Tài liệu Báo cáo khoa học: An investigation of the substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina pdf

An investigation of the substrate specificity of the
xyloglucanase Cel74A from Hypocrea jecorina
Tom Desmet
1
, Tineke Cantaert
1
, Peter Gualfetti
2
, Wim Nerinckx
1
, Laurie Gross
2
, Colin Mitchinson
2
and Kathleen Piens
1
1 Department of Biochemistry, Physiology and Microbiology, Faculty of Sciences, Ghent University, Belgium
2 Genencor International Inc., Palo Alto, CA, USA
The tropical soft rot fungus Hypocrea jecorina (for-
merly Trichoderma reesei) secretes one of the most effi-
cient and best characterized mixtures of cellulolytic
enzymes, including at least five endoglucanases (EG;
EC 3.2.1.4) and two exoglucanases or cellobiohydro-
lases (CBH, EC 3.2.1.91). These enzymes are divided
into different glycoside hydrolase (GH) families on the
basis of sequence similarities and consequent conserva-
tion of fold, and stereochemical outcome of the reac-
tion: inversion (single displacement) or retention
(double displacement) of the anomeric configuration
[1,2]. A regularly updated overview of the different
GH families can be found at />CAZY/index.html [3].

Several new genes coding for biomass-degrading
enzymes have been identified in H. jecorina [4]. One of
these enzymes is Cel74A, formerly EG VI [5], which is
coregulated with known cellulases [4] and has recently
been characterized as a xyloglucanase [6]. Specific xylo-
glucanases represent a relatively new class of enzymes
for which important issues, such as nomenclature and
EC numbering, still have to be addressed [6]. Little is
known about the structural factors that contribute to
xyloglucanase activity, as only one enzyme–xyloglucan
complex has so far been studied by crystallography [7].
Although EGs from various GH families have been
shown to display substantial activity on xyloglucan,
specific xyloglucanases can be found in GH families 5,
12, 16, 26 and 74. The last of these currently comprises
24 sequences, including Cel74A, but only eight mem-
bers have been characterized [6–13]. An inverting
mechanism has been demonstrated for GH family 74
[11], and the three-dimensional structures of two repre-
sentative enzymes have been published [7,14].
Xyloglucan is a hemicellulose composed of a b-1,4-
d-GlcP backbone with regularly distributed a- d-XylP
Keywords
Cel74A; glycoside hydrolase; Hypocrea
jecorina; Trichoderma reesei; xyloglucanase
Correspondence
T. Desmet, Department of Biochemistry,
Physiology and Microbiology, Faculty of
Sciences, Ghent University, K. L.
Ledeganckstraat 35, B-9000 Ghent, Belgium

Fax: +32 9 264 5332
Tel: +32 9 264 5272
E-mail:
(Received 2 August 2006, revised 21
October 2006, accepted 9 November 2006)
doi:10.1111/j.1742-4658.2006.05582.x
The substrate specificity of the xyloglucanase Cel74A from Hypocrea jeco-
rina (Trichoderma reesei) was examined using several polysaccharides and
oligosaccharides. Our results revealed that xyloglucan chains are hydro-
lyzed at substituted Glc residues, in contrast to the action of all known
xyloglucan endoglucanases (EC 3.2.1.151). The building block of xyloglu-
can, XXXG (where X is a substituted Glc residue, and G is an unsubstitut-
ed Glc residue), was rapidly degraded to XX and XG (k
cat
¼ 7.2 s
)1
and
K
m
¼ 120 lm at 37 °C and pH 5), which has only been observed before
with the oligoxyloglucan-reducing-end-specific cellobiohydrolase from Geo-
trichum (EC 3.2.1.150). However, the cellobiohydrolase can only release
XG from XXXGXXXG, whereas Cel74A hydrolyzed this substrate at both
chain ends, resulting in XGXX. Differences in the length of a specific loop
at subsite + 2 are discussed as being the basis for the divergent specificity
of these xyloglucanases.
Abbreviations
CNP, 2-chloro-4-nitrophenol; DSC, differential scanning calorimetry; EG, endoglucanase; EndoH, endoglycosidase H; GH, glycoside hydrolase;
HPAEC-PAD, high-pressure anion exchange chromatography with pulsed amperometric detection; OXG-RCBH, oligoxyloglucan reducing-end-
specific cellobiohydrolase; Xyl, xylose.

