Tài liệu Báo cáo khoa học: The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a b-glycosidase docx

The role in the substrate specificity and catalysis of
residues forming the substrate aglycone-binding site
of a b-glycosidase
Lu
´
cio M. F. Mendonc¸a and Sandro R. Marana
Departamento de Bioquı
´
mica, Instituto de Quı
´
mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil
The b-glycosidases from family 1 of the glycoside
hydrolases are widely distributed among living organ-
isms, being found in bacteria, archea and eukaria.
These enzymes are involved in a high diversity of physi-
ological roles [1,2]. b-glycosidases catalyze the hydro-
lytic removal of the monosaccharide from the
non-reducing end of b-glycosides [2,3]. Their active site
may be divided into subsites, which are of sufficient size
to bind a monosaccharide unit. The monosaccharide
forming the non-reducing end of the substrate, called
glycone, is bound at subsite )1, whereas the remaining
part of the substrate, called aglycone, interacts with the
aglycone-binding site, which may be composed of
several subsites identified by positive numerals. The
substrate is cleaved between subsites )1 and +1 [4].
b-glycosidases are active upon a broad range of sub-
strates, as evidenced by a total of 15 different EC
numbers grouped in family 1 of the glycoside hydro-
lases. Fucose, glucose, galactose, mannose, xylose,
6-phospho-glucose and 6-phospho-galactose are recog-

nized by the b-glycosidase subsite )1. Nevertheless, the
diversity of aglycones is higher, including monosaccha-
rides, oligosaccharides and aryl and alkyl moieties [2].
Furthermore, the aglycone specificity is an important
factor for determining the physiological functions of
b-glycosidases.
Keywords
aglycone; catalysis; glycoside hydrolase;
specificity; b-glycosidase
Correspondence
S. R. Marana, Departamento de Bioquı
´
mica,
Instituto de Quı
´
mica, Universidade de Sa˜o
Paulo, CP 26077, Sa˜o Paulo, 05513-970 SP,
Brazil
Fax: +55 11 3815 5579
Tel: +55 11 3091 3810
E-mail:
(Received 1 February 2008, revised 4 March
2008, accepted 13 March 2008)
doi:10.1111/j.1742-4658.2008.06402.x
The relative contributions to the specificity and catalysis of aglycone, of
residues E190, E194, K201 and M453 that form the aglycone-binding site
of a b-glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investi-
gated through site-directed mutagenesis and enzyme kinetic experiments.
The results showed that E190 favors the binding of the initial portion of
alkyl-type aglycones (up to the sixth methylene group) and also the first

glucose unit of oligosaccharidic aglycones, whereas a balance between
interactions with E194 and K201 determines the preference for glucose
units versus alkyl moieties. E194 favors the binding of alkyl moieties,
whereas K201 is more relevant for the binding of glucose units, in spite of
its favorable interaction with alkyl moieties. The three residues E190, E194
and K201 reduce the affinity for phenyl moieties. In addition, M453 favors
the binding of the second glucose unit of oligosaccharidic aglycones and
also of the initial portion of alkyl-type aglycones. None of the residues
investigated interacted with the terminal portion of alkyl-type aglycones. It
was also demonstrated that E190, E194, K201 and M453 similarly contrib-
ute to stabilize ES
à
. Their interactions with aglycone are individually
weaker than those formed by residues interacting with glycone, but their
joint catalytic effects are similar. Finally, these interactions with aglycone
do not influence glycone binding.
Abbreviations
BglB, b-glycosidase from Paenibacillus polymyxa; SbDhr1, b-glycosidase from Sorghum bicolor;Sfbgly, b-glycosidase from
Spodoptera frugiperda; ZmGlu1, b-glycosidase from Zea mays.
2536 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS
Structural studies of complexes between b-glycosid-
ases and substrates or inhibitors revealed residues that
are present in subsite )1 and interact through hydro-
gen bonds with the glycone hydroxyls. In addition, the
role of these interactions and residues in determining
glycone specificity has been characterized through
site-directed mutagenesis, enzyme kinetics and bio-
energetics [5–15].
Previous studies using oligocellodextrins showed
that diverse b-glycosidases have a different number

of subsites forming their aglycone-binding sites [15].
Nevertheless, just recently the structures of b-glyco-
sidases (ZmGlu1 from Zea mays, SbDhr1 from
Sorghum bicolor and BglB from Paenibacillus poly-
myxa) containing ligands in their aglycone-binding
site were obtained [13,16,17]. These data revealed
that the subsite +1 is formed by two walls that
embrace the aglycone moiety. Subsites +1 of
ZmGlu1 and BglB are narrow, whereas subsite +1
of SbDhr1 has a wider opening [13,17,18]. One of
these walls (also called the basal platform) is formed
by the side chain of a conserved tryptophan (W378
in ZmGlu1, W376 in SbDhr1 and W328 in BglB),
whereas the residues forming the other wall (also
called the ceiling) are highly variable among these
enzymes. In ZmGlu1 the subsite +1 is formed by
bulky and apolar amino acid residues T194, F198,
F205 and F466, whereas T192, V196, L203 and S462
are found in subsite +1 of SbDhr1 [13]. These
residues correspond to C170, L174, H181 and A411
in BglB, which, together with Y169, N223, E225,
Q316 and W412, form subsite +1. Data from the
BglB–cellotetraose complex also revealed the residues
forming subsites +2 and +3 [13].
The type of residues forming subsite +1 of
ZmGlu1 indicate a prevalence of hydrophobic interac-
tions with the aglycone, which are interactions that
present less restriction about the positioning of its
participant. Hence, this could explain the broad agly-
cone specificity of ZmGlu1. On the other hand, the

presence of hydrogen bond-forming residues would
determine a more restricted aglycone positioning in
SbDhr1, which is only active on dhurrin [13,16]. In
subsite +1 of BglB, which is active on oligocellodext-
rins, in spite of the presence of hydrophobic residues
L174 and A411, a network of hydrogen bonds hold
glucose units at subsites +1, +2 and +3 [17].
Therefore, in addition, to facilitate the identifica-
tion of amino acid residues composing the aglycone-
binding site, the structural data of b-glycosidase
complexes have been used to infer the molecular basis
of the specificity for aglycone. Indeed, the balance
between hydrophobic interactions and hydrogen
bonds seems to be important for the aglycone speci-
ficity. However, this model, which is a general
description of the interactions involved in the recog-
nition of aglycone, is not complete, as demonstrated
by the site-directed mutagenesis experiments intending
to exchange the aglycone specificity between SbDhr1
and ZmGlu1 [15,19]. Therefore, this model should be
developed to establish the contribution of each resi-
due in the aglycone-binding site to the binding of
diverse types of aglycones. Furthermore, these data
may be of particular importance in understanding the
physiological role of b-glycosidases and in designing
inhibitors.
In addition, another important issue in understand-
ing the aglycone specificity is the contribution of the
interactions with the aglycone to the catalysis in
b-glycosidases. Mutational studies with ZmGlu1 and

