Biochemical identification of the catalytic residues of a glycoside hydrolase family 120 β-xylosidase, involved in xylooligosaccharide metabolisation by gut bacteria

Article abstract of DOI:10.1016/j.febslet.2015.08.012

The β-xylosidase B from Bifidobacterium adolescentis ATCC15703 belongs to the newly characterized family 120 of glycoside hydrolases. In order to investigate its catalytic mechanism, an extensive kinetic study of the wild-type enzyme and mutants targeting the three highly conserved residues Asp³⁹³, Glu⁴¹⁶ and Glu³⁶⁴ was performed. NMR analysis of the xyloside hydrolysis products, the change of the reaction rate-limiting step for the Glu⁴¹⁶ mutants, the pH dependency of E416A activity and its chemical rescue allowed to demonstrate that this GH120 enzyme uses a retaining mechanism of glycoside hydrolysis, Glu⁴¹⁶ playing the role of acid/base catalyst and Asp³⁹³ that of nucleophile.

Full text of DOI:10.1016/j.febslet.2015.08.012

FEBS Letters 589 (2015) 3098–3106
journal homepage: www.FEBSLetters.org
Biochemical identification of the catalytic residues of a glycoside
hydrolase family 120 b-xylosidase, involved in xylooligosaccharide
metabolisation by gut bacteria
⇑
Davide A. Cecchini, Régis Fauré, Elisabeth Laville, Gabrielle Potocki-Veronese
Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France
INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France
CNRS, UMR5504, F-31400 Toulouse, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The b-xylosidase B from Bifidobacterium adolescentis ATCC15703 belongs to the newly characterized
family 120 of glycoside hydrolases. In order to investigate its catalytic mechanism, an extensive
kinetic study of the wild-type enzyme and mutants targeting the three highly conserved residues
Received 11 May 2015
Revised 4 August 2015
Accepted 6 August 2015
Available online 20 August 2015
393
416
364
Asp , Glu
change of the reaction rate-limiting step for the Glu
activity and its chemical rescue allowed to demonstrate that this GH120 enzyme uses a retaining
and Glu
was performed. NMR analysis of the xyloside hydrolysis products, the
4
16
mutants, the pH dependency of E416A
Edited by Miguel De la Rosa
4
16
393
mechanism of glycoside hydrolysis, Glu
nucleophile.
playing the role of acid/base catalyst and Asp
that of
Keywords:
b-Xylosidase
Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Glycoside hydrolase family 120
Site-directed mutagenesis
Catalytic residue
Retaining mechanism
Bifidobacterium adolescentis
1
. Introduction
In the last few years an increasing number of studies high-
of the composition and/or activity of the intestinal microbiota by
the administration of functional foods arouse an increasing interest
to positively impact human health and digestive comfort. Func-
tional foods are either probiotics (healthy bacteria), prebiotics
(oligo- or polysaccharides compounds not digested by human
enzymes), or symbiotics (mixtures of probiotics and prebiotics),
which are able to specifically stimulate beneficial microorganisms,
like bifidobacteria [5]. Together with fructooligosaccharides and
transgalactooligosaccharides, xylooligosaccharides (XOS) are ones
lighted the deep interactions that exist between the human body,
its gut microbiota, and dietary fibers. Only to cite few examples,
the gut microbiota has been shown to be involved in the stimula-
tion of the immune system, in providing resistance to pathogens
and to be associated with human diseases such obesity [1], in
type-2 diabetes [2,3] or atherosclerosis [4]. Thus, the modulation
of the most studied prebiotics. Made up by
D-xylopyranosyl
(
D
-Xylp) residues linked by b-(1,4) glycosidic linkages, and
Abbreviations: 3,4-dNP, 3,4-dinitrophenol; 3,4-dNP-b-
-xylopyranoside; GH, glycoside hydrolase; LG, leaving group; 4-MU, 4-
methylumbelliferone; 4-MU-b- -Xylp, 4-methylumbelliferylb- -xylopyranoside;
-NP, 2-nitrophenol; 2-NP-b- -Xylp, 2-nitrophenyl b- -xylopyranoside; 4-NP, 4-
nitrophenol; 4-NP-b- -Xylp, 4-nitrophenyl b- -xylopyranoside; N -b- -Xylp, b-
xylosyl azide; XOS, xylooligosaccharides; -Xylp, -xylopyranosyl; BaXylB,
D-Xylp, 3,4-dinitrophenyl-
b-
D
obtained from the degradation of xylan, an hemicellulose
compound of fruits and vegetables, XOS consumption has been
shown to stimulate bifidobacteria growth and to decrease cecal
and fecal pH [6,7]. XOS also have immunomodulatory activity
D
D
2
D
D
D
D
3
D
D-
D
D
[
8,9], inhibitory effect on precancerous colon lesions [6] and
b-xylosidase B from Bifidobacterium adolescentis ATCC15703; TS, transition state;
TsXylC, b-xylosidase C from Thermoanaerobacterium saccharolyticum JW/SL-YS485
inhibitory effects on the growth of sulphite-reducing clostridia
[10], whose metabolic end product, hydrogen sulfide, is a cytotoxic
compound that could be implicated in inflammatory bowel
diseases [11–14]. However little is known about the species and
enzymes involved in their degradation. One of the major reason
for this is the difficulty in isolating XOS degrading bacteria species,
Author contributions: DAC and GPV conceived and supervised the study; DAC, RF and
GPV designed experiments; DAC and RF performed experiments; DAC, RF, EL and
GPV analyzed data; DAC, RF and GPV wrote the manuscript.
