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INTRODUCTION |
Many enzymes that hydrolyze insoluble
polysaccharides contain discrete carbohydrate-binding modules
(CBMs)1 that can increase the
effective enzyme concentration on the polysaccharide surface (1, 2).
CBMs are similar to lectins in that they can discriminate between
different polysaccharides, and exhibit binding affinities in the
micromolar range. Like their cognate catalytic modules, CBMs are
classified into families of related amino acid sequences
(afmb.cnrs-mrs.fr./~cazy/CAZY/index.html). A
particularly interesting group of CBMs are those that recognize cellulose, which is arguably the most important polysaccharide on the
planet because of its source and abundance, it is the main structural
polysaccharide in plant cell walls.
Although the cellulose molecule is a simple polymer of up to 10,000 glucose residues linked by
-1,4-glucosidic bonds, natural cellulose
is relatively resistant to enzymatic hydrolysis. Chains longer than six
glucose residues are insoluble. In natural cellulose many molecules
associate in parallel to form fibrils, which in turn associate to form
fibers. Cellulose is heterogeneous: current biophysical techniques used
to study the structure of cellulose show regions of highly ordered
cellulose chains (crystalline cellulose), pseudo-ordered chains
(para-crystalline cellulose), and disordered chains (so
called "amorphous" or noncrystalline cellulose) (3). It is thought
that the varied structure of cellulose contributes to its recalcitrance
to complete hydrolysis by individual enzymes; the efficient degradation
of cellulose is only achieved by the concerted action of complex
microbial enzyme systems. It is known that some CBMs bind
preferentially to the crystalline regions, others to the amorphous
regions, and that the two types do not compete when binding (4). Those
binding amorphous but not those binding crystalline cellulose can also
bind soluble cellooligosaccharides. Surprisingly, some CBMs known to be
specific for noncrystalline cellulose do not compete when binding,
suggesting that they are discriminating between different regions of
the amorphous cellulose and making it evident that the interaction of
CBMs with noncrystalline cellulose may be more complex than originally
thought (5). It is significant in this context that the
three-dimensional structure of amorphous cellulose is still unclear.
Cellulase Cel5A from Bacillus sp. 1139, like several other
enzymes in this family, contains a family 5 catalytic module and two
CBMs, one from family 17 the other from family 28, both of which bind
to amorphous cellulose. The combination of similarity in amino acid
sequence and secondary structure analyses clearly established the
structural relationship between family 17 and family 28 CBMs
(8). However, the amino acid residues involved in binding are
poorly conserved between these two families (8) suggesting that the
CBMs might have different binding characteristics that could shed
further light on the heterogeneity of noncrystalline cellulose. This
paper shows that family 17 and family 28 CBMs do not compete for sites
on noncrystalline cellulose, emphasizing the presence of cellulose
chains with physical presentations that are distinguishable by the
CBMs. Furthermore, the discrimination of these cellulose regions by the
CBMs in Cel5A appears to influence cellulose hydrolysis by this enzyme.
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MATERIALS AND METHODS |
Chemicals--
Microcrystalline cellulose (AvicelTM
PH101; FMC International, Little Island, County Cork, Ireland), Oregon
GreenTM succinimidyl ester (Molecular Probes,
Eugene, OR), cellohexaose (Seikagaku, Tokyo, Japan), and
2',4'-dinitrophenyl-
-D-cellobioside (Sigma) were
obtained from the sources indicated. Regenerated cellulose (RC) was
prepared by phosphoric acid treatment of AvicelTM as
reported previously (6). Bacterial microcrystalline cellulose (crystalline cellulose I) was prepared from cultures of
Acetobacter xylinum (ATCC 23769) as described previously
(7).
Purification of CBMs--
Gene fragments encoding CBMs were
cloned, expressed, and the products purified as described elsewhere (8,
9). The CBMs used in this study were CcCBM17, the C-terminal
module from Clostridium cellulovorans Cel5A (10);
BspCBM28, the C-terminal module from Bacillus sp.
1139 Cel5A; and BspCBM17/CBM28, the tandem of C-terminal CBMs from Bacillus sp. 1139 Cel5A. The concentration of
purified proteins was determined by UV absorbance (280 nm) using
calculated molar extinction coefficients (11) of 31,010 M
1 cm
1, 32,290 M
1 cm
1, and 70,900 M
1 cm
1 for CcCBM17,
BspCBM28, and BspCBM17/CBM27, respectively.
Binding Studies--
Isothermal titration calorimetry was
performed essentially as described previously (6) using a MCS
isothermal titration calorimetry (MicroCal Inc., Northampton, MA).
