structure of a psychrophilic malate dehydrogenase (6), it was
observed that the relative B-factors for residues interacting with
substrate were higher than for a thermophilic counterpart. This
suggests that the relative flexibility of these residues is higher in
the psychrophile, which may lead to an increased catalytic efficiency.
The relative B-factors for the aromatic residues lining the different
subsites in pXyl and CelA are shown in Table
IV. Absolute B-values can vary because of
differences in data quality or refinement procedures, and the
usefulness of relative B-factors resides in the fact that this bias is
removed. It is clear that the highest flexibility is observed for
Tyr-378 in both enzymes, and compared with CelA, a relatively higher
B-factor is observed for Trp-124, Tyr-381, and Phe-280 in pXyl. No
significantly increased flexibility in terms of relative B-values is
observed for the aromatic residues lining subsites
Taking all these features together, we can therefore conclude that the
main cold-adaptation features in pXyl are a drastically reduced number
of ionic pairs, an increased exposure of hydrophobic residues, and
possibly an optimization of the electrostatic potential at the active
site and an increased flexibility of the aromatic residues lining the subsites.
Substrate and Product Binding--
Recently, a complex was obtained between an inactive family 8 endoglucanase mutant (E95Q) and glucopentaose (38). Six different subsites were identified, but none of these subsites (nomenclature of
Davies et al., Ref. 39) corresponds to the positioned xylose residue (Fig. 5). The xylose residue that
was not modeled points toward the +3 subsite from CelA, but does not
coincide with it. Indeed, binding of the xylobiose at the new site may
highlight an extra subsite +4, which may be important for directing the leaving product.
We have attempted to obtain a complex of the mutated xylanase with
xylohexaose, but as yet no successful results have been obtained.
However, on comparing the identified subsites from CelA (38) with the
corresponding regions in the cold-adapted enzyme, it is clear that many
differences occur (Table V). The largest discrepancy occurs at subsite
The smallest number of differences occurs at subsite
Comparing the subsites also reveals the structural basis for
discrimination between glucose and xylose residues. Xylopyranose basically differs from glucopyranose by the absence of a
CH2OH group at atom C-5 and it is therefore expected that
amino acid residues interacting with this OH-6 group in CelA are not
conserved in pXyl. Indeed, interacting residues in subsites
Hydrolytic studies suggest that pXyl can hydrolyze the Catalytic Site--
Family 8 glycosyl hydrolases are inverting
enzymes that cleave
Fig. 6A shows some important
interactions and distances between the different residues in the
catalytic site of pXyl. The prediction of a hydrogen bond between
Asp-144 and Glu-78 depends on the program used, but the two carboxyl
groups are very close to each other (2.41 Å), which may mean that at
least one of the carboxyl groups is neutral in charge. It can be seen
that Asp-144 also forms a 2.93 Å hydrogen bond with Arg-284.
As Asp-144 is strictly conserved among the family 8 glycosyl
hydrolases, a mutant of pXyl was produced in which this residue was
replaced by Asn. In agreement with the results for the family 8 endoglucanase K from Bacillus sp. KSM-330 (9), mutation of this residue resulted in a reduction of the catalytic efficiency (kcat/Km). However, while a
1820-fold reduction was obtained in the case of the endoglucanase K
mutant (9), the catalytic efficiency of the pXyl mutant (0.051 ± 0.006 ml·mg
It has also been suggested that Asp-281 is the catalytic base in family
8 enzymes (38). Structural analysis of CelA has recently identified
Glu-78 and Asp-281 (pXyl numbering) as the general acid and general
base residues in this enzyme, respectively. It was also shown that
Asp-144 plays a role in hydrolysis, as it stabilizes the sugar ring in
subsite
Curiously, the geometry of the catalytic site residues in CelA
resembles more that of the D144N mutant than that of wild type pXyl
(Fig. 8). Glu-78 and Asp-144 (pXyl numbering) in CelA are not involved
in a hydrogen bond (Fig. 6, B and C), and the two residues adopt the same conformation as in the D144N mutant xylanase. Furthermore, the Asp-281 residue in CelA forms a hydrogen bond with
Asn-200, which replaces Asp-200 (pXyl numbering).