⇑
Corresponding author at: Université de Toulouse, INSA, UPS, INP, LISBP, 135
Avenue de Rangueil, F-31077 Toulouse, France. Fax: +33 5 61 55 94 00.
E-mail address: veronese@insa-toulouse.fr (G. Potocki-Veronese).
http://dx.doi.org/10.1016/j.febslet.2015.08.012
0
014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

D.A. Cecchini et al. / FEBS Letters 589 (2015) 3098–3106
3099
mainly because they live in a very complex ecosystem composed of
0% uncultivated bacteria, and because they establish complex
2.2. Cloning of BaXylB encoding gene
7
trophic interactions with other microorganisms, that are difficult
to reproduce in vitro. To address this issue, and circumvent the dif-
ficulty of bacterial cultivation, activity based functional metage-
nomics was previously used to target any possible Carbohydrate
Active enZymes (CAZymes) [15] involved in the degradation of
all commercialized prebiotics, including XOS, by ileal and colonic
bacteria [16]. Among them was identified an enzyme classified in
the glycoside hydrolase (GH) family GH120 of the CAZy classifica-
tion (www.cazy.org) [15], taxonomically assigned to an unculti-
vated Bifidobacterium adolescentis species. This GH120 sequence
was retrieved in the metagenome of numerous European and
American subjects, indicating that this enzyme and its nearest
homologs probably play a key role in the metabolization of XOS,
derived either from prebiotics or from the hemicellulosic part of
dietary fibers. Even truncated from the N-terminal residues in
the metagenomic DNA insert, this sequence presents 98% protein
sequence identity with that of BaXylB, a b-xylosidase B (EC
The coding sequence of BaXylB was amplified from the
genomic DNA of B. adolescentis ATCC15703D-5 using the following
0
0
primers: forward 5 -AAGTTTGAATACCATGTCAAACCAACCGGC-3
0
0
and reverse 5 -TCAGTTGTTCCATTCCCAAATTCGGATGTG-3 . Using
the Gateway technology (Invitrogen), and following the manufac-
turer instruction, the amplified gene was first cloned into the
pCR8/GW/TOPO entry vector and then transferred into the T7 poly-
merase expression vector pDEST17, downstream of a (His)
sequence.
6
2.3. Mutagenesis
Site directed mutagenesis was performed from the pDEST17-
BaXylB construct using the Phusion High-Fidelity DNA Polymerase
New England BioLabs) and mutagenic primers designed to include
(
the mutation and, when possible, a restriction site to allow easy
identification of the mutation. The mutagenic primers were as fol-
lows (the mutated nucleotide are shown in bold within the under-
lined NNN mutagenic codons and the italic restriction sites):
3
.2.1.37) from B. adolescentis ATCC15703, which is highly specific
for short XOS (i.e. polymerisation degree range 3–6) [17]. GH120
BaXylB associated with GH43 BaXylC and GH8 BaRexA are part of
the B. adolescentis enzymatic toolbox with different but comple-
mentary substrate specificities to completely hydrolyse XOS
0
0
E364X, 5 -ATGGNNNGTCGCTGGCATCAAATT C-3 (forward, with
0
Gly, GGG; Ala, GCG; Asp, GAT; Gln, CAG) and 5 -AGCGACNNNCCA
0
[
17,18]. The bacterium Thermoanaerobacterium saccharolyticum
TCCGAAGAATTC-3 (reverse, with Gly, CCC; Ala, CGC; Asp, ATC;
JW/SL-YS485 also exhibits such as arsenal including three
b-xylosidases, non-classified TsXylA, GH30 TsXylB and GH120
TsXylC. Today, the novel GH120 family gathers 67 sequences, all
from bacteria. BaXylB and TsXylC are the sole GH120 enzymes that
have been functionally characterized [17,19]. In addition, the
crystallographic structure of TsXylC, solved in 2012, constitutes
the sole structural data for GH120 enzymes [20]. TsXylC comprises
two domains: the core domain that folds into a right-handed
parallel b-helix and a small flanking region that folds into a
b-sandwich domain. Both are involved in the active site formation
and provide interactions for substrate binding. From the inspection
0
Gln, CTG); D393X (AvrII), 5 -TGCTCCCTAGGCATGTGGATGNNNTG
0
GCAG-3 (forward, with Gly, GGT; Ala, GCT; Asn, AAT; Gln, GAA)
0
0
and 5 -CTGCCANNNCATCCACATGCCTAGGGAGCA-3 (reverse, with
Gly, ACC; Ala, AGC; Asn, ATT; Gln, TTC); E416X (NcoI), 5 -ATGATT
0
0
NNNGTGAGCCATGGGCC-3 (forward, with Gly, GGC; Ala, GCA;
0
0
Asp, GAT; Gln, CAA) and 5 -GGCCCATGGCTCACNNNAATCAT-3
(reverse, with Gly, GCC; Ala, TGC; Asp, ATC; Gln, TTG). After muta-
genesis, samples were treated with DpnI to digest the parental
plasmid and then used to transform Escherichia coli TOP10 cells.