BspCBM17/CBM27 (wild-type or W453/W500 mutant) was at pH 7.0 in 50 mM K-phosphate buffer. Titrations were performed by
injecting 10-µl samples of cellohexaose solution (5 mM in
K-phosphate buffer saved from the dialysis of BspCBM17/CBM27) isothermal titration calorimetry sample cell
(volume = 1.3528 ml) containing 275 µM
BspCBM17/CBM27. Heats of dilution of the titrant were
assessed by titration of the carbohydrates into buffer lacking CBM;
these were found to be negligible. Two or three independent titration
experiments were performed. Stoichiometries, enthalpies, and
equilibrium association constants were determined by fitting the
corrected data to a bimolecular interaction model.
Adsorption isotherms using RC or AvicelTM were obtained as
described previously (4, 12). Data were analyzed by nonlinear regression of the data to a Langmuir-type two binding site model (Equation 1),
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(Eq. 1)
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where [B] and [F] are the bound and
free concentrations of CBM, respectively.
[N1]o and
[N2]o refer to the density of
binding sites on the cellulose for site 1 and site 2. Ka1 and Ka2 are the association constants for
binding sites 1 and 2. The Scatchard form of the isotherm data was also
analyzed by nonlinear regression of the data to a modified McGhee-von
Hipple (13) model that treats the cellulose as a one-dimensional
lattice of overlapping binding sites and accounts for the possibility that not all of the lattice sites can accommodate CBMs (14) (Equation 2).
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(Eq. 2)
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FA is the fraction of lattice units
accessible to CBM; n is the number of lattice units occupied
by a single CBM; v is the ratio of bound CBM to the
concentration of lattice units (i.e. v = [B]/[Lat], where [Lat] is the concentration of lattice units). Several sizes of lattice units were tried: a glucose unit, cellobiose unit, a cellopentaose unit, and a cellohexaose unit. This
produced identical fits only with alterations in the regressed value of
n, as would be expected. The Scatchard form of the data was
also analyzed by a model related to Equation 2 but incorporating a
dimensionless term for cooperativity, w (15) (Equations 3 and 4).
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(Eq. 3)
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(Eq. 4)
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This model does not account for a fraction of inaccessible
lattice units, so v was calculated using estimates of the
functional lattice unit concentration from the fits of Equation 2.
Goodness-of-fit of the models to the data was assessed by runs tests of
the residuals and p value analysis.
CBMs were labeled with Oregon GreenTM 514 carboxylic acid
succinimidyl ester, which labels the amine groups of lysines and the N
terminus. This was done according to the instructions from the supplier. Fluorescence isotherms were obtained as described previously (4, 5). Competition displacement assays were done as follows. A
constant concentration of RC or AvicelTM (1 or 10 mg/ml,
respectively) was mixed with a constant concentration of Oregon
GreenTM-labeled CBM. The concentration of Oregon
GreenTM-CBM chosen was sufficient to nearly or completely
saturate the sites bound with high affinity. Unbound Oregon
GreenTM-CBM at equilibrium was quantified by fluorescence
spectroscopy in the presence of increasing concentrations of an
unlabeled competing CBM and reference to a standard curve relating
Oregon GreenTM-CBM concentration to fluorescence intensity:
bound Oregon GreenTM-CBM = total Oregon
GreenTM-CBM
unbound Oregon GreenTM-CBM.
Displacement and, therefore, binding competition was determined graphically by plotting the concentration of bound Oregon
GreenTM-CBM versus the concentration of
competing CBM.
Cloning, Production, and Purification of Cel5A and
Mutants--
Escherichia coli BL21(DE3) was used for
cloning and protein purification. The plasmid used was pET28a
(Novagen). E. coli cultures were grown routinely in TYP
medium (16) containing 50 µg of kanamycin ml
1, at
37 °C for plasmid preparation or 30 °C for protein production.
Plasmid preparation, bacterial transformation, and agarose gel
electrophoresis were described previously (16). Restriction endonucleases were used as recommended by the suppliers. DNA fragments were purified from the agarose gels with the Qiaex II kit (Qiagen). Oligodeoxynucleotide primers were synthesized by the Nucleic Acid and
Protein Service (NAPS) Unit of the University of British Columbia, using an Applied Biosystems model 380A DNA synthesizer, and purified by
extraction with 1-butanol. DNA was sequenced by the NAPS using the
AmpliTaq dye termination cycle sequencing protocol and an Applied
Biosystems model 377 sequencer.