SbDhr1 indicate that residues forming subsite +1 con-
tribute to the catalysis, probably by stabilizing the ES
à
complex of the glycosylation step [15,19]. Besides that,
structural data from the complexes of ZmGlu1 and
SbDhr1 with their natural substrates (dimboa-glc and
dhurrin, respectively) and an inhibitor (glucotetrazole)
suggested that the interactions with the aglycone could
affect the glycone positioning within subsite )1 [13].
Nevertheless, details about the role of different resi-
dues of the aglycone-binding site in the stabilization
of ES
à
and the interdependence between the binding of
aglycone and the positioning of glycone in subsite )1
are not known.
In this study, the role in the substrate specificity
and catalysis of four residues (E190, E194, K201 and
M453) forming the aglycone-binding site of a digestive
b-glycosidase from Spodoptera frugiperda (fall army-
worm) (Sfbgly; EC 3.2.1.21; GenBank accession no.:
AF052729) was evaluated through site-directed muta-
genesis and enzyme kinetic experiments. The roles of
E190, E194, K201 and M453 in aglycone binding
were characterized using competitive inhibitors with
different types of aglycones (oligosaccharides, and
alkyl and phenyl moieties), whereas p-nitrophenyl
b-glycosides (fucosides, glucosides, galactosides and
xylosides) were used to evaluate the catalytic contri-
bution of E190, E194, K201 and M453. Sfbgly, which

is classified in family 1 of the glycoside hydrolases,
has an active site composed of four subsites ()1, +1,
+2 and +3) [20]. The interactions formed between
the substrate glycone and residues Q39 and E451,
which are part of the Sfbgly active site, explain the
preference of this enzyme for b-glucosides, b-galacto-
sides and b-fucosides [10,14]. Nevertheless, the molec-
ular basis of the broad aglycone specificity of Sfbgly
had not been studied previously.
L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase
FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2537
Results and Discussion
Role of residues E190, E194, K201 and M453 in
aglycone binding
The spatial structures of ZmGlu1, SbDhr1 and BglB
revealed groups of amino acid residues that formed the
binding site of the substrate aglycone. These aglycone-
binding sites share a common structural element, a
basal platform formed by a tryptophan residue (W378
in ZmGlu1, W376 in SbDhr1 and W328 in BglB), but
they also have a variable portion (called the ceiling).
This portion is formed by T194, F198, F205 and F466
in ZmGlu1, and by V196, L203 and S462 in SbDhr1.
These residues correspond to C170, L174, H181 and
A411 in BglB, which, together with Y196, N223, E225,
Q316 and W412, form the aglycone-binding site
(Fig. 1). Hence, these residues are potentially involved
in aglycone binding [13,17]. In addition, other residues
form a ‘layer’ that helps in the positioning of the resi-
dues that interact directly with the aglycone (basal

platform and ceiling) [15]. Nevertheless, the relative
contributions of the aglycone-binding residues in the
substrate binding and catalysis are not known for any
b-glycosidase.
Sequence alignment and structural comparison indi-
cated that residues E190, E194, K201 and M453,
which correspond to T194, F198, F205 and F466 in
ZmGlu1, may be part of the aglycone-binding site of
Sfbgly (Fig. 1). Thus, in order to establish the relative
contribution of these residues to the interaction with
different types of aglycone, they were replaced through
site-directed mutagenesis, generating the mutants
E190A, E190Q, E194A, K201A, K201F and M453A.
Replacements with A were made to remove side chains
that could interact with the aglycone. Mutations
E190Q and K201F were planned taking into account
the residues found in the b-glycosidase from
A
B
E
D
C
Fig. 1. Comparison of the aglycone-binding site of some b-glycosidases. (A) Aglycone-binding site of ZmGlu1. The aglycone and glycone of
the substrate (dimboa-Glc) are also indicated. (B) Aglycone-binding site of Sfbgly. Residues E190, E194, K201 and M453 are structurally
equivalent to T194, F198, F205 and F466. The substrate (dimboa-Glc) was included to facilitate the visualization of the active site. (C) Agly-
cone-binding site of SbDhr1, including the substrate dhurrin. (D) Aglycone-binding site of BglB, including the inhibitor thiocellobiose.
(E) Sequence alignment of some b-glycosidases showing residues (boxes) forming the aglycone-binding site.
Molecular basis for specificity of a b-glycosidase L. M. F. Mendonc¸a and S. R. Marana
2538 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS
Tenebrio molitor (AF312017) [21], which is closely

related to Sfbgly (45% identity; 65% similarity). These
insect b-glycosidases were previously characterized and
showed differences in their aglycone specificity [20,21].
The mutant enzymes were expressed in bacteria and
purified through hydrophobic chromatography (sup-
plementary Fig. S1). Then, dissociation constants (K
i
)
for the complex between these mutant enzymes and
different competitive inhibitors were determined and
compared with those from the wild-type Sfbgly
(Table 1).
A series of alkyl b-glucosides (hexyl b-glucoside to
nonyl b-glucoside) was initially used to characterize
the Sfbgly mutants (Table 1). Molecular models of
these alkyl b-glucosides indicate that each four methy-
lene groups of their aglycone may cover an area simi-
lar to that of one glucose unit. Thus, this series of
alkyl b-glucosides is useful for probing mutational
effects along the aglycone-binding site.
Based on the K
i
values (Table 1), the binding energy
(DG
0
) for the pentyl moiety formed by the methylene
groups 2 to 6 of an alkyl-type aglycone was calculated
by subtracting the DG
0
for methyl b-glucoside from

that for hexyl b-glucoside. These data indicated a
favorable binding (DG
0
= )2.9 kJÆmol
)1
) between the
pentyl moiety and the wild-type Sfbgly, whereas the
energy of that interaction was reduced by mutations
E194A, K201A and M453A. Mutation K201F did not
affect the binding of that aglycone moiety (Fig. 2).
However, the binding of the pentyl moiety in the agly-
cone-binding site was unfavorable for the mutants
E190A (DG
0
= +3.5 kJÆmol
)1
) and E190Q (DG
0
=
+3.3 kJÆmol
)1
) (Fig. 2). Indeed, these mutations
caused the most drastic effects on the binding of the
initial pentyl moiety (converting a favorable interaction
to an unfavorable one). In addition, the binding energy
of the terminal portion of the alkyl-type aglycone
(from the seventh to the ninth methylene group) was
determined by subtracting the D G
0
for hexyl b-gluco-