Single colonies were selected and grown overnight in LB
of TsXylC structures in complex with xylobiose and
carboxylic residues were identified in the active site. Since they are
located near the anomeric C-1 carbon of the non-reducing
D
-xylose, three
supplemented with ampicillin (100 lg/mL), and the plasmids were
purified via mini-preparation technique (Qiagen Plasmid Mini kit).
The mutated genes were finally sequenced to confirm that only the
desired mutations were introduced.
D
-xylosyl unit or close to the glycosidic oxygen, and because their
mutation into alanine abolished the activity in preliminary assays
using 4-nitrophenyl b- -xylopyranoside (4-NP-b- -Xylp) as
were suggested as candidates for
nucleophile and general acid/base catalytic residues respectively,
D
D
2
.4. Overexpression and purification of the N-terminal (His)
6
-tagged
3
82
405
substrate, Asp
and Glu
BaXylB wild-type and mutated enzymes
3
53
while Glu
would be involved in substrate binding. However,
For the production and purification of the N-terminal
His) -tagged wild-type as well as mutated enzymes, the following
no further evidences were presented in this pioneer work to
(
6
support this assumption. TsXylC Asp3 and Glu
82
405
thus appear as
procedure was used. A preculture of chemical competent BL21-AI
cells transformed with the correct pDEST17-XylB construct was
grown overnight in LB supplemented with ampicillin (100 lg/
mL) at 37 °C and 140 rpm (orbital diameter: 25 mm). The grown
preculture was then diluted to an OD600 of 0.01 into 200 mL of
inferred catalytic residues in the CAZY database.
In the present work, the equivalent essential catalytic residues
of BaXylB were identified and their role in catalysis was confirmed
by site-directed mutagenesis, detailed kinetic analysis, pH
dependency profiles and chemical rescue. This biochemical study,
which complements TsXylC crystallographic analysis, allowed to
unequivocally identify the two catalytic residues of this retaining
b-xylosidase belonging to family GH120.
ZYM5052 supplemented with ampicillin, 1 mM CaCl
2
and 0.02%
(
w/v) -arabinose as inductor, and grown at 16 °C and 180 rpm.
L
After 20 h induced cells were harvested, resuspended into binding
buffer (20 mM Tris–HCl, pH 7.4, containing 150 mM NaCl and
2
0 mM imidazole) and disrupted by sonication. The cells extract
2
. Materials and methods
was then centrifuged to remove cells debris at 10000ꢀg for
2
0 min at 4 °C.
2.1. Substrates
The enzyme was purified by loading the soluble cell extract into
a gravity-flow chromatography column packed with 4 mL bed vol-
ume of TALON Cobalt affinity resin. Non-specific bound proteins
were removed by washing the resin with 4 bed volumes of binding
buffer. The bound enzyme was eluted in 10 mL of elution buffer
(20 mM Tris–HCl, pH 7.4, containing 150 mM NaCl and 150 mM
imidazole). The eluted protein was finally dialyzed against
The substrates 4-NP-b-
D
-Xylp, 2-nitrophenyl b-
D
-xylopyranoside
-xylopyranoside
(
(
b-
2-NP-b-
4-MU-b-
-xylopyranoside (3,4-dNP-b-
as described by Ziser et al. [21].
D-Xylp) and 4-methylumbelliferyl b-
D
D-Xylp) were purchased from Sigma. 3,4-dinitrophenyl
D
D-Xylp) was synthesized in-house

3
100
D.A. Cecchini et al. / FEBS Letters 589 (2015) 3098–3106
2
0 mM sodium phosphate buffer pH 6.0 at 4 °C. Enzyme concentra-
wavelength monitored for each of the substrate used were:
ꢁ1
ꢁ1
tion was determined by measuring the absorbance at 280 nm using
a Nanodrop instrument (Wilmington, DE), and using a theoretical
3,4-dinitrophenol (3,4-dNP),
D
e
= 1.30 mM cm
,
k = 405 nm;
ꢁ
1
ꢁ1
4-nitrophenol (4-NP),
nitrophenol (2-NP),
D
e
= 1.60 mM cm
,
k = 405 nm; 2-
k = 405 nm; 4-
ꢁ1
ꢁ1
ꢁ1
ꢁ1
ꢁ
extinction coefficient of 141670 M cm
.5. Determination of hydrolysis stereochemistry by H NMR
Enzyme-mediated hydrolysis of 4-NP-b- -Xylp was monitored
.