The DNA fragment corresponding to nucleotides 91-2287 (encoding amino
acids 31-762) of the cel5A gene was amplified from
Bacillus sp. 1139 genomic DNA (prepared as described
previously (8)) by PCR. This was inserted via introduced 5' and 3'
NheI and HindIII restriction endonuclease sites,
respectively, into appropriately digested pET28a (Novagen, Madison,
WI). This construct was verified by DNA sequencing. The remaining Cel5A
mutants (see text) were constructed using standard gene cloning techniques.
E. coli BL21(DE3) containing the pET28aCel5A constructs were
grown in 2.5 liters of TYP medium containing 50 µg of kanamycin ml
1 at 30 °C. The cultures were induced with 0.1 mM isopropyl-1-thio-
-D-galactopyranoside, either overnight at low optical density (0.1 A600) or 4 h at high optical density (0.6 A600). The cells were harvested and resuspended in 30 ml of 20 mM Tris-HCl buffer, pH 7.9, 0.5 M NaCl, and ruptured by two passages through a French
pressure cell. Debris in the cell extracts was removed by
centrifugation for 30 min at 27,000 × g at 4 °C.
Proteins were purified from the clarified cell extract by immobilized
metal affinity chromatography according to the manufacturer's
protocols (Novagen, Madison, WI). The concentrations of purified
proteins were determined by UV absorbance (280 nm) and calculated molar
extinction coefficients (11) of 145,800 M
1
cm
1, 134,420 M
1
cm
1, 102,740 M
1
cm
1, and 91,360 M
1
cm
1 for Cel5A, Cel5A17(
), Cel5A
28, and
Cel5A17(
)
28, respectively.
Cellulose Hydrolysis--
The activity of Cel5A and its mutants
on RC was assayed at pH 7 and 37 °C by measuring the liberated
reducing sugars with the hydroxybenzoic acid hydrazide reagent (17).
The reaction mixture contained 20 nmol of enzyme and 20 mg of RC in 20 ml of 50 mM sodium citrate buffer, pH 7.0, with 0.02%
sodium azide (i.e. 1 µM enzyme and 1 mg
ml
1 RC). Duplicate samples were incubated at 37 °C for
96 h with slow end-over-end mixing on a tube roller. Samples of
0.3 ml were removed at intervals, then centrifuged twice for 5 min at
10,000 × g. Samples of 10 to 100 µl of
the supernatants were assayed for reducing sugars (17). Reducing sugar
concentrations were obtained by reference to a standard curve prepared
with glucose.
Heat-inactivated samples were analyzed by analytical high performance
liquid chromatography using a CarboPac PA-100 column. Samples were
loaded onto the column in 10 mM citrate buffer and eluted
with a gradient of 0-100 mM sodium acetate in 100 mM sodium hydroxide. Eluted carbohydrates were detected by
pulsed amperometry. Sample peaks were identified and quantified by
comparison with the elution profiles of cellooligosaccharide standards.
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RESULTS |
Adsorption of CcCBM17 and BspCBM28 to Cellulose--
Neither
CcCBM17 nor BspCBM28 bound to highly crystalline
cellulose I (bacterial microcrystalline cellulose) or highly
crystalline cellulose II (mercerized cotton fibers) (not shown) but
both bound to RC and AvicelTM (Fig.
1, a and b). The
adsorption isotherms were inconsistent with a single binding site
Langmuir isotherm. Scatchard plots were concave confirming the complex
nature of the binding (Fig. 1, c-f). Descending isotherms
showed the binding of CcCBM17 and BspCBM28 to be
completely reversible (not shown).

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Fig. 1.
Depletion binding isotherms of CcCBM17
( ) and BspCBM28 ( ) on regenerated cellulose
(A) and AvicelTM (B). Solid
lines are the best fit lines to a two-binding site model.
Error bars represent the standard deviations of four binding
experiments. Panels C and D show plots of the
isotherms data in the Scatchard form. Panels C and
D show the CcCBM17 RC and AvicelTM
data, respectively. Panels E and F show the
BspCBM28 RC and AvicelTM data, respectively.
Solid lines show the fits to a Langmuir-type two-binding
site model (Equation 1). Dashed lines show the fits to
McGhee-von Hipple overlapping binding site model (Equation 2). Because
they overlapped the fits to Equation 2, fits to the McGhee-von Hipple
overlapping binding site model incorporating a cooperativity term
(Equations 3 and 4) were not included.