side from that for nonyl b -glucoside (Fig. 2). The
favorable interaction (DG
0
= )4.9 kJÆmol
)1
) observed
for the wild-type Sfbgly was not significantly affected
by any of the mutations, except for K201F, which
increased the energy of that interaction (DG
0
=
)8.1 kJÆmol
)1
).
Table 1. Inhibition data for the wild-type and mutant Sfbgly. All inhibitors were simple linear competitive. K
i
values were calculated using
ENZFITTER software. The data were obtained with at least five different concentrations of substrate (methylumbeliferyl b-glucoside) in the
presence of at least five different concentrations of inhibitor. heptylbglc, heptyl b-glucoside; hexylbglc, hexyl b-glucoside; methylbglc, methyl
b-glucoside; nonylbglc, nonyl b-glucoside; octylbglc, octyl b-glucoside; phenylbglc, phenyl b-glucoside; wt, wild-type.
Enzyme
E190A E194A K201A M453A K201F E190Q wt
Inhibitor K
i
(mM)
Phenylbglc 30 ± 2 36 ± 1 74 ± 4 52 ± 1 48 ± 1 78 ± 1 49 ± 2
Methylbglc 8.0 ± 0.2 10.1 ± 0.1 15.6 ± 0.4 10.2 ± 0.4 10 ± 1 12.1 ± 0.3 5.1 ± 0.7
Cellobiose 17 ± 1 1.2 ± 0.1 81 ± 3 5.2 ± 0.3 30 ± 1 13.7 ± 0.4 2.9 ± 0.3
Cellotriose 2.3 ± 0.2 0.070 ± 0.004 0.41 ± 0.01 0.09 ± 0.01 0.04 ± 0.01 8.6 ± 0.3 0.27 ± 0.01
Cellotetraose 2.4 ± 0.1 0.14 ± 0.01 0.54 ± 0.03 0.21 ± 0.01 0.25 ± 0.03 19.6 ± 0.7 0.20 ± 0.02

Cellopentaose 2.0 ± 0.1 0.20 ± 0.01 2.3 ± 0.1 0.26 ± 0.01 0.30 ± 0.02 16.2 ± 0.3 0.25 ± 0.02
Hexylbglc 32 ± 2 8.0 ± 0.4 10.0 ± 0.6 5.6 ± 0.2 3.3 ± 0.2 44.9 ± 0.7 1.57 ± 0.05
Heptylbglc 21 ± 1 3.6 ± 0.1 4.2 ± 0.2 2.6 ± 0.1 1.12 ± 0.06 21.8 ± 0.5 0.7 ± 0.1
Octylbglc 10.3 ± 0.6 1.49 ± 0.03 2.52 ± 0.09 1.30 ± 0.06 0.42 ± 0.03 11.0 ± 0.2 0.44 ± 0.02
Nonylbglc 5.9 ± 0.2 0.83 ± 0.02 1.16 ± 0.04 0.50 ± 0.03 0.13 ± 0.01 5.7 ± 0.3 0.22 ± 0.03
Fig. 2. Binding energies for the interactions between different
types of aglycone and the wild-type and mutant Sfbgly. Negative
values correspond to favorable interactions, whereas positive val-
ues represent unfavorable binding. Black bars indicate an aglycone
formed by a phenyl moiety; white bars indicate the initial portion
of an alkyl-type aglycone (pentyl moiety formed by methylene
groups 2 to 6); and gray bars indicate the terminal portion of an
alkyl-type aglycone (moiety formed by methylene groups 7 to 9).
wt, wild-type.
L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase
FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2539
Therefore, these results indicate that residues E190,
E194, K201 and M453 are part of the aglycone-bind-
ing site of Sfbgly and interact with the initial pentyl
moiety formed by methylene groups 2 to 6 of the
alkyl-type aglycones. On the other hand, the binding
of alkyl moieties in the more external portion of the
aglycone-binding site, which is occupied by methylene
groups 7 to 9, is not influenced by those residues.
The binding energies for an aglycone formed by a
phenyl moiety were calculated using the K
i
values for
phenyl b-glucoside and methyl b-glucoside (Table 1).
These data showed an unfavorable binding of the phe-

nyl-type aglycone with the wild-type and all-mutant
Sfbgly (Fig. 2). Mutations E190 and E194A resulted in
only small reductions (about 40%) of that unfavorable
interaction, but they did not convert it to a favorable
binding. Thus, by contrast with the binding of alkyl-
type aglycones, single mutations were unable to cause
large changes in the interaction of phenyl-type agly-
cones with Sfbgly.
In addition to the analysis of the interaction with
alkyl-type and phenyl-type aglycones, the dissociation
constants (K
i
) of the complexes between oligocellodext-
rins and the wild-type and mutant Sfbgly were also
determined (Table 1). Based on these results, the bind-
ing energies for each glucose unit of the aglycone
(DG
0
⁄ glucose unit) were calculated (Fig. 3). For orien-
tation purposes, the glucose unit forming the
non-reducing end of the aglycone was called the ‘first
glucose unit’ and the other glucose units were sequen-
tially named in the direction of the reducing end of the
aglycone. Thus, mutations E190A, E194A, K201A and
K201F affected the binding of the first glucose unit of the
aglycone (DDG
0
= +3.3, )3.9, +5.5, +4.1 kJÆmol
)1
,

respectively), whereas M453A did not influence that
interaction. Mutations M453A, K201A and K201F
increased the affinity for the second glucose unit of the
aglycone (DDG
0
= )4.2, )7.3, )10 kJÆmol
)1
, respec-
tively), whereas E190A and E194A did not affect that
interaction. It is noteworthy that E190 and E194 inter-
act with the first glucose unit of the aglycone, whereas
M453 interacts with the second glucose unit of the
aglycone. However, K201 interacts with both glucose
units. In addition, mutations E194A, K201A, K201F
and M453A also significantly affected the binding of
the third glucose unit of the aglycone (DDG
0
= +2.5,
+1.4, +5.3, +2.8 kJÆmol
)1
, respectively). The fourth
glucose unit of aglycone is not bound by any mutant
sfbgly. The same result has been previously observed
for the wild-type sfbgly. The exception to these trends
is the mutant E190Q, which showed a very low affinity
for any glucose unit of the aglycone.
Previously, it has been proposed that each glucose
unit of the aglycone interacts with a specific portion of
the aglycone-binding site, which was named subsite [4].
Taking into account this definition and combining the

results presented above it may be proposed that resi-
dues E190 and E194 are positioned in an internal
region of the aglycone-binding site, probably subsite
+1, because they influence the binding of the first glu-
cose unit of the aglycone. On the other hand, M453
may be positioned in subsite +2 because these residues
influence the binding of the second glucose unit of the
aglycone. Residue K201 may be part of both subsites
+1 and +2 or may be located in the interface between
them as it simultaneously affects the binding of the
first and the second glucose unit of the aglycone. This
proposal is in agreement with the binding data for
alkyl-type aglycones (Fig. 2), because the pentyl moiety
formed by the methylene groups 2 to 6 is large enough
to fill subsite +1 and to occupy part of subsite +2.
As a result, residues E190, E194, K201 and M453 can
still interact with that initial portion of an alkyl-type
aglycone, even though they interact with different units
of an oligosaccharidic aglycone.
Figure 3 also showed that mutations K201A and
K201F affected the interactions with the first three glu-
cose units of the aglycone, whereas M453A caused
modifications in the interaction with the second and
third glucose units. This suggests that modifications in
the interactions with a specific glucose unit of the agly-
cone may alter the conformation and ⁄ or freedom of the
other glucose units, which are part of the aglycone,
affecting its interactions with the aglycone site.
Fig. 3. Binding energies for the interactions between glucose units
of the aglycone and the wild-type and mutant Sfbgly. Negative