Conclusion--
The xylanase from the psychrophilic organism
P. haloplanktis is a cold-adapted family 8 glycosyl
hydrolase displaying an (
Compared with CelA, the substrate binding region differs appreciably.
The subsites are not conserved, with large differences occurring at
subsites +3 and /
)6 barrel. Various parameters that may explain the
cold-adapted properties were examined and indicated that the protein
has a reduced number of salt bridges and an increased exposure of
hydrophobic residues. The crystal structures of a complex with
xylobiose and of mutant D144N were obtained at 1.2 and 1.5 Å resolution, respectively. Analysis of the various substrate binding
sites shows that the +3 and
3 subsites are rearranged as compared to
those of a family 8 homolog, while the xylobiose complex suggests the
existence of a +4 subsite. A decreased acidity of the substrate
binding cleft and an increased flexibility of aromatic residues lining the subsites may enhance the rate at which substrate is bound.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-1,4-D-glycosidic bonds in xylan, a major component of
plant hemicellulose. A classification system for glycosyl hydrolases
has been introduced by Henrissat (8) and, at present, at least 89 different families have been identified, with xylanases usually being
grouped into families 10 and
11.1 Family 10 enzymes have
an (
/
)8 barrel fold (10) while the family 11 members
have a
-jelly roll fold (11). The psychrophilic xylanase, on the
contrary, can be classified into glycosyl hydrolase family 8 on the
basis of its primary structure (EMBL nucleotide sequence data base
AJ427921), sharing 20-30% sequence identity with its members (12).
The latter family mainly contains endoglucanases (EC 3.2.1.4), but also
chitosanases (EC 3.2.1.132) and lichenases (EC 3.2.1.73).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Km. The pH activity profile and
an estimate of the apparent Km of the wild type and
mutant xylanase were determined at 25 °C as already described
(12).
Data collection statistics for the xylanase/xylobiose
complex and the xylanase mutant D144N
-anomer and 35%
-anomer, typical values for xylose in solution (18).
Refinement statistics for the native cold-adapted xylanase, the
xylobiose complex, and the D144N mutant
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helices and 13
-strands. The helices form a
barrel with roughly 6 pairs of helices surrounding the central axis,
and the structure can be described as a distorted
(
/
)6 barrel (Fig. 1,
A and B). A stereodiagram of the C
atom
positions is shown in Fig. 2. This protein folding topology is common among inverting glycosidases and is
observed in family 8 and 9 endoglucanases (25, 26), family 15 glucoamylases (27), and family 48 cellobiohydrolases (28). The inner
helices of the barrel have many hydrophobic residues that constitute
the core of the protein, and an extra
-helix in comparison to common
(
/
)6 barrel proteins, comprising residues 19-25, is
present. One 2-stranded sheet is present at the bottom side in Fig.
1A, while the other
-strands occur at the top side,
forming either small or irregular sheets. On the top side, an acidic
cleft is clearly visible (Fig.
3A), indicative of the
catalytic site. Both the N and C terminus of the protein lie at the
bottom side and are separated by a distance of 39 Å. Two cysteine
residues are present, forming one disulfide bridge. Cys-324 is at the
end of helix 10, while Cys-339 lies before helix 11.
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Fig. 1.
Overview of the structures of the
cold-adapted xylanase and the C. thermocellum
endoglucanase. -Helices are in red and
-strands in blue while an extra
-helix in the
psychrophilic xylanase is shown in green. A,
side view of the (
/
)6 barrel of the native
psychrophilic xylanase. B, top view of the
barrel in A. C, top view of the
(
/
)6 barrel of C. thermocellum CelA
showing that this barrel has a more circular cross-section.
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Fig. 2.
Stereo drawing of the C
atoms in the native cold-adapted xylanase. The orientation
of the molecule is the same as in Fig. 1A.
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Fig. 3.