De
= 0.31 mM cm
,
1
ꢁ1
methylumbelliferone (4-MU),
D
e
= 1.68 mM cm , k = 360 nm.
1
2
The quantity of released leaving group (LG) was calculated using
appropriate standard curves and negative controls containing all of
the reactants except the enzyme, in order to remove background
noise issued from spontaneous hydrolysis of the substrates. The val-
D
1
by collecting H NMR using a Bruker Avance II 500.13 MHz
spectrometer equipped with a 5 mm-BBI probe and TopSpin 3.0
software, performing reactions in standard 5 mm NMR tube
containing 500
use, 4-NP-b- -Xylp was freeze-dried from D
wild-type enzyme was diluted by 10-fold in D
concentration using an Amicon Ultra filter (regenerated cellulose,
cutoff 10kDa, Millipore) system; these operations being performed
twice. Next, hydrolysis was initiated by the addition of an aliquot
of the deuterated enzyme solution (0.05 IU) and reactions were
performed at 30 °C. Upon enzyme addition and mixing, the NMR
tube was immediately transferred to the spectrometer, and after
temperature stabilization, spectra were recorded continuously
over 5.52 min during 2 h, thus providing the first spectrum
ues of kcat and K
using the program SigmaPlot 11.0. In cases where the K
toohightobeestimated,kcat/K valueswerecalculatedfromthereac-
tion rates at low substrate concentration. Indeed, when [S] ꢂ K the
reaction rates are given by the equation v = kcat[E] [S]/K and thus
can be calculated by dividing the slope of the plot of v = f
m
were determined by non-linear regression analysis
m
values were
l
L (final volume) of 5 mM substrate in D
O (99.90%) and the
O, followed by
2
O. Prior
m
D
2
m
2
0
m
kcat/K
m
([S]) by the enzyme concentration.
m
2.7. pH dependency of kcat and kcat/K values
The study of pH dependency of kcat and kcat/K
ried out following the same method used for the kinetic studies,
using 4-NP-b- -Xylp as substrate and the following buffers:
100 mM sodium citrate buffer (pH 4.0–6.0) and 100 mM sodium
phosphate buffer (pH 6.0–8.0). The pK values assigned to the ion-
m
values was car-
D
7
.25 min after enzyme addition. Each NMR spectrum was acquired
using an excitation flip angle of 90° at a radiofrequency field of
9.4 kHz, and the residual water signal was pre-saturated (65 dB
a
3
isable groups were determined using the program SigmaPlot 11.0.
attenuation) during the relaxation delay at a radiofrequency field
of 19 Hz. The following acquisition parameters were used: 6 s for
the relaxation delay, 32 scans and 4 dummy scans. Before enzyme
2.8. Chemical rescue of the E416A mutant activity
1
addition, a H NMR spectrum of the reaction mixture was acquired,
The reaction was performed with 10 mM of 2-NP-b-D-Xylp and
serving as the starting point of the reaction. Coupling constants (J)
are reported in Hz, chemical shifts (d) are given in ppm with mul-
tiplicity reported as follows: d = doublet. The reference used was
the original residual water peak, calibrated at d = 4.71 ppm at
1 M of sodium azide in 100 mM sodium citrate pH 5.0, supple-
mented with 0.1% BSA (w/v), in presence of 0.24 mg/mL of purified
E416A mutant. The reaction medium was then incubated at 30 °C
during 2 h. Reaction was stopped by heating the reaction medium
at 95 °C for 5 min. The denaturated proteins were removed by
filtration with a Centiprep concentrator (cutoff 10kDa, Amicon),
centrifuged at 10000ꢀg and 4 °C. The reaction medium devoided
3
0 °C [22].
2.6. Kinetic studies
of enzymes was then analyzed by LC–ELSD, LC–MS and by 1
NMR after lyophilisation.
H
All kinetic studies were carried out by discontinuous assays in
glass tubes at 30 °C as follows. Different substrate concentrations
were solubilised in 450 L of 100 mM sodium citrate buffer, pH 5.0,
supplemented with 0.1% BSA (w/v). Initial hydrolysis rates were
determined by adding 50 L of properly diluted enzyme to the reac-
tion mix. Enzyme concentrations used for kinetic measurements are
mentioned in Table 1. At time intervals, 50 L of samples were taken
and thereaction stopped bythe addition of250 L of 1 M Na CO . The
released chromogenic groups were determined in micro-plates by
reading 200 L of the stopped reaction mix using a Sunrise
l
2.9. Characterization of the chemical rescue reaction product
l
LC–ELSD analysis was performed with a Dionex UltiMate 3000
LC System (Thermo scientific) connected to a Varian 380-LC
Evaporating Light Scattering Detector (Agilent Technologies). Com-
l
l
2
3
pounds were separated with a Hypercarb (100 ꢀ 4.6) 5
lm column
(Thermo Scientific) at 10 °C, using a flow rate of 1 mL/min. Elution
was done with water as eluent A and acetonitrile as eluent B, using
the following gradient program: 0–10 min, 100:0 (A:B, v/v);
l
spectrophotometer (Tecan). The extinction coefficient used and
1
2
0–20 min, from 100:0 to 50:50; 20 to 30 min, from 50:50 to
0:80; 30 to 35 min, 20:80; from 35 to 40 min, reequilibration
Table 1
Kinetic parameters of 4-NP-b-D-Xylp hydrolysis by wild-type BaXylB and its Glu364,
at 100:0. Conversions of 2-NP-b-
b- -xylosyl azide (N -b- -Xylp) were monitored with the ELSD
D-Xylp into D-xylose and/or
393
416
D
D
Asp
and Glu
mutants.