values represent favorable interactions, whereas positive values
correspond to unfavorable binding. Glucose units were numbered
from the non-reducing to the reducing end of the aglycone. wt,
wild-type.
Molecular basis for specificity of a b-glycosidase L. M. F. Mendonc¸a and S. R. Marana
2540 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS
Following the characterization of the binding of
different types of aglycone, a comparative analysis of
the mutational effect on their binding revealed that
residues E190, E194, K201 and M453 have different
roles in the determination of the Sfbgly specificity for
the substrate aglycone. Thus, although the bulky and
apolar phenyl moieties may form favorable interac-
tions with the basal platform (W378 in Sfbgly), the
binding of these moieties is dominated by unfavorable
interactions with the polar residues E190, E194 and
K201, whereas M453 has little influence on this. The
replacement of E190, E194 and K201 with A, which
has a small side chain, reduced those unfavorable
interactions (Fig. 2), and, interestingly, the more com-
pact alkyl moieties overcame those unfavorable inter-
actions. Indeed, residues E190, E194, K201 and M453
contributed to the favorable binding of the initial pen-
tyl moiety of the aglycone. Of these residues, E190 is
the most important for that interaction because muta-
tions E190A and E190Q resulted in the largest
decrease of affinity for the pentyl moiety (DDG
0
= 6.4
and 6.2 kJÆmol

)1
, respectively); E194, K201 and M453
contribute similarly to the binding of pentyl moieties
given that the replacement of those residues by A
resulted in similar decrease (when taking into account
the experimental errors) of the affinity (DDG
0
$
2kJÆmol
)1
) for pentyl moieties. However, these same
residues give different contributions to the binding of
the glucose units of the aglycone. Thus, M453 does
not affect the binding of the first glucose unit
(DDG
0
$ 0; Fig. 4); in fact, it interacts with the second
glucose unit of the aglycone (Fig. 3). E194 has an
unfavorable interaction with the first glucose unit,
given that replacement of E194 with A increased, by
3.9 kJÆmol
)1
, the affinity for that glucose unit (Fig. 4).
Conversely, E190 and K201 may form interactions
(probably hydrogen bonds) that are important for the
binding of the first glucose units of the aglycone
because their replacement with A caused a large
decrease in the affinity for glucose (DDG
0
= +3.9 and

+5.6 kJÆmol
)1
, respectively; Fig. 4). In agreement,
K201 corresponds to H181 in BglB, a residue that inter-
acts through a hydrogen bond with glucose units at sub-
site +1 of that enzyme [17]. Moreover, E190
corresponds to T194 in ZmGlu1, which may form a
hydrogen bond with the aglycone of dimboa-Glc.
It is noteworthy that, regarding aglycone binding,
mutation K201F drastically reduced the glucose binding
without affecting the affinity for alkyl moieties. In addi-
tion, mutation K201F also increased the affinity for the
terminal portion of alkyl-type aglycones (Fig. 2). Resi-
due K201 is replaced by F in the b-glycosidases from
Tenebrio molitor (Tmbgly; AF312017) and Cavia porce-
lus (Cpbgly; U50545). Moreover, F179 is part of the
aglycone-binding site of Cpbgly [22]. In order to com-
pare the aglycone-binding sites of these b-glycosidases,
the effect of the aglycone size on the binding of several
alkyl b-glucosides was measured. Interestingly, these
binding data showed that each aglycone methylene
moiety of the aglycone increases in 1.6 kJÆmol
)1
the
affinity of the wild-type Sfbgly for alkyl b-glycoside
(Fig. 5), whereas mutation K201F increased that
parameter to 2.6 kJÆmol
)1
, which was similar to that of
Cpbgly (3.0 kJÆmol

)1
) and higher than that of Tm bgly
(1.0 kJÆmol
)1
) [21,23]. Therefore, residues in posi-
tion 201 (and its equivalent in other b-glycosidases)
may be an important factor in the determination of sub-
site +1 preference for alkyl-type aglycones.
In summary, the triad formed by E190, E194
and K201 controls the specificity for the substrate
Fig. 4. Mutational effect (DDG
0
) on the binding energy of alkyl moi-
eties and glucose units of the aglycone. Black bars correspond to
alkyl moieties and white bars correspond to glucose units. Positive
DDG
0
values correspond to a decrease in affinity, whereas negative
DDG
0
values represent an increase in affinity.
–10
–15
–20
–25
98 7
6 5
Aglycone carbon number
Binding energy (kJ·mol
–1

)
Fig. 5. Effect of the aglycone size on the binding between alkyl b-
glucosides and the wild-type and mutant Sfbgly. (
), wild-type; ( ),
mutant K201F.
L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase
FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2541
aglycone. This triad reduces the affinity for phenyl
moieties. Residue E190 drives the binding of both alkyl
moieties (the initial portion up to the sixth methylene
group) and the first glucose unit of oligosaccharidic
aglycones, whereas a balance between interactions with
E194 and K201 determines the specificity for glucose
units versus alkyl moieties. E194 favors the binding of
alkyl moieties, whereas K201 is more relevant for the
binding of glucose units, in spite of its favorable interac-
tion with alkyl moieties. Therefore, the replacement of
E194 and K201 may be an important mechanism for
changing the specificity of Sfbgly for aglycone.
The relative contribution to aglycone specificity of
residues corresponding to E190, E194, K201 and
M453 is probably different in other b-glycosidases,
especially in view of the broad variety of residues
occupying those positions. Nevertheless, the differen-
tial participation of several residues in the definition of
the aglycone specificity may be a general trend.
Catalytic role of residues E190, E194, K201 and
M453 from the Sfbgly aglycone-binding site
Steady-state kinetic parameters for the hydrolysis of
p-nitrophenyl b-glycosides and methylumbelliferyl

b-glucosides by the wild-type and mutant Sfbgly were
determined (Table 2). In order to evaluate the role of
residues E190, E194, K201 and M453 on the catalytic
Table 2. Steady-state kinetic parameters for the hydrolysis of p-nitrophenyl b-glycosides and methylumbeliferyl b-glucoside by wild-type and
mutant Sfbgly. Experiments were carried out using at least 10 different substrate concentrations. Parameters were calculated using
ENZFITTER
software. MUbglc, methylumbeliferyl b-glucoside; NPbfuc, p-nitrophenyl b-fucoside; NPbgal, p-nitrophenyl b-galactoside; NP bglc, p-nitrophe-
nyl b-glucoside; NPbxyl, p-nitrophenyl b-xyloside.
Enzyme Substrate K
m
(mM) k
cat
(s
)1
) k
cat
⁄ K
m
(s
)1
ÆmM
)1
) Relative k
cat
⁄ K
m
(%)
E190A NPbfuc 0.59 ± 0.05 0.348 ± 0.009 0.58 ± 0.05 100
NPbglc 1.05 ± 0.05 0.59 ± 0.01 0.56 ± 0.02 90
NPbgal 8.2 ± 0.2 0.072 ± 0.001 0.0087 ± 0.0002 1.5