Comparison of the molecular surfaces and
active site clefts (GRASP representations) of the cold-adapted xylanase
(A) and the C. thermocellum endoglucanase
(B). Positive potentials are shown in blue
and negative potentials are displayed in red. A decreased
negativity and accessibility of the substrate binding cleft is visible
for the cold-adapted xylanase.
/
)6 fold is also observed in this enzyme; however, there are many differences between the two structures (Fig.
1). CelA has only 12 helices and also fewer
-strands. The barrel is
less distorted than in pXyl and has a more circular cross-section.
Furthermore, 5 cysteine residues are present, none of which occur in
disulfide bridges. The acidic cleft is larger than in the case of pXyl
and has a greater accessibility (Fig. 3). The full-length CelA also
contains a C-terminal dockerin domain, not present in the crystal
structure, which serves to anchor the protein to the cellulosome (29).
In addition, the N and C terminus of the catalytic domain are
separated from each other by only 8.13 Å, in contrast with 39 Å for
the pXyl.
Parameters affecting stability and flexibility in the cold-adapted
xylanase (pXyl) and the C. thermocellum endoglucanase (CelA)
-amylase (4) and citrate synthase (3) from Antarctic bacteria and
for trypsin from Arctic salmon (31).
3 and +3. In
addition, no conclusions can be drawn concerning relative B-factors on
comparison of their values for the catalytically important residues
Glu-78, Asp-144, and Asp-281 (Table IV).
Relative B-factors for catalytically important residues and aromatic
residues lining the subsites of the cold-adapted xylanase (pXyl) and
the Clostridium thermocellum endoglucanase (CelA)
-1,4-Linked xylobiose was
soaked into existing xylanase crystals and an omit map showed the
position of the ligand in the structure (Fig.
4A). However, final
2Fo
Fc electron
density maps only allowed a clear positioning of the xylose at the
reducing end, indicating that the other residue is flexible. The
positioned xylose adopts a normal 4C1 chair
conformation, forms various hydrogen bonds with the enzyme either
directly or indirectly through water molecules, and partly stacks
against the aromatic side chain of Tyr-378 (Fig. 4).
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Fig. 4.
Binding of xylobiose to pXyl. A,
omit map of a complex of the enzyme with xylobiose, showing the
positioning of the reducing end xylose in the xylanase structure. The
map is contoured at 2 (pink), 3
(blue), and
4
(green). The refined coordinates for the xylosyl
residue and for Tyr-378 are shown. B, LIGPLOT diagram
showing the interactions between the xylosyl residue and protein
residues/water molecules (dark gray spheres). Hydrogen bonds
are represented by dashed lines. The side chain of Tyr-378
is in hydrophobic contact with the C-5 atom of the xylosyl
residue.
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Fig. 5.
Protein-carbohydrate stacking interactions
for the C. thermocellum endoglucanase
(red) and superposition of the corresponding residues
in the cold-adapted xylanase (dark blue).
Conformation B of Tyr-381 and Phe-280 in the psychrophilic xylanase are
shown in light blue. The glucopentaose substrate, as deduced
from an enzyme-substrate complex (35) is shown in yellow to
indicate the position of the subsites. The position of a glucosyl
residue occupying subsite +3 in the same complex is shown in
gray, and the xylosyl residue found in the pXyl-xylobiose
complex is displayed in green. The xylosyl residue that was
not modeled in the latter complex points toward the +3 site. Residue
numbering is as for the cold-adapted xylanase.
3. The aromatic residue Trp-205 from
CelA, which stacks against a glucose residue of the substrate, is
replaced by Tyr-194, whose side chain points in a different direction
(Fig. 5). Furthermore, none of the residues that participate in
hydrogen bonding to the substrate are conserved. At subsite +3, the
aromatic residue Tyr-378 (pXyl numbering), which stacks against the
glucose residue in CelA, adopts approximately the same conformation in
pXyl, although none of the residues involved in hydrogen bonding are
conserved. The fact that the +3 subsite is not conserved may explain
why one xylose residue is very flexible in the pXyl-xylobiose complex.
However, a complex between a larger substrate or product and pXyl is
needed to unambiguously describe the +3 subsite of the xylanase.