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CcCBM17 and BspCBM28 bind to soluble
cellooligosaccharides (8, 9). Therefore, they probably bind to
individual chains in noncrystalline regions of insoluble cellulose but
not to crystalline cellulose I and II. They have up to five
glucose-binding subsites (8, 9) so the molecules to which
CcCBM17 and BspCBM28 bind can be considered as a
linear array of potentially overlapping binding sites. This could
result in nonlinear Scatchard plots, as could the presence of multiple,
independent classes of binding sites with differing affinities.
Therefore, the binding isotherms for CcCBM17 and
BspCBM28 were evaluated by nonlinear regression against
binding models for noncooperative binding to two or more classes of
binding sites (Equation 1), noncooperative binding with steric
exclusion of overlapping binding sites (Equation 2), and cooperative
binding with steric exclusion of overlapping binding sites (Equations 3
and 4). The fits to the modified McGhee-von Hipple models (Equations 2,
3, and 4) were poor and were rejected (Fig. 1). In contrast, fits using
the two-binding site model (Equation 1) were better (Fig. 1). Whereas
it is possible that there were more than two classes of binding sites,
fits to three- or four-binding site models were not statistically
better, so they were rejected on the basis that binding of this
complexity could not be resolved with data of this accuracy.
There was little difference between the association
constants for CcCBM17 and BspCBM28 binding to RC
and AvicelTM. Values of ~1 × 106
M
1 (
G ~8.3 kcal/mol) were
obtained for the high affinity association constant
(Ka1) and ~2 × 104
M
1 (
G ~5.9 kcal/mol) for the
low affinity Ka2 (Table
I). It must be noted that the
Ka values and binding site densities
([N]o) for the low affinity interactions are
only estimates because the data do not extend to saturation of these
binding sites. The association constants were 2-5-fold higher than
originally reported for these CBMs, probably because of the use of a
two-binding site model to fit the data, rather than the one-site model
that was used originally. In general, the density of the low affinity sites ([N2]o) was 2-10-fold
higher than that of the high affinity sites
([N1]o) for a given CBM and
cellulose preparation (Table I). The densities of both classes of
binding sites on both celluloses were 2-8-fold larger for
CcCBM17 indicating a significant difference in the way the
two CBMs recognize cellulose.
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Table I
Adsorption parameters for the binding of CcCBM17 and BspCBM28 to
AvicelTM and RC in 50 mM potassium phosphate, pH
7.0, at 25 °C
Errors represent the standard deviations of four binding experiments.
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Neither CBM (20 µM) adsorbed to RC (0.5 mg/ml) in the presence of excess cellopentaose (1 mM), a
specific competitor for the CBM-binding site. This ratio of CBM
concentration to RC was sufficient to saturate the high affinity sites
and extend into the low affinity interactions. Scatchard plots of
isotherms obtained in the presence of bovine serum albumin, a
noncellulose binding protein used as a "blocking" agent, and using
fluorescently tagged Oregon GreenTM-CBMs to quantify free
and bound protein also showed two classes of binding sites (not shown).
This suggests specific binding to both classes of binding site,
although a potential contribution of CBM-CBM interactions at the
cellulose surface cannot be entirely excluded.
Competition Binding--
BspCBM28 did not compete with
CcCBM17 for binding to AvicelTM or RC (Fig.
2). Likewise, CcCBM17 was
unable to compete with BspCBM28 for binding to sites on
AvicelTM or RC. Adsorption isotherms with labeled CBMs
showed that the fluorescent label did not interfere with binding (not
shown). As expected, unlabeled CBM competed with the same labeled CBM for binding (data not shown). Therefore, there appear to be separate and distinct high affinity binding sites for CcCBM17 and
BspCBM28 in noncrystalline cellulose. The quantities of
protein required to do these experiments precluded a similar approach
to examining competition for the low affinity class of sites.

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Fig. 2.
Competition binding studies where the bound
concentrations of Oregon GreenTM-labeled
CcCBM17 ( ) or BspCBM28 ( ) on
regenerated cellulose (A) or AvicelTM
(B) were measured in the presence of increasing
amounts of the other unlabeled CBM. Error bars
represent the standard deviations of experiments performed in
duplicate. Dashed lines show the theoretical concentrations
of bound material if complete competition is assumed.
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Interaction of the BspCBM17/CBM28 Tandem with
Noncrystalline Cellulose--
The BspCBM17/CBM28 tandem
from Bacillus sp. 1139 Cel5A bound very tightly to RC and
recognized two classes of binding sites (not shown). Nonlinear
regression of the data with a two-site binding model gave a high
affinity interaction Ka1 and [N1]o of 2.1 (±0.8) × 107 M
1 and 3.5 (±0.9) µmol/g
RC, respectively, and a low affinity interaction Ka2 and
[N2]o of 1.1 (±0.5) × 106 M
1 and 4.7 (±0.7) µmol/g
RC, respectively.