NPbxyl 4.9 ± 0.4 0.00188 ± 0.00009 0.00038 ± 0.00003 0.06
MUbglc 6.3 ± 0.6 0.020 ± 0.001 0.0031 ± 0.0003 0.5
E194A NPbfuc 0.38 ± 0.03 0.98 ± 0.01 2.5 ± 0.2 100
NPbglc 1.20 ± 0.07 0.56 ± 0.01 0.46 ± 0.02 18
NPbgal 9.0 ± 0.3 0.189 ± 0.003 0.0210 ± 0.0007 0.8
NPbxyl 4.8 ± 0.2 0.0127 ± 0.0003 0.0026 ± 0.0001 0.1
MUbglc 2.6 ± 0.1 0.2674 ± 0.0004 0.102 ± 0.003 3.9
K201A NPbfuc 0.71 ± 0.05 2.43 ± 0.03 3.4 ± 0.2 100
NPbglc 2.6 ± 0.2 0.66 ± 0.02 0.25 ± 0.02 7.5
NPbgal 4.9 ± 0.3 0.058 ± 0.001 0.0118 ± 0.0007 0.3
NPbxyl 2.4 ± 0.1 0.0073 ± 0.0001 0.0030 ± 0.0001 0.09
MUbglc 2.6 ± 0.2 0.032 ± 0.001 0.0123 ± 0.0001 0.3
M453A NPbfuc 0.65 ± 0.04 4.15 ± 0.05 6.3 ± 0.4 100
NPbglc 1.26 ± 0.03 1.52 ± 0.01 1.20 ± 0.02 18
NPbgal 4.4 ± 0.2 0.174 ± 0.004 0.039 ± 0.002 0.6
NPbxyl 3.6 ± 0.3 0.0238 ± 0.0009 0.0066 ± 0.0006 0.1
MUbglc 1.7 ± 0.1 0.62 ± 0.01 0.36 ± 0.02 5.7
K201F NPbfuc 0.62 ± 0.06 8.9 ± 0.1 14 ± 1 87
NPbglc 1.2 ± 0.1 4.0 ± 0.1 3.3 ± 0.2 20
NPbgal 6.2 ± 0.3 4.03 ± 0.09 0.65 ± 0.03 3.9
NPbxyl 1.04 ± 0.06 0.089 ± 0.001 0.085 ± 0.005 0.5
MUbglc 0.08 ± 0.01 1.32 ± 0.08 16 ± 2 100
E190Q NPbfuc 0.66 ± 0.04 0.668 ± 0.009 1.00 ± 0.06 100
NPbglc 2.10 ± 0.09 0.65 ± 0.01 0.30 ± 0.01 30
NPbgal 9.9 ± 0.5 0.153 ± 0.003 0.0154 ± 0.0008 1.5
NPbxyl 4.7 ± 0.4 0.0211 ± 0.0008 0.0044 ± 0.0004 0.4
MUbglc 6.2 ± 0.4 0.114 ± 0.004 0.018 ± 0.001 1.8
wt NPbfuc 0.67 ± 0.03 7.14 ± 0.06 10.6 ± 0.4 100
NPbglc 0.90 ± 0.04 2.26 ± 0.03 2.5 ± 0.1 23
NPbgal 2.9 ± 0.1 0.269 ± 0.003 0.092 ± 0.003 0.9

NP
bxyl 1.56 ± 0.05 0.0302 ± 0.0003 0.0193 ± 0.0006 0.02
MUbglc 2.3 ± 0.1 0.231 ± 0.003 0.100 ± 0.004 0.9
Molecular basis for specificity of a b-glycosidase L. M. F. Mendonc¸a and S. R. Marana
2542 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS
steps, these data were used to calculate the effect of
mutations of these residues on the stability of the ES
à
complex for p-nitrophenyl b-glucoside, p-nitrophenyl
b-fucoside, p-nitrophenyl b-galactoside and p-nitrophe-
nyl b-xyloside hydrolysis. Figure 6 shows that all
mutations destabilized the ES
à
complex for all sub-
strates tested, except mutation K201F, which resulted
in stabilization of the ES
à
complex for p-nitrophenyl
b-galactoside and p-nitrophenyl b-xyloside hydrolysis.
Destabilization of ES
à
indicates a reduction in the rate
of substrate hydrolysis, whereas the opposite is valid
for the stabilization of ES
à
. Hence, the existence of
these mutational effects on ES
à
stability indicate that
residues E190, E194, K201 and M453 participate in

the catalysis. Considering that the catalytic mechanism
of b-glycosidases is divided into two steps (glycosyla-
tion and deglycosylation), and that the aglycone is
released in the first step [2], the interactions of these
residues with the aglycone probably contribute to sta-
bilizing the ES
à
complex of the glycosylation step.
Interestingly, a comparison of these mutational
effects with those resulting from the mutation of resi-
dues Q39 and E451, which are involved in the glycone
binding in Sfbgly [10], showed that the ‘ES
à
destabiliz-
ing effects’ resulting from mutations of the aglycone-
binding residues are usually smaller than those from
mutations at subsite )1 (Q39A and E451A) (Fig. 6).
Nevertheless, the total effect of the mutations of the
aglycone-binding residues (20, 20 and 16 kJÆmol
)1
for
p-nitrophenyl b-fucoside, p-nitrophenyl b-glucoside
and p-nitrophenyl b-galactoside hydrolysis, respec-
tively) is similar to the combined effect of mutations
Q39A and E451A (33, 31 and 32 kJÆmol
)1
for p-nitro-
phenyl b-fucoside, p-nitrophenyl b-glucoside and
p-nitrophenyl b-galactoside hydrolysis, respectively).
Hence, the energy of the non-covalent interactions

available to stabilize ES
à
tend to be concentrated in a
few residues in subsite )1 (for instance Q39 and E451),
whereas these ‘ES
à
-stabilizing’ interactions are more
homogeneously distributed among the aglycone-bind-
ing residues. Besides, as the contributions of the agly-
cone-binding residues to the stability of ES
à
are
relevant, they should be considered in the design of
b-glycosidase inhibitors.
Figure 6 also shows that the mutational effects
(DDG
à
) tend to be similar regardless of the substrate
when considering the aglycone-binding residues. This
trend is not observed for mutations of residues (Q39
and E451) directly involved in the binding of glycone,
or for mutation K201F. Taking into account that the
substrates share a common aglycone, but differ in the
glycone, the results obtained for the mutants E190A,
E194A, K201A and M453A are those expected for res-
idues that interact with the substrate aglycone but do
not participate in the binding of the substrate glycone.
Therefore, an implication of these results is that the
interactions with aglycone do not affect the binding of
the glycone within the Sfbgly active site.