356 FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS
(Xyl ¼ xylose) substitutions at C6. A specific letter
code has been proposed, in which the unsubstituted
and substituted Glc residues are represented as G and
X, respectively [15]. Further substitution with b- d-Gal P
at C2 of a Xyl residue results in a trisaccharide repre-
sented as the letter L. The repeating unit in xyloglucan
from Tamarindus indica has been identified as XXXG,
with Gal substitutions occurring only at the middle two
positions [16]. Cel74A from H. jecorina has been repor-
ted to hydrolyze this polymer at unsubstituted Glc resi-
dues, resulting in the release of XXXG units [6].
However, the hydrolysis of this building block by the
GH family 74 oligoxyloglucan reducing-end-specific
cellobiohydrolase (OXG-RCBH) from Geotrichum sp.
M128 [8] has prompted us to examine the specificity
of Cel74A using xyloglucan oligosaccharides. In this
article, hydrolysis at substituted Glc residues is clearly
demonstrated, and a new interpretation of the end-
products of xyloglucan hydrolysis is presented. A
subsite map of Cel74A is proposed, and the structure–
function implications for GH family 74 xyloglucanases
are discussed.
Results and Discussion
Protein characterization
Cel74A was purified to apparent homogeneity from an
engineered strain of H. jecorina by a combination of
gel filtration and affinity chromatography. The single
band observed with SDS ⁄ PAGE corresponds to a
molecular mass of about 100 kDa (Fig. 1), in contrast

to the theoretical value of 85.070 kDa for the mature
protein (SwissProt Q7Z9M8), consisting of a catalytic
domain, a linker region, and a C-terminal carbohy-
drate-binding module. A high apparent molecular mass
for intact Cel74A (105 kDa) was also reported by
Grishutin et al. [6]. Their enzyme sample, however,
also contained lower molecular mass species (75–
90 kDa), whereas heterogeneity was not observed in
our Cel74A preparation.
It has long been known that glycosylated proteins
can run with aberrantly low mobility on SDS ⁄ PAGE
[17]. There are two potential N-glycosylation sites in
Cel74A (N213 and N417), but the purified protein was
not extensively N-glycosylated, as treatment with endo-
glycosidase H (EndoH) did not result in a reduction of
the apparent molecular mass (Fig. 1). Chymotryptic
peptide mapping and MS ⁄ MS not only identified the
protein as full-length Cel74A, giving over 80% cover-
age, including the expected N-terminus and C-termi-
nus, but also revealed that both N-glycosylation sites
carry a single GlcNAc after EndoH treatment (data
not shown). MS of the EndoH-treated Cel74A yielded
a molecular mass of 86.552 kDa. Taking into account
the two GlcNAc residues, this observed molecular
mass exceeds the calculated molecular mass by
1.075 kDa, a mass consistent with six hexose residues.
GHs containing a carbohydrate-binding module are
routinely O-glycosylated in the linker region between
the catalytic domain and the carbohydrate-binding
module. Regions 725–739 and 764–782 of the Cel74A

linker were not identified in the chymotryptic map
and, presumably, contain the sites of O-glycosylation,
but we have not identified the specific site(s) or the
form of the O-glycans.
The CD spectrum of Cel74A looks a lot like that of
an unordered protein (Fig. 2) [18]. Addition of sub-
strate does not affect the overall secondary structure
but decreases the ellipticity around 230 nm that is
diagnostic of tryptophan exciton coupling [19]. The
structure of GH family 74 enzymes has recently been
shown to consist of two b-propeller domains [14]. Such
a fold has been recognized in a wide range of proteins,
including other GHs (clans E, F and J) [3] and hemo-
pexin [20]. The CD spectrum of the latter protein [21]
is very similar to the one observed here; therefore, the
unusual spectrum is probably diagnostic of the b-pro-
peller fold. Thermal melting can be monitored at
212 nm and reveals a T
m
of 64.1 °C at pH 5.5, coinci-
dent with the value of 64.0 °C determined by differen-
tial scanning calorimetry (DSC) (data not shown). The
pH of optimum stability as determined by DSC is 5
(data not shown).
123 4
200
116
97
66
45