In CcCBM17 tryptophan residues 88 and 135 are crucial to
cellulose binding (9). Analogous tryptophan to alanine mutations at
residues 453 and 500 in the CBM17 module of BspCBM17/CBM28 to give BspCBM17(
)/CBM28 (residue numbers
correspond to that of the whole Cel5A enzyme) resulted in a
stoichiometry of 1.1 (±0.1) for binding cellohexaose in contrast to a
stoichiometry of 2 mol of cellohexaose to 1 mol of wild-type
BspCBM17/CBM28. The binding parameters of BspCBM17(
)/CBM28 were virtually identical to
those for the isolated BspCBM28 module
(Ka = 3.90 (±0.05) × 104
M
1 and
H =
58.24 (±1.0)
kJ/mol for BspCBM17(
)/CBM28 and
Ka = 4.04 (±0.05) × 104
M
1 and
H =
62.24 (±0.5)
kJ/mol for BspCBM28 (8)). Thus, the mutations effectively
destroyed binding by the CBM17 module.
Cellulose Hydrolysis by Bacillus sp. 1139 Cel5A--
Three mutants
of Cel5A were made in which CBM28 was deleted or CBM17 inactivated by
the Trp453/Trp500 double mutation (Fig.
3). The kinetics of
2'4'-dinitrophenyl-
-D-cellobioside hydrolysis were the
same, within experimental error, for wild-type Cel5A and the three
mutants. Therefore, the catalytic activity of the enzyme was unaffected
by the mutations (not shown).

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Fig. 3.
Modular representations of Cel5A from
Bacillus species 1139 and the derivatives used in this
study. Modules are colored gray or white
with the identity of module given below. The numbering
above Cel5A shows the amino acid positions of the module
boundaries as determined by sequence similarity searches. represents the W453A and W500A mutations in the given enzyme.
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The initial rates of reducing sugar release from RC were: Cel5A, 300 (±15) µmol liter
1 h
1;
Cel5A17(
), 252 (±9) µmol liter
1
h
1; Cel5A
28, 205 (±7) µmol liter
1
h
1; Cel5A17(
)
28, 170 (±15) µmol
liter
1 h
1 (Fig.
4A). The enzyme concentrations
used (1 µM) were such that only high affinity sites were
bound. The rates of hydrolysis slowed with time until at ~20 h all
four enzymes were working at similar rates (indicated by constant
differences between Cel5A, Cel5A17(
), Cel5A
28, and
Cel5A17(
)
28; Fig. 4B). None of the
reactions went to completion; only ~20-25% of the insoluble
cellulose was converted to soluble sugars. After 48 h of
incubation the addition of fresh RC to hydrolysis reactions resulted in
a burst of reducing sugar release for all four Cel5A derivatives (not
shown) indicating that the wild-type and mutant enzymes were stable
beyond 48 h of incubation. Furthermore, although the rates of
sugar release in this burst were ~3-fold lower than at the initial
time points, the relative rates of the enzymes remained consistent (not
shown).

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Fig. 4.
Hydrolysis of regenerated cellulose.
Panel A, release of reducing sugar from RC by Cel5A ( ),
Cel5A17( )( ), Cel5A 28 ( ), and
Cel5A17( ) 28 ( ) measured by the HBAH reducing sugar
assay (17). Error bars represent the standard deviation of
readings performed in quadruplicate. Panel B, data
normalized to the activity of the catalytic module alone
(Cel5A17( ) 28) and expressed as a fraction of the
maximum of that sugar released by Cel5A17( ) 28,
i.e. the difference between the sample and
Cel5A17( ) 28 at any time divided by the
reducing sugars released by Cel5A17( ) 28 at
96 h.
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Soluble Sugar Release--
Glucose (G1), cellobiose (G2), and
cellotriose (G3) were the only soluble products released from RC by the
wild-type and mutant enzymes during the first 10 min of hydrolysis
(Fig. 5A). As hydrolysis proceeded, G1 and G2 continued to accumulate, but G3 decreased after
2 h, presumably by its hydrolysis. The rates of G1, G2, and G3
release were subtly different (Fig. 5, B and C).