This hypothesis was further investigated by deter-
mining the influence on the glycone binding of the
Sfbgly interactions with the aglycone. Thus, the effect
of the alteration of the glycone structure in the stabil-
ity of the ES
à
complex (DDG
à
) for the wild-type and
mutant Sfbgly was determined using the k
cat
⁄ K
m
for
the hydrolysis of p-nitrophenyl b-glucoside, p-nitrophe-
nyl b-fucoside and p-nitrophenyl b-galactoside. These
DDG
à
values represent the manner in which the inter-
actions within subsite )1 are affected by the modifica-
tion of the glycone structure, in particular the spatial
positioning of its hydroxyl groups. Hence, two b-glyco-
sidases, presenting exactly the same interaction pattern
within subsite )1, should be equally affected by the
alteration of the substrate glycone generating the same
DDG
à
. Thus, a plot of these DDG
à
values for a pair of

b-glycosidases presenting identical subsites )1 would
be a line showing a correlation coefficient and slope
equal to 1. Indeed, comparison between wild-type and
mutant Sfbgly using such plots revealed correlation
coefficients and slopes close to 1 for all mutants except
K201F, which presented a correlation coefficient of
0.58 (Table 3). These results indicate that despite the
mutations in the aglycone-binding site, the interactions
with the glycone remained very similar for the mutants
of Sf bgly analyzed, except for K201F. Therefore, at
least for the Sfbgly activity upon p-nitrophenyl b-gly-
cosides, the interactions with aglycone do not affect
glycone binding. In addition, the catalytic contribu-
tions of residues E190, E194, K201 and M453 result
exclusively from interactions with the aglycone that
stabilize ES
à
. Conversely, mutation K201F influenced
the interaction pattern of the p-nitrophenyl b-glyco-
Fig. 6. Mutational effect on the stability of ES
à
(DDG
à
) involving dif-
ferent substrates. Dark gray bars, p-nitrophenyl b-fucoside; white
bars, p-nitrophenyl b-glucoside; black bars, p-nitrophenyl b-galacto-
side; light gray bars, p-nitrophenyl b-xyloside.
L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase
FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2543
sides within subsite )1 through modifying the interac-

tions with their aglycones, suggesting that in mutant
K201F the binding of glycone and aglycone are inter-
dependent. These results are not observed for mutant
K201A, revealing that such mutational effects are
related to the type of residue occupying position 201.
Interestingly, the residue corresponding to K201 in
BglB is H181, which delineates a channel that could be
the pathway for the release of the aglycone in the glyco-
sylation step and the entrance of the water molecule
involved in the hydrolysis of the covalent intermediate
[17]. Thus, the unusual effects of the K201F mutation
on the Sfbgly catalysis could be the result of a drastic
alteration in that putative channel, which would change
the rate of the glycosylation and deglycosylation steps.
Materials and methods
Site-directed mutagenesis
Site-directed mutagenesis experiments were performed as
described in the instructions of the kit ‘QuikChange site-
directed mutagenesis’ (Stratagene, La Jolla, CA, USA) using
a plasmid pT7-7 [24] encoding the wild-type Sfbgly as the
template. A pair of mutagenic primers was used to produce
each desired mutation. Only the primers corresponding to
the sense strand are listed, as follows: mutation E190A,
5¢-caacgagcctagagcgatttgctttgagg-3¢; mutation E190Q, 5¢-caa
cgagcctagacagatttgctttgagg-3¢; mutation E194A, 5¢-gagagattt
gctttgcgggttatggatctgc-3¢; mutation K201A, 5¢-gttatggatctgct
accgcggctccgatcctaaacg-3¢; mutation K201F, 5¢-ggttatggatctg
ctacttcgctccgatcctaaacgc-3¢; and mutation M453A, 5¢-ggacaa
ctttgaatgggcggagggttatattgag-3¢. The incorporation of muta-
tions was verified by DNA sequencing.

Expression of recombinant Sfbgly
BL21 DE3 cells (Novagen, Darmstadt, Germany) were
transformed with pT7-7 plasmids encoding the wild-type
and mutant Sfbgly. Transformed bacteria were cultured
(37 °C, 150 r.p.m.) in 500 mL of Luria–Bertani (LB) broth
containing carbenecillin (50 lgÆmL
)1
) until an attenuance
(D) of 0.6–0.8 at 600 nm was reached. Then, the production
of recombinant Sfbgly was induced (25 °C, 6 h, 150 r.p.m.)
by adding 1 mm isopropyl thio-b-d-galactoside. The
induced bacteria were harvested by centrifugation (7000 g,
30 min, 4 °C) and resuspended in 50 mm Hepes buffer
containing 150 mm NaCl, 0.02% (w ⁄ v) of hen egg-white
lysozyme and 0.1% (v ⁄ v) of Triton X-100. This suspension
was incubated at room temperature for 45 min with slow
shaking at 30 r.p.m. Then, the suspension was exposed to
four pulses (45 s each) of ultrasound using a Branson soni-
fier (at output 4.0) adapted with a microtip. Cell debris was
harvested by centrifugation (7000 g,4°C, 30 min) and the
supernatant was sequentially filtered through cheesecloth
and 0.22-lm Millex filters (Millipore, Billerica, MA, USA).
Purification of recombinant Sfbgly
The soluble material resulting from the lysis of the induced
bacteria was mixed with 200 mm sodium phosphate
(pH 7.0) containing 3.4 m (NH
4
)
2
SO

4
(2 : 1, v ⁄ v). This mix-
ture was incubated at 4 °C, without shaking, for 16h. The
precipitated material obtained after centrifugation (7000 g,
4 °C, 30 min) was discarded,and the supernatant was
filtered through cheesecloth and 0.22-lm Millex filters
(Millipore). Then, this supernatant was loaded onto a
Resource ETH column (GE HealthCare, Chalfont, St Giles,
UK). Non-retained proteins were eluted with 50 mm
sodium phosphate (pH 7.0) containing 1.27 m (NH
4
)
2
SO
4
,
whereas retained proteins were eluted using a linear gradi-
ent of (NH
4
)
2
SO
4
(from 1.27 to 0 m) prepared in 50 mm
sodium phosphate (pH 7.0). Fractions of 1.0 mL were col-
lected and analyzed for b-glycosidase activity using 4 mm
p-nitrophenyl b-fucoside prepared in 50 mm citrate phos-
phate buffer (pH 6.0).
Fractions containing b-glycosidase activity were pooled,
mixed with (NH

4
)
2
SO
4
as described above and then loaded
onto a Resource ISO column (GE HealthCare). Non-
retained proteins were eluted with 50 mm sodium phos-
phate (pH 7.0) containing 0.95 m (NH
4
)
2
SO
4
, whereas
retained proteins were eluted using a linear gradient of
(NH
4
)
2
SO
4
(from 0.95 to 0 m) prepared in 50 mm sodium
phosphate (pH 7.0). Fractions of 1.0 mL were collected and
analyzed for b-glycosidase, as previously described. To
ascertain the purity of Sfbgly, fractions containing b-glyco-
sidase activity were pooled and submitted to SDS-PAGE
analysis followed by silver staining [25,26].
The protein concentrations were determined by using
absorbance at 280 nm in the presence of 6 m guanidium

