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INTRODUCTION |
The 1,3-1,4-
-glucanase (1,3-1,4-
-D-glucan
4-glucanohydrolase, EC 3.2.1.73) from barley (Hordeum
vulgare, cv. Alexis) degrades mixed linked
-glucans in the cell
walls of the starchy endosperm in grains (1). It acts as an
endohydrolase on a long linear polysaccharide with about 30%
-1,3
and 70%
-1,4 glycosyl linkages (2). The scissile bond is the
-1,4-linkage of an O-3-substituted glucose in the mixed
linked
-glucan. The crystal structure of the tetragonal form of the
1,3-1,4-
-glucanase (isoenzyme
EII)1 is known at 2.2-Å
resolution as well as that of the homologous 1,3-
-glucanase from
barley (3). EII is glycosylated at Asn190 by a branched
oligosaccharide of mixed composition (4). This oligosaccharide may
function in increasing the thermostability of the protein (5).
1,3-1,4-
-Glucanases occur also in bacterial strains (6). Both
native and engineered enzymes have been structurally characterized (7,
8) including the 1,3-1,4-
-glucanases MAC from Bacillus macerans and LIC from Bacillus licheniformis (9, 10).
The crystal structure of H(A16-M), a hybrid
-glucanase with residues 1-16 derived from the Bacillus amyloliquefaciens enzyme
(AMY) and residues 17-214 from MAC, was solved at 1.6-Å resolution
(11). The crystal structures of MAC, LIC, and H(A16-M) are sufficiently similar overall and around the active sites to suggest that these 1,3-1,4-
-glucanases, as well as AMY and SUB, the enzyme from Bacillus subtilis, share a common mode of substrate binding
and hydrolysis.
There is neither sequential nor structural homology between the plant
and the bacterial enzymes, and following the universally adopted
nomenclature (6), they belong to the families 17 and 16 of glycosyl
hydrolases, respectively. Despite this difference they cleave the same
substrate at the same cutting site and are inhibited by the same
covalently binding inhibitors,
3,4-epoxyalkyl-
-D-cellobiosides (12). However, the
barley 1,3-1,4-
-glucanase binds the epoxide preferentially with a
propyl linker, whereas SUB prefers a butyl linker (13). Because both
endohydrolases follow the same stereochemical pathway in glycosyl bond
cleavage with retention of the
-configuration at the anomeric carbon
(14, 15), the differences in inhibitor binding are surprising at first
sight and warrant a structural explanation. A cleavage mechanism with
overall retention of configuration requires the presence in the
catalytic site of a general acid separated by about 5.5 Å from a
nucleophilic residue (6). These roles have been assigned to
Glu288 and Glu232 of EII, respectively (3).
Here we present the structure analysis of monoclinic crystals of the
barley 1,3-1,4-
-glucanase (isoenzyme EII) at 2.0-Å resolution. We
discuss structural differences between three molecules of EII in two
space groups and compare the active center structures of plant and
Bacillus 1,3-1,4-
-glucanases. Structural reasons
for inhibitor binding preferences are considered, and possible
conformations and positions of a hexameric
-glucan fragment are
investigated by molecular dynamics calculations.
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EXPERIMENTAL PROCEDURES |
Diffraction Data and Structure Analysis by Molecular
Replacement--
The purification, crystallization, and x-ray
diffraction data of barley 1,3-1,4-
-glucanase, isoenzyme EII, have
been described (16). The diffraction data set was collected from one
crystal at room temperature with a 180-mm MarResearch imaging plate
system on an Enraf Nonius FR571 rotating anode x-ray generator (45 kV, 90 mA). The images were evaluated with MOSFLM, AGROVATA, and ROTAVATA from the CCP4 suite (20). Relevant crystallographic parameters in the
monoclinic space group P21 are summarized in Table
I. Space group and unit cell dimensions
are consistent with the presence of two protein molecules per
asymmetric unit. The 40,727 unique reflections correspond to 99.3% of
the observations expected for 13.65 to 2-Å resolution, and the
completeness in the outermost resolution shell from 2.09 to 2.0 Å is
96.6%. The crystal and x-ray diffraction data for EII in the
tetragonal space group P43212 (Ref. 3; PDB
entry 1ghr) are added to Table I for comparison.
