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-

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

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

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

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

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

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
,

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

)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

)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

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

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

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.

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-

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
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

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]:

)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

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.

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

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.

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.