Fig. 1. SDS ⁄ PAGE analysis of Cel74A: lane 1, molecular mass
markers (kDa); lane 2, Cel74A; lane 3, Cel74A treated with EndoH;
lane 4, EndoH.
T. Desmet et al. Hypocrea jecorina Cel74A
FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS 357
Substrate specificity
The hydrolytic activity of Cel74A on cellotetraose
was optimal at pH 5 and 60 °C (data not shown).
The kinetic parameters for the hydrolysis of carboxy-
methyl cellulose, b-glucan and xyloglucan (Table 1)
differ from those reported by Grishutin et al. [6],
although the strong preference of Cel74A for the last
of these substrates is clear in both studies. A possible
reason for the quantitative differences is the presence
of truncated protein in the previously reported
enzyme sample.
The considerable increase in activity on cellotetraose
and 2-chloro-4-nitrophenyl cellotrioside (GGGCNP)
(CNP, 2-chloro-4-nitrophenol) in comparison to cello-
triose and GGCNP, respectively (Table 2), suggests an
active center composed of at least four subsites
() 2 ⁄ + 2). The preferential release of GCNP from the
chromogenic substrates excludes the use of direct spec-
trophotometric assays. The strong contribution of all
four subsites to ligand binding is reflected in the higher
degree of inhibition of activity on XXXG by cello-
tetraose (60% decrease in activity) compared to cello-
triose (5% decrease in activity) (for conditions, see
Experimental procedures). The accommodation of a
b-1,3-bond between subsites ) 1 and + 1 is unusual

for a b-1,4-glucanase but not unprecedented [22],
and is supported by the inhibition of Cel74A by lami-
naritriose (10% decrease in activity on XXXG).
Mixed-linkage oligosaccharides, however, seem to be
preferentially cleaved at b-1,4-bonds (Table 2).
Hydrolysis of xyloglucan chains
The end-products of xyloglucan hydrolysis by Cel74A
have been reported to be XXXG, XXLG ⁄ XLXG and
XLLG (see introduction for nomenclature), indicating
hydrolysis at nonsubstituted Glc residues [6]. However,
we demonstrate by high-pressure anion exchange chro-
matography with pulsed amperometric detection
(HPAEC-PAD; Fig. 3A) and ESI-MS (not shown) that
XXXG is readily hydrolyzed to XX and XG by Cel74A.
Moreover, this enzyme exhibits 30-fold higher activity
on XXXG (k
cat
¼ 7.2 s
)1
and K
m
¼ 120 lm) than on
GGGG (Table 2). Its active center must therefore con-
tain specific binding sites for the Xyl residues at subsites
) 2 to + 1 (Fig. 4), as has been proposed for the GH
family 74 exoglucanase from Geotrichum [8].
With such an active site composition, one would
expect the end-products of xyloglucan hydrolysis by
Cel74A to be XGXX, XGXL ⁄ LGXX and LGXL
instead of their structural isomers XXXG, XXLG ⁄

XLXG and XLLG, respectively. Unfortunately, these
isomers cannot be discriminated by MS and are
undoubtedly very hard to separate by HPLC. They
would be indistinguishable on the chromatograms
reported by Grishutin et al., as the two octasaccharide
isomers present in their product mixture also coeluted
[6].
Further proof of hydrolysis at substituted Glc resi-
dues by Cel74A was obtained with the oligosaccharide
XXXGXXXG (Figs 3 and 5). Although this com-
pound has been treated with b-galactosidase by the
supplier, some b-1,2-GalP residues are still present,
Fig. 2. CD spectra of Cel74A at 25 °C and pH 5.0, with (dashed
line) and without (solid line) the addition of 0.1 mgÆmL
)1
tamarind
xyloglucan. Data were collected every 1 nm for 5 s, and three
spectra were averaged.
Table 1. Kinetic parameters for the hydrolysis of b-glucans by Cel74A (37 °C, pH 5). ND, not determined (very low activity); PASC, phos-
phoric acid-swollen cellulose; CMC, carboxymethyl cellulose; HEC, hydroxyethyl cellulose.
Substrate Backbone k
cat
(s
)1
) K
m
(mgÆmL
)1
) k
cat