Wild-type Cel5A and Cel5A17(
) had better initial rates of
glucose release than Cel5A
28 and Cel5A17(
)
28, which
were essentially the same (Fig. 5B). The initial rates of G2
and G3 production were similar for wild-type Cel5A,
Cel5A17(
), and Cel5A
28, perhaps with wild-type
favoring G2 production slightly (Fig. 5, C and
D). All produced G2 and G3 more rapidly than
Cel5A17(
)
28.

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Fig. 5.
Release of soluble sugars from RC measured by
high performance liquid chromatography. Panel A, high
performance liquid chromatography traces of soluble sugars released
after 10 min of treatment with enzyme. Top trace shows the
elution profile of cellooligosaccharide standards where the peaks
correspond to the following: 1, glucose (G1); 2,
cellobiose (G2); 3, cellotriose (G3); 4,
cellotetraose (G4); 5, cellopentaose (G5); 6,
cellohexaose (G6). Peaks at ~2 and ~18 min in the samples are
attributed to buffer components. Remaining traces are labeled with the
enzyme that was used. Panels B-D, release of
glucose (B), cellobiose (C), and cellotriose
(D) from regenerated cellulose by Cel5A ( ),
Cel5A17( )( ), Cel5A 28 ( ), and
Cel5A17( ) 28 ( ). Insets show data
normalized to the activity of the catalytic module alone
(Cel5A17( ) 28) and expressed as a fraction of the
maximum of that sugar released by Cel5A17( ) 28.
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DISCUSSION |
The Binding Sites for CcCBM17 and BspCBM28 Are Not the
Same--
CcCBM17 and BspCBM28 are unable to bind to
crystalline preparations of cellulose and, therefore, must recognize
only the regions of RC and AvicelTM that are not
crystalline. The crystal structure of CcCBM17 revealed a
binding site architecture well designed to accommodate only individual
glucan chains providing a rationale for this binding specificity (9).
Examples of CBMs in families 4, 6, 17, 28, and 29, now form a class of
cellulose-specific modules that bind glucan chains rather than
crystalline surfaces (8, 18-22). The presence of high and low affinity
binding sites for CcCBM17 and BspCBM28 in
noncrystalline cellulose is a new and unexpected finding. It suggests
multiple different physical presentations of cellulose chains that are
sufficiently different to be discriminated by CBMs. Differences in
physical presentation of a cellulose chain may involve chain
conformation, the presence of local microstructure (e.g.
proximity of other chains), or even differences in solvation. Distinct
binding sites are also present on crystalline cellulose: CfCBM2a from Cellulomonas fimi Xylanase 10A
recognized high affinity and low affinity binding sites in bacterial
microcrystalline cellulose (12). In this case, heterogeneity in the
structure of crystalline cellulose was proposed to influence binding.
CcCBM17 and CfCBM4-1 from C. fimi
Cel9B bind to distinct sites in noncrystalline preparations of
cellulose because, like CcCBM17 and BspCBM28,
they do not compete for binding (5). This led to the proposal that
cellulose chains in noncrystalline cellulose could have two or more
different "physical forms" or "substructures." Our observations
are unlikely to be an artifact of unseen oddities in the binding
equilibria, such as the apparent irreversibility of CfCBM2a
(5), because 1) for both CBMs the binding was completely reversible, 2)
cellopentaose was as an effective binding competitor, and 3) each CBM
competed with a fluorescently tagged itself for binding. Furthermore,
there was a 2-3-fold disparity in the densities ([N]o) of the high affinity binding sites for
the two CBMs (Table I). Because these CBMs are a similar size (190 amino acids), similar shape (
-sandwich fold), and bind optimally to
cellohexaose (8, 21) differences in the "footprint" size of the
CBMs on a cellulose molecule cannot explain the differences in
capacity. Thus, there is compelling evidence that CcCBM17
and BspCBM28 recognize different high affinity sites in the
noncrystalline regions of AvicelTM and RC. We are again led
to the conclusion that noncrystalline cellulose contains chains with
sufficiently different physical presentations to be discriminated by CBMs.
Noncrystalline cellulose has traditionally been referred to as
amorphous cellulose, literally shapeless cellulose, because we fail to
detect regular structural elements. It is well known that insoluble and
soluble polysaccharides adopt conformations with energetic minima (23).
Even the conformation of cellooligosaccharides in solution is
stabilized by intramolecular hydrogen bonds to a consistent helical
axis between 2- and 3-fold (24). Accordingly, it is likely that the
molecules in noncrystalline regions of cellulose can adopt different
conformations of minimum energy.