Since the structure of EII is known for the space group
P43212, the phases of the structure factors
could be determined by molecular replacement using XPLOR (17). Two
molecules in the asymmetric unit (Mol1 and Mol2) were found by rotation
and translation search. The search model 1ghr (3) gave two single peaks
10
and 9
above the average in the Patterson correlation function. After translation search (15.7
and 14.8
), rigid body, and packing refinement against data between 13.65 and 3 Å the R value
was 31.4%.
Structure Refinement--
For refinement, 5% of the reflections
were set aside as test set for the calculation of
Rfree (18). Rfree was
used to monitor convergence throughout. After 100 steps of minimization
with the standard repel nonbonded energy function followed by 50 steps of Powell Lennard-Jones minimization (Rwork = 28.3%, Rfree = 32.4%), a simulated annealing
run following the standard slow-cooling protocol (19) with structure
amplitudes F > 2
(F) between 13.65 and
2.0 Å, restrictions for non-crystallographic symmetry (NCS) converged
with Rwork = 27.2% and
Rfree = 31.2% (no NCS:
Rwork = 26.3%, Rfree = 31.1%). In the following steps, NCS restrictions were used for all but
those residues making crystallographic or non-crystallographic
intermolecular contacts. Subsequent B-value refinement
resulted in Rwork = 25.2% and
Rfree = 28.5%. Electron density maps were
calculated with XPLOR and CCP4 (20) programs and displayed in O (21).
Further refinement was performed with XPLOR/O combining positional
refinement with overall and atomic B-value refinement. After
several cycles of positional and B-value refinement and
manual revision of the model via electron density and difference
density maps, water molecules were added until most significant peaks
(>4
) disappeared from the difference electron density map. Ending
up with 418 water molecules in the asymmetric unit and reduced NCS
restraints for both molecules, the final Rwork
was 17.1% and Rfree 21.2%.
Molecular Dynamics Modeling of Substrate Binding--
Substrate
binding to a bacterial 1,3-1,4-
-glucanase has been modeled (11).
Because the substrate specificity and the stereochemical course of
-glucan hydrolysis are identical for Bacillus and barley 1,3-1,4-
-glucanases, we used the hexameric
-glucan fragment proposed earlier (11) as start model in simulating substrate binding to
EII. The glucose moieties are numbered from 1 to 6 beginning at the
non-reducing end. They bind to enzyme subsites p4, p3, p2, p1, p1
, and
p2
, and cleavage occurs at the catalytic site between p1 and p1
.
Manual primary positioning of the hexamer in the binding cleft using O
(21) took into account restraints deduced from chemical probing with
barley 1,3-1,4-
-glucanase (22) and inhibitor binding to the
structurally very similar 1,3-
-glucanase of barley (1ghs; Ref.
23).
To start the molecular dynamics simulation, a FORTRAN program written
for this purpose but not specific for the glucanase molecule first
filled a P1 cell with dimensions 66.379 × 60.241 × 52.643 Å with TIP3 waters provided by the XPLOR package (24). After this
procedure, molecule Mol1 of EII was oriented with its inertia
equivalent ellipsoid axes parallel to the P1 cell axes. The overlapping
water molecules were removed so that Mol1 finally was embedded in 5,069 non-overlapping water molecules. The water box is large enough to cover
the substrate by a water shell of at least 13-Å thickness. The charged
residues of the protein are made net neutral to mimic the effects of
solvation and counterions. The nonbonded interaction cutoff was
specified to 8.5 Å. The molecular dynamics calculation, done by XPLOR,
preceded by 120 steps of standard repel non-bonded energy minimization
and 80 steps of Powell Lennard-Jones minimization in vacuum, started
with the fixed enzyme-substrate complex to relax the water molecules
around the macromolecule. The solvent molecules were relaxed further during 120 steps of energy minimization followed by molecular dynamics
over 5 ps at 100 K, 5 ps at 200 K, 20 ps at 300 K with 0.2-ps
reassignment using the CHARMM force field. The energy was then
minimized for the solute (100 steps Powell) followed by several steps
of molecular dynamics for the solute (5 ps at 100 K, 5 ps at 200 K, 5 ps at 300 K). Finally, molecular dynamics were run for 80 ps at 300 K
(with temperature coupling) for the whole system, followed by 80 steps
of Powell Lennard-Jones minimization. Over the entire simulation the
EII molecule remained fixed, but those side chains in the binding cleft
containing atoms in a 4-Å surface shell, the hexaglucan, and the
waters were allowed to move. Alternative protocols without water
relaxation at 100, 200, and 300 K and without temperature coupling
resulted in similar sugar conformations and positions.