⁄ K
m
(mLÆmg
)1
Æs
)1
)
Avicel b-1,4- ND ND ND
PASC b-1,4- 7 ± 2 0.8 ± 0.2 9
CMC b-1,4- 52 ± 8 1.3 ± 0.3 40
HEC b-1,4- 138 ± 11 4.8 ± 1.2 29
Laminarin b-1,3- 193 ± 23 2.1 ± 0.6 92
b-Glucan b-1,3 ⁄ 1,4- 306 ± 9 0.5 ± 0.1 612
Xyloglucan b-1,4- 805 ± 13 0.3 ± 0.1 2683
Hypocrea jecorina Cel74A T. Desmet et al.
358 FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS
and therefore the substrate and several products
appear as a cluster of peaks on the chromatograms
(Fig. 3B–D). Attempts to treat the sample more exten-
sively with b-galactosidase from Aspergillus niger
resulted in general degradation, presumably by con-
taminating enzyme activities. Nevertheless, this sub-
strate mixture is still very useful for the qualitative
analysis of the degradation pattern, and will be
referred to as XXXGXXXG for simplicity.
From the results presented here, it is obvious that
XXXGXXXG is rapidly hydrolyzed to XX, XGXX
and XG. Indeed, the release of the small products
(XG ⁄ XX) allows the heptasaccharide product to be
identified as XGXX (Fig. 3C). The unambiguous detec-

tion by ESI-MS of XGXXXG as intermediate product
(Fig. 5) confirms that the substrate is not hydrolyzed
to its repeating unit XXXG. Moreover, it can be con-
cluded that Cel74A is not able to hydrolyze xyloglucan
at unbranched Glc residues, as XGXX does not disap-
pear after hours of incubation with Cel74A (data not
shown), in agreement with the size of the end-products
of xyloglucan hydrolysis reported by Grishutin et al.
[6]. As a comparison, XXXGXXXG was treated with
H. jecorina Cel12A (EG III), which is known to hydro-
lyze xyloglucan at unbranched Glc residues, thus
releasing XXXG units [23]. Indeed, this cellulase is
not able to hydrolyze XXXG (data not shown)
and only produces a heptasaccharide product from
XXXGXXXG (Fig. 3D). Cel12A and Cel74A clearly
display different degradation patterns on xyloglucan
chains.
Mode of action
The recent elucidation by X-ray crystallography of the
structure of two GH family 74 enzymes has suggested
a structural basis for the difference between an exoglu-
canase (OXG-RCBH from Geotrichum) [14] and an
EG (XGH74A from Clostridium thermocellum) [7].
Both enzymes have an active site located in an open
cleft, but a specific loop segment that blocks the
entrance of the cleft in OXG-RCBH at subsite + 2
could provide this enzyme with exoglucanase activity
by preventing binding to the middle of a xyloglucan
Table 2. Relative activity of Cel74A towards oligosaccharides
(37 °C, pH 5).

Substrate
a
Relative activity Final products
GGCNP 0.24 G + GCNP
GGGCNP 0.68 GG + GCNP
GGGGCNP 0.84 GGG + GCNP
GGG 0.32 G + GG
GGGG 0.76 GG + GG
GGGGG 1 GGG + GG
G3G3G 0.38 G + G3G
GG3G 0.52 G + G3G
GGG3G 0.83 GG + G3G
XXXG 27 XX + XG
XXXGXXXG 39 XX + XGXX + XG
a
X, Glc substituted with a-D-XylP at C6; CNP, 2-chloro-4-nitro-
phenol. The number 3 indicates a b-1,3 bond; all other backbone
linkages are b-1,4.
Fig. 3. HPAEC-PAD profiles of xyloglucan oligosaccharides: XXXG
(pure) after incubation with Cel74A (A), and XXXGXXXG (impure)
before (B) and after incubation with Cel74A (C) or with Cel12A (D).
The numbering of the peaks is as follows: 1, XG; 2, XX; 3, XXXG;
4, XGXX; 5, XXXGXX ⁄ XGXXXG; 6, XXXGXXXG. Some of the com-
pounds in (B)–(D) appear as a cluster of peaks, due to partial substi-
tution with Gal residues.
Fig. 4. Schematic representation of the active site () 2 ⁄ +2) of
Cel74A from H. jecorina.
T. Desmet et al. Hypocrea jecorina Cel74A
FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS 359
chain. The ‘exo-loop’ sequence is absent in XGH74A