Comparison of the subsites and the protein-carbohydrate interactions of
the C. thermocellum endoglucanase (CelA) and the cold-adapted
xylanase (pXyl)
1 where most of
the hydrogen bonds involving the catalytically important residues
Asp-144, Glu-78, and Asp-281 (pXyl numbering, see below) are found.
2,
1,
and +3 of CelA do not occur in the pXyl structure. Furthermore, the main chain of Pro141 in subsite
2 would sterically clash against an
OH-6 group, showing that the binding of cellulose to pXyl is impossible. On the other hand, residues in subsites +1 and +2 that
interact with OH-6 are conserved in the two structures.
-1,4 linkage
that precedes (at the non-reducing end) a
-1,3 linkage, and that it
can only cleave
-1,4 bonds positioned at least three linkages after
a
-1,3 bond (12). The first suggestion indicates that a
-1,3
linkage occurs between subsites +1 and +2, resulting in the +2 sugar
being orientated differently within the +2 subsite. Nevertheless, its
location within the enzyme can be similar to a normal case where only
-1,4 linkages occur and, as a consequence, has a profound effect on
the +3 xylosyl residue, which now points away from the "normal" +3
subsite. As for the second suggestion, a
-1,3 linkage occurs just
outside of subsite
3. Because of the absence of a
4 subsite, this
is structurally tolerated. The first hypothesis also makes sense as
subsite +3 is one of the least conserved subsites where no amino acid
residues that interact with substrate can be proposed. Detailed
structures of complexes of an inactivated xylanase with various
substrates are needed to unambiguously prove this hypothesis.
-1,4-glycosidic linkages between consecutive
sugar residues. This results in an inversion of anomeric configuration
at C1 and the formation of a hydroxyl group at O-4. The catalytic
mechanism normally requires two carboxylic acids, separated by a
distance of ~9.5 Å. One acts as a general base, removing a proton
from water, the other acts as a general acid donating a proton to the leaving group. In family 8 enzymes Glu-78 has been shown to be the
catalytic acid, while both Asp-144 and Asp-281 (pXyl numbering) have
been proposed as the catalytic base (9, 38).
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Fig. 6.
Schematic representation of the geometry
of the catalytic center of the wild type cold-adapted xylanase
(A), the psychrophilic xylanase mutant D144N
(B), and the native C. thermocellum
endoglucanase CelA (C). Dashed lines
indicate hydrogen bonds. Distances between residues not implicated in
hydrogen bonding are shown by a double arrow. Hypothetical
charges are shown as + or . All distances shown are in Å. For
clarity, water atoms implicated in hydrogen bonds are not
included.
1·s
1) is only 182 times
lower than that of the wild type xylanase (9.3 ± 0.9 ml·mg
1·s
1). Furthermore, while
realizing the limitations of the test used, it appears that the
apparent Km of the pXyl mutant (35.6 ± 7.1 mg/ml) is unchanged (or perhaps very slightly increased) as compared
with the wild type enzyme (28 ± 4.5 mg/ml). This is also in good
agreement with the results obtained for the endoglucanase K mutant (9)
and indicates that Asp-144 is not important for substrate affinity.
Analysis of the pH activity profile in the presence of substrate shows
that the acidic limb is affected in the mutant (Fig.
7), indicating a slight increase in the
pKa of the catalytic base and/or nucleophile.
These results suggest that Asp-144 may act as the catalytic base in
pXyl, yet the pKa change observed is small and
the distance (2.41 Å) between Glu-78 and Asp-144 is too short for
inverting enzymes.
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Fig. 7.
Profiles of pH versus
activity for the wild type cold-adapted xylanase (circles,
solid line) and the D144N mutant (triangles, dashed
line). Activity was measured at 25 °C with 3%
soluble birchwood xylan as substrate.
1 in a strained boat conformation (38). The distance between
Glu-78 and Asp-281 in CelA is short for an inverting enzyme (6.35 Å for the native enzyme and 6.5 Å for the complexed CelA), and in the
pXyl structure this distance is even less (4.5 Å), but may change upon
substrate binding. Analysis of the catalytic site of D144N (Fig.