 |
RESULTS AND DISCUSSION |
Refinement Results--
After combining working and test
data sets, final positional and B-value refinement yielded
an R-value of 17.0%. The R-value was 16.5% for
all observations with a low resolution cutoff at 6 Å as used in the
previous structure analysis (3). Two residues of
N-acetyl-D-glucosamine (NAG) per molecule of the
barley 1,3-1,4-
-glucanase EII were located at the glycosylation
site, Asn190. The side chains of Asp183 and
Ile304 in Mol1 and Gln41 in Mol2 were refined
in two alternative positions with 2/3 and 1/3 occupancy, and two
acetate molecules were identified in non-related positions. In the
Ramachandran diagram, more than 91% of the non-glycine and
non-proline residues are within the most favored regions, and all other
residues are within additional allowed regions. Two
cis-peptides preceding Pro137 and
Ala276, identical to those in 1ghr, are present in both
Mol1 and Mol2. The average correlation of the calculated
Fc map with the 2Fo
Fc electron density map is better than 0.9 for both
molecules according to an analysis with O (21). The correlation
coefficient drops below 0.7 for the highly flexible side chains of
Arg100, Arg197, and Arg261, which
have B values of about 50 Å2 and are not
sterically restricted by hydrogen bonds or van der Waals contacts in
both molecules.
Stereochemical parameters of the main and side chains are strongly
restrained by standard weights and are better than or inside of the
bandwidth defined in PROCHECK (25) for structures with comparable
resolution. The final atomic coordinate set representing the monoclinic
form of EII was scrutinized with WHATCHECK (26), PROCHECK, and several
programs of the CCP4 suite. The results are summarized in Tables I and
II. The experimental data and the refined
atomic coordinates of both molecules in the asymmetric unit were
submitted to the Brookhaven Protein Data Bank (27) entry code
1aq0.
Overall Structure and Crystal Packing--
The overall structure
and the active site of EII have been described by Varghese et
al. (3) and are very similar in the monoclinic crystal form. The
overall shape of the 1,3-1,4-
-glucanase can be approximated by its
inertia equivalent ellipsoid with half-axes of 29.1 × 25.2 × 16.4 Å. The global folding pattern belongs to the

8 barrel type, although
-strand number 8 of the
barrel is truncated to just two residues (Fig.
1). The active site is located in an open
cleft at the bottom of the barrel defined by the C-terminal ends of the
parallel intra-barrel
-strands. It is about 36 Å long and 8 to 9 Å deep, allowing the binding of oligosaccharide substrates.

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Fig. 1.
Stereographic cartoon of the molecular
structure of the barley 1,3-1,4- -glucanase, isoenzyme EII. The
view is through the active site channel of the enzyme.
Arrows indicate -strands, and wound ribbons
indicate -helices. No arrow is drawn for -strand number 8 consisting of only two residues. The two catalytic glutamic acid residues 232 and 288 of EII are drawn in ball-and-stick
representation, as well as the carbohydrate attached to residue
Asn190. Drawn with MOLSCRIPT (39).