and all other characterized GH family 74 EGs, but
part of it (seven out of 11 amino acids) is conserved in
H. jecorina Cel74A (SwissProt Q7Z9M8).
OXG-RCBH hydrolyzes XXXG to XX and XG,
just like Cel74A, but it cleaves XXXGXXXG exclu-
sively at the reducing end [8], in contrast to the pres-
ently studied enzyme (Table 2). Cel74A has been
shown to lower the average molecular mass of xyloglu-
can very slowly, which is typical for an exo mode of
action [6]. However, this exo-like behavior is probably
not absolute [6] nor reducing-end-specific, in light of
the fast hydrolysis of XXXGXXXG at both chain
ends (Figs 3 and 5). Indeed, the exo-loop cannot com-
pletely block the active site cleft of Cel74A at subsite
+ 2, as its end-products of xyloglucan hydrolysis
have a backbone degree of polymerization of four
instead of two. Furthermore, its preference for soluble
(carboxymethylcellulose ⁄ hydroxyethyl cellulose) over
crystalline (Avicel) and amorphous (phosphoric acid-
swollen cellulose) is typical of EG activity (Table 1).
A possible conformation of XXXG in subsites
) 2 ⁄ + 2 of OXG-RCBH is shown in Fig. 6. The exo-
loop is located roughly above the postulated + 2 sub-
site, where it is believed to restrict binding of a
branched Glc residue [14]. Docking experiments on
OXG-RCBH indicate that the shorter loop length in
Cel74A can be expected to increase access of an oligo-
saccharide to potential subsites + 3⁄ + 4, and may be
the basis for the unique mode of action of this enzyme.
More definitive statements await the solution of the

Cel74A structure, which is currently underway.
Conclusions
The xyloglucanase Cel74A from H. jecorina is shown
in this study to release XGXX units from xyloglucan
chains, in contrast to a recently published report [6].
Assuming hydrolysis at unsubstituted Glc residues,
those authors have tentatively identified the heptasac-
charide product of xyloglucan hydrolysis as XXXG.
Indeed, analysis by HPLC and ESI-MS does not
discriminate between these heptasaccharide isomers.
However, the fast hydrolysis of XXXG by Cel74A
(k
cat
¼ 7.2 s
)1
and K
m
¼ 120 lm) and the release of
both XX and XG from XXXGXXXG (Figs 3 and 5)
100
%
0
100
%
0
554.0
1076.2
A
BD
C

1157.8
629.1
554.0
497.0
782.1
929.2
1076.2
1000500 m/z
Fig. 5. ESI-MS analysis of XXXGXXXG, before (A) and after (B) incubation with Cel74A, and the proposed cleavage pattern (C) with the
theoretical m ⁄ z value (z in superscript) of the most important fragments (the position of the Gal residues is arbitrary) detected as Na
+
adducts (D).
Fig. 6. Possible conformation of XXXG in subsites ) 2 ⁄ + 2 of OXG-
RCBH from Geotrichum (Protein Data Bank 1sqj). The glucosyl unit
in subsite ) 1 was minimized in a skew-boat
1
S
3
-conformation, by
analogy with the substrate distortion often observed in crystallo-
graphic studies of b-glucanase complexes [2].
Hypocrea jecorina Cel74A T. Desmet et al.
360 FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS
revealed in our study clearly indicate cleavage at sub-
stituted Glc residues.
An active site composed of at least four subsites
() 2 ⁄ + 2) and containing additional interaction sites
for the Xyl residues is proposed for Cel74A (Fig. 4), in
accordance with OXG-RCBH from Geotrichum [8].
The latter enzyme hydrolyzes xyloglucan oligosaccha-