6B) shows that the mutation has an effect on the positions
of both Asn-144 and Glu-78. The hydrogen bond between residue 144 and
Arg-284 is lost, and the side chain of Glu-78 is turned away from
Asn-144. Instead, Glu-78 now forms hydrogen bonds with the side chains
of Tyr-380 and Arg-284 (Fig. 6B). Thus, if Asp-281 is indeed
the true catalytic base, Asp-144 is also a critical residue, playing a
role in the positioning of Glu-78 and perhaps also in facilitating
hydrolysis and regulating the pKa of the
nucleophile and/or catalytic base. This may lead to a decrease in
activity and to the alteration of the pH activity profile as observed
in this study. Furthermore, analysis of the local geometry of the
catalytic site of pXyl (Fig. 8) indicates
that Asp-281 is better positioned than Asp-144, in relation to the
general acid and the expected positioning of substrate, to act as the
general base. Further studies, including site-directed mutagenesis of
Asp-281, as well as structural studies of a complex with substrate, are
necessary to clarify this point. In addition, because of the pronounced
effects of the D144N replacement, the neutral residue in the
Glu-78-Asp-144 pair is likely to be Glu-78. This corresponds well with
the role of this particular glutamate as a general acid.
View larger version (19K):
[in a new window]
Fig. 8.
Stereo view of the active site region of the
wild type cold-adapted xylanase (blue), D144N
(green) and the C. thermocellum
endoglucanase (red) in complex with
glucopentaose. Glucosyl residues in subsites 1 (top) and +1
(bottom) are shown in ball-and-stick representation. The
general acid (Glu-78) and the putative general bases (Asp-144, Asp-281)
of the psychrophilic xylanase are shown, indicating the better
positioning of Asp-281 to act as the general base.
/
)6 fold. Compared with a
thermophilic endoglucanase from the same family, the structure is
destabilized by a drastically reduced number of salt bridges and an
increased exposure of hydrophobic residues. The rate at which substrate
binds may be enhanced by a decreased acidity of the substrate binding
site and by an increased flexibility of aromatic residues lining the
different subsites. Contrary to expectations, however, is a decreased
accessibility of the substrate binding region. Other cold adaptations
are not evident, or are more discrete, and this supports the idea that every cold-adapted enzyme has a unique means of dealing with lower environmental temperatures.
3, as well as the appearance of a potential +4
subsite. The selectivity of pXyl for xylan over cellulose is due to the
absence of residues that interact with the glucose OH-6 group in CelA
and the steric hindrance, which would occur with an OH-6 group in
subsite
2.
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ACKNOWLEDGEMENTS |
---|
We thank access to beamline X13 at the EMBL, Hamburg Outstation, beamline BM14 at the ESRF, Grenoble, and the protein crystallography beamline at ELETTRA, Trieste.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the European Union (Network Contract No. CT97-0131), the Region Wallone (Contract BIOVAL 981/3860), and the Fond National de la Recherche Scientifique (Contract 2.4515.00) (to T. C., M. A. M., C. G., and G. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1H12, 1H13, and 1H14) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Both authors contributed equally to this work.
¶ Research Fellow of the Fund for Scientific Research-Flanders.
** Recipient of Research Grant G006896 from the Fund for Scientific Research-Flanders and Project 12050198 from the Research Council of the University of Gent. To whom correspondence should be addressed. Tel.: 32-0-9-264-51-09; Fax: 32-0-9-264-53-38; E-mail: Jozef.VanBeeumen@rug.ac.be.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M206862200
1 P. M. Coutinho and B. Henrissat, Carbohydrate-Active Enzymes server at URL: afmb.cnrs-mrs.fr/~cazy/CAZY/index.html.
3 T. Collins, M. A. Meuwis, C. Gerday, and G. Feller, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: pXyl, cold-adapted xylanase from P. haloplanktis; CelA, endoglucanase from C. thermocellum; FOM, figure-of-merit; SAD, single wavelength anomalous dispersion.
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