The conformation of oligosaccharides is known to play a role in the
binding specificity of CBMs. Family 4 CBMs can discriminate between the
loose, looping helical molecules of
-1,3-glucans and the linear
molecules of cellulose (25) and the position in space of a single
tryptophan residue in family 2 CBMs determines specificity for
crystalline cellulose or the 3-fold helix of xylan (26). Discrimination
of different cellulose chain conformations in noncrystalline cellulose
may explain the observations made with CcCBM17,
BspCBM28, and CfCBM4-1. However, the
conformations of cellooligosaccharides bound in crystals of
CcCBM17 and CfCBM4-1 are essentially identical to
one another and to that of cellopentaose in solution (9, 25). The only
apparent difference in how these CBMs bind cellooligosaccharides is the
depth of their binding sites and the orientations of the chains in the
binding sites. CcCBM17 has a very shallow groove that binds
the cellooligosaccharide with the planes of the sugar rings
approximately parallel to the protein surface (9), whereas
CfCBM4-1 has a deep groove that binds the
cellooligosaccharide edge-on (25). It is possible that the
conformations of cellooligosaccharides in the crystals may not
represent the conformation of molecules in noncrystalline cellulose
that are optimally bound by CBMs. It is significant that both of these
CBMs bind noncrystalline cellulose with affinities approximately an
order of magnitude larger than their affinities for
cellooligosaccharides (9, 27). Nonetheless, these structural observations do suggest that the presentation of the cellulose chains,
e.g. orientation of chains relative to potentially
interfering microstructures, rather than their conformation may play a
role in how CBMs bind them.
Fine-tuning Cellulose Recognition with Tandem
CBMs--
Unfortunately, despite considerable effort,
BspCBM17 could not be independently produced and
characterized for comparison with BspCBM28.
CcCBM17 was chosen as a substitute for BspCBM17 because it is well characterized and it has 55% amino acid identity and 70% similarity with BspCBM17 (Fig.
6). Most of the sequence divergence
between CcCBM17 and BspCBM17 is in loop regions
removed from the binding site (Fig. 6). All of the amino acid side
chains comprising the cellulose-binding site of CcCBM17 are
conserved in BspCBM17 and the residues in CcCBM17
that interact directly with the carbohydrate are identical in the two
molecules (Fig. 6). Thus, we believe that the structures and binding
functions of BspCBM17 and CcCBM17 are very
similar making CcCBM17 an appropriate substitute for
BspCBM17. The following conclusions are based on this
assumption. Although unlikely, it is formally possible that undetected
subtle differences in BspCBM17 and CcCBM17 do
lend them different specificity for noncrystalline cellulose.

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Fig. 6.
Conservation of structural and functional
elements between CcCBM17 and
BspCBM17. Panel A shows the
structure of CcCBM17 in a tube representation of the
polypeptide backbone. Residues identical in BspCBM17 are
colored blue, similar residues are colored pink,
and variant residues in khaki. The bound cellotetraose
(gray and red) and residues that interact with it
(color corresponds to conservation as above) are shown in a ball and
stick representation. The bound Ca2+ is shown as a
green sphere. This figure was prepared with MOLMOL (39).
Panel B shows an amino acid alignment of CcCBM17
and BspCBM17. Conserved residues, identical or similar,
corresponding to those in panel A are highlighted. Aromatic
amino acids involved in the binding of CcCBM17 to
cellotetraose are indicated above the sequences by .
Polar amino acids involved in binding are indicated by .
Asterisks (*) mark blocks of 10 amino acids. This alignment
was prepared using ClustalW (40).
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The high affinity of BspCBM17/CBM28 relative to its
constituent modules (20-fold larger Kas than the
individual modules) is another example of cooperativity between the
modules in tandem CBMs that leads to nonadditive enhancements in
affinity, termed avidity (20, 28, 29). This must result from the
simultaneous interaction of the covalently linked modules with binding
sites in the cellulose that are proximal in three-dimensional space. A
two-step mechanism for the adsorption of tandem CBMs to crystalline cellulose is appropriate to describe the high affinity interaction, Ka1, of BspCBM17/CBM28 with
noncrystalline cellulose (29) (Fig. 7).
Using the high affinity binding constants of CcCBM17 and
BspCBM28 as estimates of
Ka,17 and
Ka,28, the unimolecular fractions of
Ka1,
Ku,17 and Ku,28 can be calculated to be ~10
(dimensionless units). This equilibrium strongly favors the doubly
bound species (both modules of the tandem bound) over the singly bound
species 10 to 1 until binding sites with an arrangement appropriate for
occupation by both modules of BspCBM17/CBM28 are depleted
(i.e. [N1]o = 3.5 µmol/g RC). The [N1]o for
BspCBM17/CBM28 compares remarkably well with
[N1]o for BspCBM28
binding to RC suggesting that the high affinity interaction of
BspCBM17/CBM28 is limited by the number of CBM28-binding
sites. Only some 40% of the high affinity CBM17-binding sites
participate in this interaction.