hydrochloride prepared in sodium phosphate (pH 6.5).
Extinction coefficients (e
280 nm
) were calculated based on
the mutant Sfbgly sequences [27,28].
Enzyme kinetic analysis
The initial rate of hydrolysis of at least 10 different concen-
trations of p-nitrophenyl b-fucoside, p-nitrophenyl b-gluco-
side, p-nitrophenyl b-galactoside, p-nitrophenyl b-xyloside
Table 3. Parameters of the linear free energy relationships (LFER)
between the wild-type and mutant Sfbgly.
LFER
parameter
Mutant
E190A E194A K201A M453A K201F E190Q
Slope 1.0 0.98 1.0 1.0 0.58 0.9
Correlation
coefficient
0.98 0.99 0.99 0.99 0.99 0.99
Molecular basis for specificity of a b-glycosidase L. M. F. Mendonc¸a and S. R. Marana
2544 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS
and methylumbeliferyl b-glucoside were separately measured
at 30 °C. Substrate concentrations ranging from 0.2 to 4 K
m
were used and their hydrolysis was detected by following the
production of p -nitrophenolate or methylumbeliferone. All
substrates were prepared in 50 mm citrate phosphate
(pH 6.0). The enzyme kinetic parameters K
m
and k

cat
were
determined by fitting these data on the Michaelis–Menten
equation using the enzfitter software (Elsevier-Biosoft,
Cambridge, UK).
The K
i
for linear competitive inhibitors (cellobiose, cello-
triose, cellotetraose, cellopentaose, methyl b-glucoside, hexyl
b-glucoside, heptyl b-glucoside, octyl b-glucoside, nonyl
b-glucoside and phenyl b-glucoside) were determined by
measuring the initial hydrolysis rate of at least four different
concentrations of methylumbeliferyl b-glucoside in the pres-
ence of at least five different concentrations of inhibitors
(ranging from 0 to 4 K
i
). The K
i
values were calculated from
replots of the inhibitor concentration versus the slope of the
lines observed in Lineweaver–Burk plots [29].
Calculation of thermodynamic parameters
Differences in the energy of the ES
à
complexes (DDG
à
)
between a pair of different enzymes (mutant and wild-type)
hydrolyzing the same substrate were calculated by the equa-
tion [30,31]:

DDG
z
¼ÀRT lnðk
cat
=K
m
Þ
mut
=ðk
cat
=K
m
Þ
wt
ð1Þ
where R is the gas constant (8.3144 JÆmol
)1
ÆK
)1
), T is the
absolute temperature (303 K) and mut and wt indicate
mutant and wild-type Sfbgly, respectively.
The binding energy between an enzyme and a competi-
tive inhibitor was calculated using the equation:
DG
0
¼ÀRT lnð1=K
i
Þð2Þ
where R is the gas constant (8.3144 JÆmol

)1
ÆK
)1
) and T is
the absolute temperature (303 K).
The binding energy corresponding to each glucose unit of
an oligocellodextrin was calculated using the equation:
DDG
0
n
¼ DG
0
n
À DG
0
ðnÀ1Þ
ð3Þ
where DDG
0
n
represents the binding energy of the ‘n’ glu-
cose unit of the inhibitor, DG
0
n
corresponds to the binding
energy of the oligocellodextrin presenting a degree of poly-
merization equal to ‘n’, and DG
0
(n)1)
is the binding energy

of the oligocellodextrin presenting a degree of polymeriza-
tion equal to ‘(n)1)’. Methyl b-glucoside was used as an
inhibitor presenting a degree of polymerization equal to 1.
Linear free energy relationships
The effect of the alteration of the substrate structure on the
stability of the complex ES
à
was evaluated using the follow-
ing equation [30,31]:
DDG
z
¼ RT lnðk
cat
=K
m
Þ
1
=ðk
cat
=K
m
Þ
2
ð4Þ
where R is the gas constant (8.3144 JÆmol
)1
ÆK
)1
), T is the
absolute temperature (303 K) and k

cat
⁄ K
m
is the rate
constant of hydrolysis of substrates 1 and 2 by the same
enzyme (wild-type or mutant Sfbgly). The substrates were
p-nitrophenyl b-fucoside, p-nitrophenyl b-glucoside and
p-nitrophenyl b-galactoside.
Then, DDG
à
values calculated for the wild-type Sfbgly
were plotted versus those for each mutant Sfbgly. The simi-
larity between the active sites of the wild-type enzyme and
each mutant enzyme is related to the linear correlation
coefficient and the line slope [32].
Structural comparison and sequence alignment
The spatial structure of Sfbgly was modeled using chain A
of the myrosinase of the aphid Brevicoryne brassicae
(1WCG) as the template [33]. The homology modeling
process was performed using the Swiss Model server. The
resulting structure was visualized using DeepView ⁄ Swiss
PDBViewer v3.7 [34]. The structure of Sfbgly, Zea mays
b-glucosidase 1 (ZmGlu1; 1E56), Sorghum bicolor (SbDhr1;
1V03) and Paenibacillus polymyxa (BlgB; 2O9R) were visu-
alized and superimposed using DeepView ⁄ Swiss PDBView-
er v3.7. Amino acid sequences of these b-glycosidases were
retrieved from the CAZy databank [2] and aligned using
clustalx [35]. Only the segment containing residues form-
ing the aglycone-binding site of ZmGlu1 and SbDhr1 was
presented.

Acknowledgements
This project is supported by FAPESP (Fundac¸ a
˜
ode
Amparo a
`
Pesquisa do Estado de Sa
˜
o Paulo) and
CNPq (Conselho Nacional de Desenvolvimento Cient-
ı
´
fico e Tecnolo
´
gico). L. M. F. Mendonc¸ a is a graduate
fellow from FAPESP and S. R. Marana is staff mem-
ber of the ‘Departamento de Bioquı
´
mica – IQUSP’
and research fellow from CNPq.
References
1 Esen A (1993) b-glucosidases. Biochemistry and Molecular
Biology. ACS Symposium Series 553, Washington, DC.
2 Coutinho PM & Henrissat B (1999) Carbohydrate
Active Enzymes Server Available at: s-
mrs.fr/CAZY.
3 Kempton JB & Withers SG (1992) Mechanism of Agro-
bacterium b-glucosidase: kinetic studies. Biochemistry
31, 9961–9969.
4 Davies GJ, Wilson KS & Henrissat B (1997) Nomencla-

ture for sugar-binding subsites in glycosyl hydrolases.
Biochem J 321, 557–559.
L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase
FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2545
5 Namchuck NM & Withers SG (1995) Mechanism of
Agrobacterium b-glucosidase: kinetic analysis of the role
of noncovalent enzyme ⁄ substrate interactions. Biochem-
istry 34, 16194–16202.
6 Wiesmann C, Hengstenberg W & Schulz GE (1997) Crys-
tal structures and mechanism of 6-phospho-b-galactosi-
dase from Lactococcus lactis. J Mol Biol 269, 851–860.
7 Burmeister WP, Cottaz S, Driguez H, Iori R, Palmieri S
& Henrissat B (1997) The crystal structure of Sinapis alba
myrosinase and a covalent glycosyl-enzyme intermediate
provide insight sinto the substrate recognition and active
site machinary of an S-glycosidase. Structure 5, 663–675.
8 Sanz-Aparicio J, Hermoso JA, Martinez-Ripoll M,
Lequerica JL & Polaina J (1998) Crystal structure of
b-glucosidase A from Bacillus polymyxa: insights into
the catalytic activity in family 1 glycosyl hydrolases.
J Mol Biol 275, 491–502.
9 Cicek M, Blanchard D, Bevan DR & Esen A (2000)
The aglycone specificity-determining sites are different
in 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one-
(DIMBOA)-glucosidase (Maize b-glucosidase) and
dhurrinase (Sorghum b-glucosidase). J Biol Chem 275,
20002–20011.
10 Marana SR, Terra WR & Ferreira C (2002) The role of
amino acid residues Q39 and E451 in the determination
of substrate specificity of the Spodoptera frugiperda