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The two molecules of EII in the asymmetric unit make numerous
intermolecular contacts with distances between 2.5 and 4 Å. 13 amino
acids of Mol1 are in contact with 14 amino acids of Mol2 through
non-crystallographic symmetry. 39 and 43 amino acids of Mol1 and Mol2,
respectively, engage in additional crystallographic contacts. These
contacts result from a tighter packing of the molecules in the
monoclinic cell (Vm = 2.41 Å3
Da
1) in comparison with tetragonal 1ghr
(Vm = 3.26 Å3 Da
1), where
44 residues make intermolecular contacts. The contacts do not include
any residue of the catalytic site and of the potential substrate-binding site (see below) of Mol1 and Mol2, as well as of
1ghr, and therefore do not exert a major influence on the protein structure within the binding cleft. This is suggested by the analysis of distances (LSQKAB, Ref. 28) between 45 equivalent residues within
the 4-Å surface of the substrate binding cleft. A comparison of Mol1,
Mol2, and 1ghr shows the residues in the cleft to be structurally
conserved to a higher degree than those in the rest of the molecules
(Table III). The differences between the
atom positions of the residues in the catalytic and substrate-binding site (Glu288, Glu232, Tyr33,
Glu93, Asn92, Val134,
Phe135, Asn168, Tyr170,
Leu173, Phe275, Glu280,
Lys283, and Trp291) are below the average
coordinate error for Mol1 and Mol2 determined to 0.2 Å from the
Luzzati (29) analysis.
Structural Heterogeneity, Subdomains, and Hydration--
The most
prominent difference between the main chains of the three molecules is
found for amino acid residues 190-200 of Mol2 compared with the other
two (Fig. 2). In this region, the structure of Mol2 is influenced by the NCS interactions of Gly199 and
Ala200 in a
-turn of Mol1 with Val196 in
Mol2, and by numerous crystallographic contacts to Mol2 itself. The
shift of the C
backbone preceding the small
-sheet by a maximum
of 4.8 Å at Thr194 indicates a certain flexibility of this
region. Statistics measuring the degree of similarity of molecules (30)
show clearly that Mol1 is more similar to 1ghr than is Mol2 (Table
III). Therefore, all considerations of general aspects of the
structures will refer to Mol1.

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Fig. 2.
Least-squares superpositions of the two
molecules of EII related by non-crystallographic symmetry in the
P21 crystal and 1ghr, the tetragonal form of the
enzyme. The view is onto the  8 barrel. The C
traces are shown as bold solid line for Mol1, dashed
line for Mol2, and thin solid line for 1ghr (3). The
1ghr trace is almost completely hidden behind the solid
line. Note the two nearly perpendicular depressions on this face
of the molecule, the -glucan-binding channel running horizontally and the domain boundary crossing in vertical orientation (see text).
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By using the program PUU (31) two structural domains were detected with
a boundary crossing the 
8 barrel perpendicular to the
binding cleft. Residues Ile1 to Ser126 and
Tyr273 to Phe306 constitute domain I; residues
Val127 to Thr272 belong to the glycosylated
domain II (right side in Fig. 3). Only two
strands link both domains at Ser126/Val127 and
Thr272/Tyr273. This assignment of structural
domains differs from that proposed earlier (3) and is supported by a
least-squares superposition of domains I of Mol1 and 1ghr yielding a
rotation by about 1° between their domains II. The rotation axis
traverses the center of mass of domain II and is inclined by about
50° against the cleft axis marked by a modeled hexaglucan substrate
(see below) in Fig. 3. Interestingly, the two
-strands crossing the
inter-domain boundary are not regularly structured. Strand
8 is truncated to just two residues in Mol1 and Mol2 and
is next to the cis-peptide preceding Ala276. The
long strand
5 is interrupted by a 310 turn
at Gln129 to Ile131 where it crosses from
domain I into domain II also reflecting some structural irregularity.
Functional implications of this domain structure will be discussed
below.

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Fig. 3.