3
detector set-up with the following parameters: evaporation
70 °C, nebulisation 40 °C and nitrogen flow 1 slm. Retention times
Enzyme Enzyme
k
cat (sꢁ1)
K
m
(mM)
kcat/K
m
(s mM
ꢁ
1
ꢁ1
concentration (
l
M)
)
for b-
.1, 14.3 and 23.3 min, respectively. LC–MS analysis was per-
formed with a Dionex UltiMate 3000 LC System (Thermo scientific)
but connected to MSQ Plus mass spectrometer (Thermo
3
D-Xylp, a-D-Xylp, N -b-D-Xylp and 2-NP-b-D-Xylp were 2.4,
Wild-
type
0.01
130.2 ± 8.9
8.4 ± 0.1
1.6 ± 1.0
15.50
3
D393A
D393E
E416G
E416A
E416Q
E416D
E364G
E364A
E364Q
E364D
3.50
1.14
1.78
6.01
0.39
0.04
5.81
6.25
6.51
0.01
<0.001
<0.001
6.85 ± 1.20
0.52 ± 0.02
0.15 ± 0.01
6.81 ± 0.21
4.20 ± 0.04
NS
NS
NS
146.2 ± 13.0 0.05
a
2.4 ± 0.1
2.8 ± 0.1
4.8 ± 0.2
1.5 ± 0.1
NS
0.22
0.05
1.42
2.80
2.37
0.41
0.01
4.96
scientific). The compounds were separated with the same column
and method as described above and analyzed with the MS instru-
ꢁ
ment operated in an electrospray negative ionization mode (ESI )
a
at 30 V and 500 °C, and scanned from 100 to 1500 ms.
1
NS
NS
H NMR spectra of the reaction mixture and standards were
3
recorded at 298 K in CD OD, calibrated to the residual solvent sig-
nal [22]. Mass spectrometry analyses of the crude reaction mixture
were also performed in positive and negative ionization mode (ESI)
407.6 ± 22.4 82.2 ± 7.8
a
NS: no saturation.

D.A. Cecchini et al. / FEBS Letters 589 (2015) 3098–3106
3101
with
a
Bruker Daltonics Esquire 3000 + mass spectrometer
carboxylic residue, the acid/base catalyst, acts as a general acid,
protonating the interglycosidic oxygen, and thereby assisting and
stabilizing the LG departure. In the second step instead, called
the deglycosylation step, the acid/base catalyst acts as a general
base, activating a water molecule which in turn attacks the
glycosyl-enzyme intermediate at its anomeric center, to release
the newly formed reducing end with retention of the anomeric
configuration.
(
PCN-ICMG, Grenoble) and revealed mass of 198 and 217 corre-
+
ꢁ
D
sponding to [M+Na] and [M+N
respectively.
3
]
-xylopyranosyl azide adducts,
3
. Results and discussion
3.1. Stereochemical outcome of the BaXylB-catalyzed hydrolysis and
The stereochemical course of the BaXylB-catalyzed hydrolytic
its mode of action
1
reaction was monitored by H NMR spectroscopy (Fig. 1). Prior to
the addition of the enzyme, the spectrum of the anomeric proton
region (4.5–5.3 ppm) displayed a doublet centered at 5.23 ppm
Members of the GH120 family have been proposed to be retain-
ing glycosidases, which achieve glycosidic bond hydrolysis via the
canonical two-step double-displacement mechanism, through the
involvement of two carboxylic amino acids [19]. As illustrated in
Scheme 1, during the first step of this general mechanism, called
the glycosylation step, one of these two carboxylic residues, the
nucleophile, attacks the substrate anomeric carbon forming a
covalent glycosyl-enzyme intermediate. Concomitantly, the second
(
4
7.4 Hz), corresponding to the signal of the axial H-1 protons of
-NP-b- -Xylp, but no signal corresponding to -xylose was detect-
D
D
able. Immediately (i.e. within the first 15 min after the addition of
the enzyme), a new doublet was observed at 4.57 ppm with a split-
ting of 7.9 Hz, corresponding to the axial H-1 of the reducing
b-D-Xylp. The equatorial H-1 of a-D-Xylp counterpart (5.18 ppm,
d, 3.7 Hz) appeared later as a result of mutarotation. This spectro-
scopic analysis unequivocally demonstrates that GH120 BaXylB
catalyzes hydrolysis with retention of the anomeric configuration
and proceeds via a two-steps displacement mechanism (Scheme 1),
being consistent with that proposed for enzymes of the GH120
family [19].