b-glycosidase. Eur J Biochem 269, 3705–3714.
11 Zechel DL, Boraston AB, Gloster T, Braston CM,
Macdonald JM, Tilbrook DMG, Stick RV & Davies
GJ (2003) Iminosugar glycosidase inhibitors: structural
and thermodynamic dissection of the binding of isofag-
omine and 1-deoxynojirimycin to b-glucosidases. JAm
Chem Soc 125, 14313–14323.
12 Gloster TM, Roberts S, Ducros VM-A, Perugino G,
Rossi M, Hoos R, Moracci M, Vasella A & Davies GJ
(2004) Structural studies of the b-glycosidase from
Sulfolobus solfataricus in complex with covalently and
noncovalently bound inhibitors. Biochemistry 43,
6101–6109.
13 Verdoucq L, Morinie
`
re J, Beavan DR, Esen A, Vasella
A, Henrissat B & Czjzek M (2004) Structural determi-
nants of substrate specificity in family 1 b-glucosidases.
J Biol Chem 277 , 31796–31803.
14 Marana SR, Andrade EHP, Ferreira C & Terra WR
(2004) Investigation of the substrate specificity of a
b-glycosidase from Spodoptera frugiperda using site-
directed mutagenesis and bioenergetic analysis. Eur J
Biochem 271, 4169–4177.
15 Marana SR (2006) Molecular basis of substrate specific-
ity in family 1 glycoside hydrolases. IUMBM Life 58,
63–73.
16 Czjzek M, Cicek M, Zamboni V, Bevan DR, Henrissat
B & Esen A (2000) The mechanism of substrate (agly-
cone) specificity in b-glucosidases is revealed by crystal

structures of mutant maize b-glucosidase-DIMBOA, -
DIMBOA-Glc and–dhurrin complexes. Proc Natl Acad
Sci USA 97, 13555–13560.
17 Isorna P, Polaina J, Latorre-Garcı
´
a L, Can
˜
ada FJ,
Gonza
´
lez B & Sanz-Aparı
´
cio J (2007) Crystal structure
of Paenibacillus polymyxa b-glucosidase B complexes
reveal the molecular basis of substrate specificity and
give new insight into the catalytic machinery of family 1
glycosidases. J Mol Biol 371, 1204–1218.
18 Czjzek M, Cicek M, Zamboni V, Burmeister WP,
Bevan DR, Henrissat B & Esen A (2001) Crystal struc-
ture of a monocotyledon (maize ZmGlu1) b-glycosidase
and a model of its complex with p-nitrophenyl b-D-thi-
oglucoside. Biochem J 354, 37–56.
19 Verdoucq L, Czjzek M, Moriniere J, Bevan DR & Esen
A (2003) Mutational and structural analysis of aglycone
specificity in maize and sorghum b-glucosidases. J Biol
Chem 278, 25055–25062.
20 Marana SR, Jacobs-Lorena M, Terra WR & Ferreira C
(2001) Amino acid residues involved in substrate bind-
ing and catalysis in an insect digestive b-glycosidase.
Biochim Biophys Acta 1545, 41–52.

21 Ferreira AHP, Marana SR, Terra WR & Ferreira C
(2001) Purification, molecular cloning, and properties of
a b-glycosidase isolated from midgut lumen of Tenebrio
molitor (Coleoptera) larvae. Insect Biochem Mol Biol 31,
1065–1076.
22 Tribolo S, Berrin J-G, Kroon PA, Czjzek M & Juge N
(2007) The crystal structure of human cytosolic b-gluco-
sidase unravels the substrate aglycone specificity of a
family 1 glycoside hydrolase. J Mol Biol 370, 964–975.
23 Gopalan V, Daniels LB, Glew RH & Claeyssensi M
(1989) Kinetic analysis of the interaction of alkylglyco-
sides with two human b-glycosidases. Biochem J 262,
541–548.
24 Tabor S & Richardson CC (1985) A bacteriophage T7
RNA polymerase ⁄ promoter system for controlled exclu-
sive expression of specific genes. Proc Natl Acad Sci
USA 82, 1074–1078.
25 Laemmli UK (1970) Cleveage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
26 Blum H, Beir H & Cross H (1987) Improved silver
staining of plant proteins, RNA and DNA in polyacri-
lamide gels. Electrophoresis 8, 93–99.
27 Gill SC & von Hippel PH (1989) Calculation of pro-
teins extinction coefficients from amino acid sequence
data. Anal Biochem 182, 319–326.
28 Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wil-
kins MR, Appel RD & Bairoch A (2005) Protein identi-
fication and analysis tools on the ExPASy server. In
The Proteomics Protocols Handbook (Walker JM, ed),

pp. 571–607. Humana Press, Totowa, NJ.
29 Segel HI (1993) Enzyme Kinetics. Behaviour and Analy-
sis of Rapid Equilibrium and Steady-State Enzyme
Systems. John Wiley and Sons, New York, NY.
Molecular basis for specificity of a b-glycosidase L. M. F. Mendonc¸a and S. R. Marana
2546 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS
30 Wilkinson AJ, Fersht AR, Blow DM & Winter G (1983)
Site-directed mutagenesis as a probe of enzyme structure
and catalysis: tyrosyl-tRNA synthetase cysteine-35 to
glycine-35 mutation. Biochemistry 22, 3581–3586.
31 Fersht A (1999) Structure and Mechanism in Protein
Science: A Guide to Enzyme Catalysis and Protein Fold-
ing. W.H. Freeman, New York, NY.
32 Withers SG & Rupitz K (1990) Measurement of active-
site homology between potato and rabbit muscle a-glu-
can phosphorylase through use of a linear free energy
relationship. Biochemistry 29, 6405–6409.
33 Husebye H, Arzt S, Burmeister WP, Hartel FV, Brandt
A, Rossiter JT & Bones AM (2005) Crystal structure at
1.1 Angstroms resolution of an insect myrosinase from
Brevicoryne brassicae shows its close relationship to beta-
glucosidases. Insect Biochem Mol Biol 35, 1311–1320.
34 Guex N & Peitsch MC (1997) Swiss-Model and Swiss-
PDBViewer: An environment for comparative protein
modeling. Electrophoresis 18, 214–272.
35 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F
& Higgins DG (1997) The Clustal_X windows interface:
flexible strategies for multiple sequence alignment aided
by quality analysis tools. Nucleic Acids Res 25, 4876–
4882.

Supplementary material
The following supplementary material is available
online:
Fig. S1. SDS-PAGE of the purified mutant Sfbgly.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase
FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2547