Domain structure of barley
1,3-1,4- -glucanase. The domains were estimated by the program
PUU (31). The red net defines the domain boundary calculated
by ANY_PLANE (JJM) and CURVE (32). A hexameric -glucan model
oriented such that the reducing end points to the right is shown
together with the catalytic site residues Glu232 and
Glu288, Tyr33, and the glycosylation site with
NAG11 and NAG21.
Presumably, the catalytic event is coupled with a movement of the left
domain against the glycosylated right domain, thereby distorting the
scissile bond of the -glucan positioned directly at the domain
boundary. Drawn with SETOR (40).
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Water molecules in 130 pairs related by NCS were identified allowing
for a maximal separation of 1.5 Å. These water-binding sites present
in both Mol1 and Mol2 are expected to have biological relevance. 37 out
of these 130 sites have identical positions within 1.5 Å with any of
the 48 water sites present in 1ghr indicating that their positions are
independent of crystal contacts and preparation conditions. The
water distribution around EII is quite asymmetric, since the molecular
surface near the C and N termini shows significantly higher hydration
than other parts of the protein molecule. This finding is true also for
the waters detected in the molecule 1ghr. Interestingly, only 12 waters
out of the 130 are found within the substrate-binding cleft of EII. The
lack of complete cleft hydration may be due to the presence of exposed
hydrophobic residues (see below).
The Glycosylation Site--
Barley 1,3-1,4-
-glucanase,
isoenzyme EII, has only one site for potential
N-glycosylation, the sequence
Asn190-Ala191-Ser192. The attached
carbohydrate moieties were analyzed by Harthill and Thomsen (4). They
identified five different branched N-glycans comprised of
different sugars contributing with different relative amounts between
15 and 30%. All have a common core sequence starting with two
-1,4-linked NAG molecules. These two residues are represented by
clear electron density (Fig. 4). They have
been identified in difference density maps at a late stage of
refinement, because they were absent in 1ghr. The sugars in the two
NCS-related protein molecules are differently well defined. The average
temperature factors are 30.0 and 60.7 Å2 for
NAG11 and NAG21 of
Mol1, and 49.1 and 63.6 Å2 for the corresponding residues
in Mol2. Whereas NAG11 and
NAG21 are well defined, a third sugar residue,
-L-fucose, attached to O-3 of
NAG12 is seen at very low electron density (0.3 e Å
3) but was not modeled. Both glycosylation sites are
sterically accessible to different degrees.

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Fig. 4.
Stereographic drawings of electron density
around the carbohydrate moiety attached to Asn190. The
2Fo Fc omit map with a cushion
of 6 Å around the carbohydrate is contoured at 0.62 e
Å 3 around NAG11 (top)
and at 0.34 e Å 3 around
NAG21 (bottom). Drawn with OPLOT
(21).
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Both disaccharides are in the energetically preferred
4C1 chair conformation, and the torsions of the
-1,4 glycosyl bonds
(H-1-C-1-O-4-C-4) and
(C-1-O-4-C-4-H-4) are 49.8° and 15.2° in
NAG11/NAG21, and
44.9° and 3.9° in
NAG12/NAG22. These
dihedrals correspond to
and
angles also found in saccharides
bound to lysozyme (PDB entry 1lzr) or influenza virus neuraminidase
(PDB entry 1nnc).
The Active Site: Structure and Hydration--
The substrate
binding cleft of barley 1,3-1,4-
-glucanase was geometrically
defined using SURFNET (32), and residue accessibilities were
characterized by NACCESS (33). The largest cleft found by SURFNET (1 Å < sphere radius < 4 Å; cutoff distance between atoms and mask
region 4 Å) has a volume of 2031 Å3 (Fig.
5, top) and comprises the
catalytic site and the potential substrate binding site as described
recently (3, 23). The global dimensions are 7.5 Å in width, 8 to 9 Å in depth around the catalytic center, and 36 Å in length. All
accessible atoms within a distance
4 Å from the cleft surface were
selected, and their polarity was estimated by NACCESS. The resulting
patterns are given in Table IV. The
"left" side of the cleft (in the orientation of Fig. 5) is mainly
decorated with apolar residues, whereas the "right" side,
especially in the vicinity of the catalytic site, is covered with polar
residues. The residues Tyr170, Glu232,
Tyr33, Glu288, and Trp291 in or
near the catalytic center are accessible, also Leu173,
Phe275, Asn92, Glu93, and
Asn168 which possibly interact with the substrate (3) as
partially proven by chemical probing (22).