3.2. Identification of the key acidic residues by site directed
mutagenesis
From the protein sequence alignment of BaXylB with TsXylC,
which shares 48% of sequence identity [17], and the inspection of
TsXylC protein structure co-crystallized with -xylose (PDB
D
number: 3VSV) and xylobiose (PDB number: 3VSU) it was possible
to list all the equivalent BaXylB residues forming the active site.
Scheme 1. Two-step displacement mechanism of retaining GHs (BaXylB number-
ing), showing the glycosylation and deglycosylation steps, and transition states. The
leaving group (LG) can be either a chromogenic compound (e.g. 4-NP), glycoside (e.
g. XOS) or carbohydrate monomer. The kinetic constants correspond to the
2
Fig. 1. Time course of 4-NP-b-D O
-Xylp hydrolysis by BaXylB. 1H NMR spectra, in D
at 303 K, of the anomeric region of the substrate (i.e. before addition of enzyme),
after 13, 24, and 62 min of incubation with the enzyme, and hydrolysis products (as
a result of mutarotation).
following
= k
equations:
/(kꢁ1 + k ).
k
cat = k
2
k /(k
3 2
3
+ k ),
K
m
= (kꢁ1 + k )k /(k
2 3 2
+ k
3
)k
1
and
kcat/K
m
1
k
2
2

3
102
D.A. Cecchini et al. / FEBS Letters 589 (2015) 3098–3106
Among them only three residues bear a carboxyl group, Glu364,
increase when mutated, Glu364 was thought to be involved in sub-
strate binding, as proposed for TsXylC [20].
3
93
and Glu , which correspond to the Glu353, Asp
416
382
Asp
Glu
and
4
05
residues (Fig. 2) that were identified in the active site of
4
16
TsXylC [20].
In order to identify which among these three putative catalytic
3.3. Kinetic analysis of wild-type and Glu
bearing different leaving groups
mutants with substrates
residues plays the role either of the nucleophile or the acid/base
catalyst, an extensive study was performed, by mutating these
three residues into glycine, alanine, asparagine or glutamine and
thus by determining the kinetic parameters of these mutants using
As in retaining glycosidases the acid/base catalyst has a double
role in catalysis, its substitution with non-acidic amino-acids
should affects the rates of both reaction steps, even though the
effect on each step will be different. The analysis of the kinetic
properties of such mutants with substrate bearing different LGs
is a common strategy for studying the mechanistic action of gly-
cosidases. Indeed, in the first step, the effect of the mutation
strongly depends on the leaving ability of the aglycone group.
Thus, the glycosylation rates of substrates with poor LGs
4
-NP-b-D
-Xylp as substrate (Table 1). Asp393 mutation displays the
most dramatic effect on BaXylB activity, since kcat value for D393A
5
was severely reduced of about 10 -fold compared to the wild-type
enzyme. As expected, activity was partially restored by replacing
3
93
Asp
m
by glutamate, while the K value increased by 17-fold. As
already reported for retaining GHs, the mutation of the nucleophile
catalyst usually has the greater impact on enzyme activity, some-
times completely abolishing it [23,24]. The magnitude of activity
decrease obtained by Asp3 mutation, which is comparable to that
a
(pK > 8), which need protonic assistance by the catalytic acid/base
residue, are more strongly affected compared to those obtained
with substrates bearing good LGs. In the deglycosylation step
instead, as the covalent glycosyl-enzyme intermediate is the same,
the effect on the hydrolytic rates might be unaffected for all sub-
strates. If the glycosylation step is rate-limiting, a correlation
between pK of the aglycone group and kcat should be observed.
a
If instead the deglycosylation step is rate limiting, kcat will be
invariant with substrate reactivity [26].
93
3
93
observed for other retaining GHs [23,25], indicates that Asp
416
would play the role of nucleophile. Mutations of Glu
also affect
activity, but to a less extent. For Glu4 mutants, kcat were 19 to
68-fold lower than that of the wild-type, while K values were
not significantly affected by these mutations. Additionally, muta-
16
8
m
3
64
tion of Glu
also affected enzyme activity, but in a different
way, since it was difficult to obtain reliable kinetic constants.
Indeed, at the highest substrate concentration used (70 mM), the
activities determined were still at the initial increasing part of
In order to gain more information on the mechanistic role
4
16
played by the Glu
residue, kinetic constants of the E416A,
E416G, E416Q and E416D mutants were determined using sub-
strates bearing different LGs and compared to those obtained for
the wild-type. Kinetic constants (Table 2), as well as Brønsted plots
the Michaelis–Menten curve, which suggest very high K
for these mutants. However, for mutant E364Q, it was possible to
determine the kcat and K values, which are 4- and 25-fold higher
than the wild-type ones, respectively. Because of this great K
m
values
m
(Fig. 3) show that the wild-type kcat and K
all the tested substrates (with pK < 8, i.e. bearing good LG) indicat-
ing that here deglycosylation is the rate-limiting step. On the con-
m
values are similar for
m
a
4
16
trary, kcat values measured for the Glu
with all the tested substrates. Moreover, kcat values decreased with
pK increase, suggesting that (i) with a substrate bearing a good LG
like 3,4-dNP-b- -Xylp (pK = 5.36), the deglycosylation step still is
mutants were reduced
a
D
a
the rate-limiting step, as for the wild-type, and (ii) with substrates
bearing ‘poorer’ but still good LGs, the glycosylation step becomes
at least partially rate-limiting. This is exactly one of the effects
expected for an enzyme incapable of proton assistance because
devoided of the general acid/base catalytic residue. Moreover, the
Table 2
Kinetic parameters of aryl b-D-xyloside hydrolysis by wild-type BaXylB and its Glu4
16
mutants.