rides at substituted Glc residues, but releases only XG
from XXXGXXXG and cannot be very active on
xyloglucan, in contrast to Cel74A (Table 1).
A specific loop at subsite + 2 is believed to be
responsible for the exoglucanase-like behavior of
Cel74A and OXG-RCBH, but the exact mechanism is
not yet clear. This ‘exo-loop’ also seems to restrict the
access of branched residues to subsite + 2, and could
therefore be the major determinant of the degradation
pattern of GH family 74 xyloglucanases. Enzymes that
hydrolyze xyloglucan at branched Glc residues obvi-
ously need a ) 1 subsite that is relatively spacious
(Fig. 6), but a spacious ) 1 subsite is also observed
in the EG XGH74A from Clostridium thermocellum,
which hydrolyzes xyloglucan at unbranched Glc resi-
dues [7].
Clearly, further studies are required to determine the
exact nature of the interactions of the loop residues in
different GH family 74 enzymes, especially with
branched substrates. Access to more crystal structures
of enzyme–ligand complexes will hopefully lead to a
better understanding of the factors that contribute to
the divergent specificity of these xyloglucanases.
Experimental procedures
Enzyme expression and purification
Cel74A was obtained from an engineered derivative of
H. jecorina strain RL-P37, in which four cellulase genes
(cbh1 ⁄ cel7a, cbh2 ⁄ cel6a, egl1 ⁄ cel7b, and egl2 ⁄ cel5a) were
disrupted as described in Bower et al. [24]. Higher levels of
Cel74A expression were observed in a derivative of this

strain, transformed with a circular plasmid carrying the cat-
alytic domain of Cel7A (carbohydrate-binding module I)
behind the cel7a promoter. The resultant strain was grown
at 25 °C in a batch-fed process with lactose as carbon
source and inducer, using a minimal fermentation medium
essentially as described in Ilmen et al. [25]. First, 0.8 L of
medium containing 5% glucose was inoculated with 1.5 mL
of spore suspension. After 48 h, the culture was transferred
to 6.2 L of the same medium in a 14 L fermenter (Bio-
lafitte, Princeton, NJ, USA). One hour after the glucose
was exhausted, a 25% (w ⁄ w) lactose feed was started in a
carbon-limiting fashion, so as to prevent its accumulation.
The pH during fermentation was maintained in the range
4.5–5.5. The final protein concentration in the culture
supernatant was 12.4 gÆL
)1
.
After concentration of the supernatant to 88 gÆL
)1
by ul-
trafiltration at 4 °C with a PTGC membrane from Milli-
pore (Billerica, MA, USA), Cel74A was purified by gel
filtration followed by affinity chromatography. A sample of
6.5 mL was applied to a 2.6 · 60 cm Superdex 75 prepara-
tive grade column (Amersham Biosciences, Piscataway, NJ,
USA) equilibrated in 0.15 m sodium acetate ⁄ acetic acid
(pH 5.5). Cel74A eluted in the void volume, with 95% pur-
ity as estimated by SDS ⁄ PAGE (not shown). Subsequent
affinity chromatography, as described in Tomme et al. [26],
succesfully removed residual b-glucosidase activity and

yielded an electrophoretically homogeneous preparation.
The final sample was concentrated with an Ultrafree-MC
Centrifugal Filter Unit (Millipore) to an A
280
of 5, and was
stored at 4 °C in MilliQ-water (Ultrapure Water System;
Millipore).
Protein characterization
The concentration of Cel74A was determined from the
absorbance at 280 nm, using an extinction coefficient of
200 900 m
)1
Æcm
)1
calculated on the basis of the amino acid
composition [27]. EndoH (Sigma-Aldrich, St Louis, MO,
USA) treatment of Cel74A was performed according to the
supplied protocol. ESI-MS was performed with the LCQ
Classic and Advantage (Thermo Electron Corporation,
Waltham, MA, USA) for the determination of the molecu-
lar mass and for peptide mapping, respectively.
CD spectra were collected from a 0.1 cm path length cell
on an Aviv 215 spectrophotometer (Proterion Corporation,
Piscataway, NJ, USA) equipped with a five-position ther-
moelectric cell holder. The CD thermal denaturation experi-
ments were performed at 212 nm, where the maximum
signal difference occurs. The data were fitted to a two-state
transition [28] using savuka software provided by O Bilsel
(University of Massachusetts Medical School, N. Worces-
ter, MA, USA). The midpoint of the transition (T