[N2]o of BspCBM17/CBM28 was very close to the concentration of the remaining 60% (or ~5.0 µmol/g cellulose) of the high affinity CBM17-binding sites.
Furthermore, the affinity of CcCBM17 for RC was identical to
Ka2 of BspCBM17/CBM28.
Presumably, the second class of binding sites (Ka2) for BspCBM17/CBM28
represents the formation of species bound only by its CBM17 module to a
high affinity CBM17-binding site.

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Fig. 7.
Schematic representation of the proposed
two-step binding of the BspCBM17/CBM28 tandem to
noncrystalline cellulose (29). The filled ellipse
represents the CBM17 module and the open ellipse represents
the CBM28 module. Equilibrium constants according to the individual
steps are labeled. Binding to a single cellulose chain is shown for
simplicity. The same effect would be observed if the individual modules
bound different cellulose chains.
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The biological significance of this form of binding cooperativity in
CBMs is unclear. We have previously suggested that in many cases this
may be a mechanism for compensating the loss of affinity at higher
temperatures in thermophilic and hyperthermophilic CBMs (20). This is
unlikely in this case, because the source organism is mesophilic, and
in other cases of cooperativity in CBMs that originate from mesophilic
organisms (28, 30). BspCBM17/CBM28 is effectively
concentrated to a specific region through its high affinity for
proximal CBM17 and CBM28-binding sites. The low affinity sites for
BspCBM17/CBM28 will only be occupied when the high affinity arrangement is completely occupied. Thus, an alternative function of
tandem CBMs may be to "fine-tune" binding specificity for cellulose chains, as in this case, or other glycans with a particular physical presentation.
Do the CBM17 and CBM28 Modules of Cel5A Influence Cellulose
Hydrolysis?--
The presence of the CBM17 and CBM28 modules in
wild-type Cel5A clearly enhances the ability of the enzyme to release
soluble sugars from noncrystalline cellulose. However, the effect is
subtle, producing only up to a 2-fold enhancement in the initial rate of sugar release and a ~40% increase in overall soluble sugar yield
relative to Cel5A lacking functional CBMs. This is consistent with the
modest 2-3-fold decreases in activity on insoluble xylan,
-1,3-glucans, or cellulose when a CBM is removed from its cognate catalytic module (31-35). Occasionally, larger decreases in catalytic activity are observed (36-38).
The enzyme loading on the cellulose at the initiation of hydrolysis can
be predicted using knowledge of the individual CBM and tandem binding
properties. 99% of Cel5A and 74% of Cel5A17(
) are
predicted to be bound to the region where CBM17- and CBM28-binding sites are in close proximity, which will be called region A. 89% of
Cel5A
28 would be distributed ~40:60 between region A and the region comprising only CBM17-binding sites, which will be called region
B. Considering only bound quantities of enzyme, one would expect
differences in the initial rates of cellulose hydrolysis (within the
first 2-3 h) that are approximately proportional to the amounts of
bound enzyme, i.e. relative activities of Cel5A > Cel5A
28 > Cel5A17(
). All were better than
Cel5A17(
)
28 supporting the general importance of the
CBMs to Cel5A activity. However, Cel5A17(
) had better
initial rates of releasing reducing sugars than Cel5A
28 (Fig. 4),
mostly attributable to glucose release (Fig. 5B), which is
not consistent with predictions based only on bound quantities of
enzyme. Although the difference is admittedly subtle, we suggest that
region A may have greater susceptibility to hydrolysis than region B
resulting in the observed differences in enzyme activity. The
fine-tuning of cellulose recognition by the CBM tandem appears to
refine noncrystalline cellulose hydrolysis by Cel5A.
Biological Implications--
Binary (crystalline versus
noncrystalline) paradigms of cellulose structure are inadequate with
respect to the enzymology of cellulose recognition and hydrolysis.
Mounting evidence indicates that CBMs discriminate distinct binding
sites within noncrystalline cellulose implying that this form of
cellulose is not truly structureless as it was thought to be. Indeed,
this form of cellulose, which is more appropriately called
noncrystalline cellulose instead of amorphous cellulose, appears
to have cellulose chains with different and distinct physical
presentations. Discrimination of these physical presentations by the
CBMs of cellulases has clear implications on the hydrolytic activities
of these enzymes.