a
ꢁ1
ꢁ1
ꢁ1
Aglycone
pK
a
Enzyme
kcat (s
)
K
m
(mM)
m
kcat/K (s mM )
3
4
,4-dNP
-NP
5.36
7.18
7.22
7.53
Wild-type
E416G
E416A
E416Q
E416D
114.07
22.23
4.42
16.81
18.45
3.4
1.3
1.8
1.6
1.8
33.55
17.10
2.46
10.50
10.25
Wild-type
E416G
E416A
E416Q
E416D
130.19
0.52
0.15
6.81
4.20
8.4
2.4
2.8
4.8
1.5
15.50
0.22
0.05
1.42
2.80
Fig. 2. Residues surrounding xylobiose in the active site of the GH120 TsXylC (PDB
accession number: 3VSU) [20]. The corresponding amino acids in BaXylB are
mentioned in parentheses. Interatomic distances are labeled in Å. Among the three
carboxylic residues within the active site structure of the Michaelis
2-NP
Wild-type
E416G
E416A
E416Q
E416D
166.59
7.88
3.41
25.00
37.54
3.8
43.84
13.05
8.56
31.29
50.32
0.60
0.40
0.79
0.75
3
53
TsXylC-xylobiose complex, Glu
anomeric carbon and the glycosidic oxygen to be a catalytic residue. Asp
properly positioned (at 3.1 Å) for a nucleophilic attack to the anomeric carbon of the
is too far (more than 5.4 Å) from both the
382
is
4
-MU
Wild-type
E416G
E416A
E416Q
E416D
134.01
0.01
0.003
0.29
1.95
1.6
83.76
0.06
0.008
1.19
distorted non-reducing
D-Xylp unit, displaying a quasi-axial orientation for the
0.10
0.39
0.25
0.20
scissile glycosidic bond. The positioning of Glu405 is somehow ambiguous. Although
well located to serve as the general acid/base catalyst, it appears slightly too far
(
3.6 Å) from the glycosidic oxygen for proton donation [32,33]. This is probably due
9.78
to the incorrect positioning of the reducing
unclear electron density map.
D-Xylp unit, as a consequence of an
a
a
pK values from [29,34].

D.A. Cecchini et al. / FEBS Letters 589 (2015) 3098–3106
3103
Fig. 3. Brønsted plots of aryl b-
and its E416A (red), E416G (green), E416Q (blue) and E416D (orange) mutants. (A)
plot of log(kcat) versus pK of the aglycone group and (B) plot of log(kcat/K ) versus
pK of the aglycone group. The values obtained from 2NP-b- -Xylp (N) have been
excluded from linear fitting.
D-xyloside hydrolysis by wild-type BaXylB (black)
Fig. 4. pH dependency of kcat/K
the wild-type BaXylB (d) and its E416A mutant (s).
m
(A) and kcat (B) for hydrolysis of 4-NP-b-
D
-Xylp by
a
m
a
D
k
cat/K
m
obtained with 2-NP-b-
with 3,4-dNP-b- -Xylp, also due to an higher impact on the K
ues for the former substrate. This suggests that with 2-NP-b-
D
-Xylp was higher than that obtained
val-
-Xylp
D
m
high negative Brønsted coefficient blg of ꢁ1.3 and ꢁ1.2, measured
for the E416G and E416A mutants respectively, indicates high
amounts of negative charges on the glycosidic oxygen in the tran-
sition state (i.e. significant cleavage of the interglycosidic bond in
the TS), consistent with relatively little proton donation to the
forming phenolate anion caused by the impaired proton assistance
of this acid/base mutants.
D
the first step is faster and, as a consequence, would led to the accu-
mulation of a higher amount of glycosyl-enzyme intermediate,
resulting in a lower K
to its pK (7.22), the leaving ability of 2-NP should be lower than
that of 3,4-dNP (pK = 5.36). Thereby, we can speculate that in
m
value. This was surprising, since according
a
a
the orto position of the phenyl ring, the nitro group would either
specifically interact with residues of the catalytic site, modifying
the overall binding mode of the substrate or taking advantage of
hydrophobic packing, towards by stabilizing the TS of the glycosy-
lation step.
m a
By plotting the log(kcat/K ) as function of pK , Brønsted coeffi-
cients of ꢁ1.1 (for E416G and E416A) were obtained for the glyco-
sylation step, values similar to that obtained by plotting the log
(kcat) – which instead reflects both the glycosylation and deglyco-
a
sylations steps – versus pK , thus suggesting that the same event
was mainly monitored.