m
)isan
apparent value, as the thermal denaturation was not rever-
sible and was accompanied by precipitation.
DSC thermograms were collected on a VP DSC instru-
ment from Microcal (Northampton, MA, USA). The ther-
mal denaturation of Cel74A was completely irreversible,
and no transition was seen in a repeat scan; therefore, an
apparent T
m
was approximated as the midpoint of the
DSC peak.
Substrate specificity
b-Galactosidase from A. niger, tamarind xyloglucan and
the derived oligosaccharide XXXG were obtained from
Megazyme (Bray, Co. Wicklow, Ireland), barley b-glucan,
laminarin, laminaritriose, carboxymethyl cellulose, hydroxy-
T. Desmet et al. Hypocrea jecorina Cel74A
FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS 361
ethylcellulose and Avicel were obtained from Sigma-
Aldrich, and the cello-oligosaccharides were obtained from
Merck (Darmstadt, Germany). The oligosaccharides
derived from b-glucan were a gift from A Planas (Universi-
tat Raman Llull, Barcelona, Spain), and the xyloglucan
oligosaccharide XXXGXXXG was a gift from B McCleary
(Megazyme). Phosphoric acid-swollen cellulose was pre-
pared according to Wood [29], and the CNP glycosides
were synthesized as in Van Tilbeurgh [30].
All reactions were performed at 37 °C in 0.1 m sodium
acetate ⁄ acetic acid buffer (pH 5), except for the determin-

ation of the pH profile (McIlvaine buffers pH 3.5–7) and
the temperature profile (30–80 °C) for cellotetraose hydroly-
sis. The increase in reducing sugars was measured by the bi-
cinchoninic acid method described by Mopper and Gindler
[31], using a Microplate Reader 550 from Bio-Rad (Hercu-
les, CA, USA). A standard curve was obtained using
0–100 lm glucose.
The kinetic parameters for the hydrolysis of the different
polymers were determined by mixing 0.01–10 mgÆmL
)1
sub-
strate with 14–140 nm enzyme. Release of soluble reducing
sugars was measured by the bicinchoninic method as above.
The relative activity on the different oligosaccharides and
chromogenic glycosides was determined using 2 mm sub-
strate and 2 lm enzyme. At regular intervals, samples were
deactivated by boiling for 2 min. After 10-fold dilution in
water, the oligosaccharides were analyzed by HPAEC-PAD
(Dionex, Sunnyvale, CA, USA), and the chromogenic gly-
cosides by normal-phase HPLC (Waters, Milford, MA,
USA). ESI-MS of the reaction products was performed
with a Q-trap mass spectrometer in positive mode (Applied
Biosystems, Foster City, CA, USA).
The kinetic parameters for the hydrolysis of 0.04–4 mm
XXXG were determined by HPAEC-PAD with 0.1 lm
enzyme. A calibration curve was obtained using 0–100 lm
substrate. The degree of inhibition by oligosaccharides was
determined by comparing the activity of Cel74A on 0.1 mm
XXXG, determined as described above, in the presence and
absence of 2 mm inhibitor. The inhibitors were not hydro-

lyzed in the time course of these experiments (10 min).
Docking experiments
Docking of the oligosaccharide XXXG into the active site
of OXG-RCBH from Geotrichum (Protein Data Bank 1sqj)
was carried out with autodock version 3.0.5 [32]. The lig-
and was drawn and minimized with hyperchem 4.5
(MM + force field; HyperCube Inc, Gainesville, USA)
[33,34]. The glucose unit in subsite ) 1 was drawn in a
skew-boat
1
S
3
conformation, by analogy with the substrate
distortion often found in crystallographic studies of b-glu-
canase complexes [2]. The graphical user interface auto-
docktools (ADT 1.1) was used for formatting of the
ligand and macromolecule, as well as for setting the grid
and docking parameters. Minor variations of the standard
Lamarckian genetic algorithm parameter settings were used:
the number of runs was set at 50, with a run termination of
7000 generations at a maximum of 25 · 10
7
energy evalua-
tions. The colored figure was prepared with pymol-osx
0.93 ().
Acknowledgements
The authors wish to thank Chris Cummings (Genen-
cor) for H. jecorina strain construction, Nicole Chow
(Genencor) for peptide mapping, Koen Sandra (Ghent
University) for ESI-MS analyses of reaction products,

and Marc Claeyssens (Ghent University) for fruitful
discussions. This work was supported in part by a sub-
contract from The Office of Biomass Program, within
the DOE Office of Energy Efficiency and Renewable
Energy. Tom Desmet holds a fellowship of the Insti-
tute for the Promotion of Innovation through Science
and Technology in Flanders (IWT Vlaanderen).
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