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Fig. 5.
Stereographic drawings of the substrate
binding cleft of barley 1,3-1,4- -glucanase and of the hybrid
Bacillus 1,3-1,4- -glucanase H(A16-M). Top,
barley 1,3-1,4- -glucanase, active site atoms of Glu232,
Tyr33, and Glu288 are labeled.
Bottom, H(A16-M). The active site residues
Glu105, Tyr94, and Glu109 are
labeled. Water molecules binding to the corresponding cleft are shown
as blue spheres. The maps defining the clefts were estimated by SURFNET (32). Drawn with BOBSCRIPT (42) and RASTER3D (43).
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Table IV
Active site residues in barley and Bacillus 1.3-1.4- -glucanases
Active site residues of EII and H(A16-M) possibly interacting
with bound substrate. Left, and right is according to Fig. 5. Attached
water molecules are indicated by w; a denotes the apolar part of the
residue; and p refers to the polar part. The catalytic site glutamic
acids and the tyrosine defining subsite p2 are emphasized by boldface.
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Water molecules are mainly located at the polar right side of the cleft
floor (Fig. 5) where several residues form hydrogen bonds to five water
molecules. Only the three water molecules at the lowest end of the
cleft are also present in the 1ghr model (3). Three water molecules
form H bonds to the Ala174 backbone and the
Tyr172 and Glu280 side chains at the left side.
Thus, the mostly polar right half of the cleft is covered by a water
layer. A comparison with the water structure in the active site of the
Bacillus 1,3-1,4-
-glucanase will follow below.
Molecular Dynamics Simulation of Substrate Binding--
A model of
a hexaglucan substrate bound to the active site of EII was constructed
to assist our understanding of the enzymology of the barley
1,3-1,4-
-glucanase. The
-glucan before and after the simulated
docking procedure is shown in Fig. 6
(bottom). In the start configuration, C-1 of glucose residue
Glc4 points to the nucleophile Glu232 of EII; Glc4 is
situated halfway between Glu232 and the general acid
Glu288; Glc3 O-6 forms hydrogen bonds to Glu280
and Lys283 (as the inhibitor in 1ghs); Glc3 O-2 makes an H
bond to Asn92 (as in 1ghs), and the face of Glc3 is in
hydrophobic contact with Tyr33 (as in 1ghs). The water
molecules localized in the cleft were removed completely, because no
fixed waters could be detected between inhibitor and
1,3-1,4-
-glucanase in the crystal structure of an enzyme-inhibitor
complex (34).

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Fig. 6.
Structural comparison of barley
(left) and Bacillus 1,3-1,4- -glucanases
(right). Top, the EII folding pattern belongs to
the  8 barrel type; H(A16-M) adopts the jellyroll
fold. The key residues for the catalytic event are marked in both
enzymes. Middle, molecular surface drawings of the
1,3-1,4- -glucanases. The hexameric -glucan models are oriented
such that the reducing ends point upwards and shown together with the
catalytic site residues Glu232 and Glu288 (EII,
left) and Glu105 and Glu109
(H(A16-M), right). Tyrosine residues Tyr33 (EII)
and Tyr94 (H(A16-M)) stacking against Glc3 of the substrate at subsite p2
are also shown. Surface colors indicate electrostatic potential (blue, positive, and red, negative) as calculated
with DELPHI (41). Note the different rotational setting of the
substrate chain in the active site cleft and the different orientation
of the protein side chains. Bottom, superposition of
-glucan model substrates for barley and hybrid Bacillus
1,3-1,4- -glucanases. The molecules are superimposed with the
glucose moiety Glc3 interacting with a tyrosine in subsite p2. The
substrate strand fitting into the active site of H(A16-M) (Ref. 11,
green) defines the start conformation used in molecular
dynamics modeling of the substrate bound to EII (red).
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The molecular dynamics simulation of substrate binding was equilibrated
after 70 ps. For further discussion we use the middle structure of the
inhibitor as determined by LSQMAN (35). Sugar residues Glc2, Glc3,
Glc4, and Glc5 are situated within the binding cleft, their root mean
square distances from the middle structure are 0.38, 0.31, 0.72, and
0.66 Å, respectively. The first residue, Glc1, is outside of the
binding cleft as defined by SURFNET (Fig. 5). The root mean square
distances of Glc1 and Glc6 are about 0.66 and 0.68 Å, respectively.
Because the position of the substrate is strongly influenced by
Tyr33 and the contacts are built in manually, most of the
interactions found in the inhibitor complex of the 1,3-
-glucanase
(1ghs; Ref. 3) are present in this model, too. The global course of the hexamer is visible in Fig. 3. The smallest root mean square
value
for Glc3 coincides with the longest retention of any glucose residue in
one position during the simulation. This is in agreement with the
results of the subsite mapping of barley 1,3-
-glucanase isoenzyme
GII (36), where the binding affinity at the second subsite toward the
non-reducing end relative to the cutting point is at the maximum. In
the Bacillus as well as in the barley
1,3-1,4-
-glucanases the first and sixth residues of the
-glucan
model substrates are not tightly bound to the ends of the clefts.
All glucose units of the modeled substrate are in the preferred
4C1 chair pucker, and the torsion angles are in
the low energy regions between 28 and 54° for
and
52 to +14°
for
in
-1,3 glycosyl bonds (37), and around
= 40°,
=
20° for
-1,4-bonds (38). Only the torsion angle
= 72.5° at
the scissile
-1,4-bond is outside the minimum, indicating strain
which may be relieved by bond cleavage. A deviation from the low energy
conformation of similar magnitude but with opposite sign was observed
in the modeled
-glucan substrate of the Bacillus
1,3-1,4-
-glucanase (11).
In rationalizing substrate binding and conversion, the domain structure
and possible structural rearrangements must be taken into account. The
general acid, Glu288, and the substrate fix point
Tyr33 are located at the same domain I, whereas the
nucleophile Glu232 at the bottom of the cleft belongs to
the glycosylated domain II. The substrate residues Glc1 to Glc4 are
attached to domain I, and the scissile bond O-4 of the modeled hexamer
is positioned at the inter-domain boundary, and residues Glc5 and Glc6
are attached to domain II (Fig. 3). A slight rotation of the domains
relative to each other may support a rearrangement of the catalytic
residues and/or the substrate position, thus providing a fine tuning of the catalytic event. A domain rearrangement of this kind is observed between Mol1 and Mol2 of EII (see above).
Comparison of Active Site Structures and Modeled Substrate Binding
by Barley and Bacillus 1,3-1,4-
-Glucanases--
Active site
directed inhibition of 1,3-1,4-
-glucanases from B. subtilis and barley (13) by covalent modification with epoxyalkyl cellobiosides reveals subtle differences in their active site geometries. Both enzymes with unrelated primary, secondary, and tertiary structures (Fig. 6) have identical substrate specificity, but
the optimum aglycon length of the cellobiosides for maximum inactivation is C-4 (SUB) and C-3 (EII), respectively, and SUB is much
more efficiently inhibited by the
(S)-epoxybutyl-
-cellobioside than by the
R-isomer, whereas the reverse is found for EII. From this,
it was concluded that the differences in inhibitor binding might be
related to a different cleavage mechanism (13). It could be shown
recently, however, that
-glucan hydrolysis follows the same
stereochemical course in the bacterial and plant enzymes (14, 15). In
addition, the covalent complex of the barley 1,3-
-glucanase with
2,3-epoxypropyl cellobioside shows binding of the S-isomer
to the nucleophile of this close homolog of EII.
A common motif in all inhibitor binding studies (14, 34) and substrate
binding models (Ref. 11; this work) is the hydrophobic "stacking"
interaction of the sugar residue at the p2 subsite with a tyrosine side
chain (Tyr33 in EII and Tyr94 in H(A16-M)). The
distances of the plane midpoints are between 4.3 and 4.7 Å in all
complexes. In H(A16-M) (2ayh), as well as in the barley glucanases
(Mol1 of EII, 1ghr, 1ghs), the distances between oxygens O
-1 and
O
-2 of the catalytic nucleophile and the corresponding tyrosine
midpoint are identical (about 4.5 and 6.5 Å). The distances between
the active O
atoms and the tyrosine midpoint are changed to 5.7 Å by the inhibitor binding to H(A16-M) and to 5.4 Å by substrate binding
modeled for EII (Mol1). Taking this distance of about 5.5 Å into
account, the additional length of 1.3 Å of the butyl linker in
comparison to the propyl linker requires a bulging out of the linker.
The molecular environment of the butyl linker of the inhibitor bound to
H(A16-M) consists of residues Trp192, Val88,
Trp184, and Phe30 at one side, and of
Tyr123, Trp103, and Phe92 at the
other. The butyl linker interacts by hydrophobic contacts with the
plane of Phe92 and covers the hydrophobic surface. Such a
large hydrophobic area does not exist in the case of EII. The only
hydrophobic residues in the vicinity of the linker are
Phe275 and Trp291, and therefore, preserving
the strong hydrophobic interaction between sugar residue Glc3 and
Tyr33, the 1.3 Å shorter propyl linker of the epoxyalkyl
cellobioside inhibitor is preferentially bound.
By analogy to the cleft determination and characterization
described above for EII, the substrate binding site of H(A16-M) was
investigated. Here, the form of the cleft is quite different from that
of EII (Fig. 5, bottom), and the volume is about 1,640 Å3. The width is comparable with the cleft width of 7-8
Å of EII near the catalytic site, but the groove is considerably
deeper (about 12.5 Å) and shorter (about 29 Å). In both cases the
clefts are somewhat branched out at the molecular surface (Fig. 5). All residues within the active site (Asp107,
Glu105, Glu109, Trp103,
Glu119, and Trp192) or interacting with the
cellobioside inhibitor (Tyr24, Asn26,
Glu63, and His99) as described (34) and
additional potential substrate binding partners (Arg65,
Tyr94, Ser90, Glu131, and
Asn121) have accessible surface fractions. These residues
are summarized in Table IV for comparison with EII. No obvious
similarity between both patterns is found. Whereas the branched cleft
in EII has a nearly isometric cross-section in the substrate binding
region, the cleft of H(A16-M) is narrow and deep. The hydration pattern of the H(A16-M) groove is quite different from the water structure in
the EII cleft (Fig. 5).
The six glucose residues cover the whole length of the cleft in
H(A16-M), but eight can be accommodated in EII (36). In any case, only
the innermost four appear tightly bound within the reaction center. In
Fig. 6 (middle), the surfaces, modeled substrates, catalytic
residues, and the hydrophobic binding centers at subsite p2
(Tyr33 of EII and Tyr94 of H(A16-M)) are shown.
The basic differences between both families of 1,3-1,4-
-glucanases
are the relative positioning of the general acid, the nucleophile, and
these tyrosine residues. Given the same orientation of the substrate
with the reducing end of Glc6 facing upward in Fig. 6
(middle), the general acid, Glu288, of EII is at
the left side of the hexameric
-glucan, but the corresponding
Glu109 is at the cleft bottom for H(A16-M). To follow the
same stereochemical pathway a global rotation is necessary between both
substrates by about 90°.
In conclusion, the active sites of the plant and Bacillus
1,3-1,4-
-glucanases are surprisingly different in view of their close functional similarity. The enzymes show nicely how the same catalytic activity can evolve on completely different protein folds and
in dissimilar local geometries.
Kim Henrick is thanked for helpful
discussions regarding the carbohydrate moieties and Yves Muller for
critically reading the manuscript.