3.4. pH dependency of the wild-type and E416A catalytic constants
The change of the reaction rate-limiting step for the Glu416
mutants, highlighted by the strong correlation between their kcat
The pH dependency profile of kinetic parameters is another way
to investigate the role of specific residues in catalysis. While the
and substrate pK
a
, strongly supports for Glu4 as the acid/base
16
catalyst.
plot of kcat versus pH reflects the pK
in the enzyme-substrate complex, that of kcat/K
pH reflects the pK values of the free enzyme. Both for the wild-
type BaXylB and its E416A mutant, the values of kcat and kcat/K
were measured in the pH range of 4.0–8.0, using 4-NP-b- -Xylp
a
values of ionisable groups
An exception of this general trend was observed with 2-NP-b-
Xylp, for which kcat values similar to the most reactive substrate
,4-dNP-b- -Xylp were obtained. This suggests that for 2-NP-b-
Xylp the deglycosylation step seems the slowest step. Furthermore,
D
-
m
as a function of
a
3
D
D
-
m
D

3
104
D.A. Cecchini et al. / FEBS Letters 589 (2015) 3098–3106
Fig. 5. Formation of the b-
D
-xylopyranosyl azide product obtained by the E416A BaXylB mutant. (A) Schematic representation for activity rescue by azide. (B) LC–ELSD
analysis of the reaction products obtained after 1 h of incubation from 2-NP-b-
spectra, at 298 K in CD OD, of the reaction mixture obtained from 2-NP-b- -Xylp hydrolysis in the presence of 1 M sodium azide and controls (substrate and hydrolysis
products, i.e. anomeric mixture of -Xylp) thereof.
D
-Xylp hydrolysis in absence and in additional presence of 1 M sodium azide. (C) 1H NMR
3
D
D
as substrate (Fig. 4). The pH dependency profile of the wild-type
values is a bell-shape curve typical of glycosidases. pK val-
ues of 4.3 and 6.5 reflects the ionization state of the two carboxylic
catalytic residues in the active site, acid pK being assigned to the
nucleophile, and basic one to the acid/base catalyst. A pK of 6.5 is
high for a carboxylic residue, but similar values have been
observed for other glycosidases [23,27,28]. The pH dependency
profile of kcat values, in the tested pH range was different. A single
ionization curve was obtained, with only a basic limb, from which
GH1 retaining b-glucosidase from an Agrobacterium sp. [29]. Unfor-
tunately, measurements of kcat at pH values lower than 4.0 were
precluded due to enzyme instability. This hampered us to know
kcat/K
m
a
a
a
if the pK assigned to the nucleophile would have been shifted to
a
a lower value, or if after the formation of the enzyme-substrate
complex, the nucleophile would not be protonated anymore, due
to drastic change in the proton exchange ‘cycle’.
In contrast with what was observed with the wild-type, the pH
dependency of E416A activity was overall drastically reduced espe-
cially at high pH values, either for the free enzyme or for its com-
plexed form, suggesting that the acid/base catalyst has been
removed. For both the free and complexed forms of E416A
a
an apparent pK of 6.5 was calculated, since no saturation of kcat
values was observed at the lowest tested pH. Such differences of
pH dependency profiles between the free enzyme and the
substrate-enzyme complex has also already been observed for a
a
mutants, the acid limb pK is 4.3, suggesting that in this case the

D.A. Cecchini et al. / FEBS Letters 589 (2015) 3098–3106
3105
introduction of the mutation may have created sufficient space
References
between the catalytic site and the surface of the enzyme to permit
proton exchange with the reaction medium.
[
1] Turnbaugh, P.J., Hamady, M., Yatsunenko, T., Cantarel, B.L., Duncan, A., Ley, R.
E., Sogin, M.L., Jones, W.J., Roe, B.A., Affourtit, J.P., Egholm, M., Henrissat, B.,
Heath, A.C., Knight, R. and Gordon, J.I. (2009) A core gut microbiome in obese
and lean twins. Nature 457, 480–484.
2] Qin, J., Li, Y., Cai, Z., Li, S., Zhu, J., Zhang, F., Liang, S., Zhang, W., Guan, Y., Shen,
D., Peng, Y., Zhang, D., Jie, Z., Wu, W., Qin, Y., Xue, W., Li, J., Han, L., Lu, D., Wu,
P., Dai, Y., Sun, X., Li, Z., Tang, A., Zhong, S., Li, X., Chen, W., Xu, R., Wang, M.,
Feng, Q., Gong, M., Yu, J., Zhang, Y., Zhang, M., Hansen, T., Sanchez, G., Raes, J.,
Falony, G., Okuda, S., Almeida, M., LeChatelier, E., Renault, P., Pons, N., Batto, J.
M., Zhang, Z., Chen, H., Yang, R., Zheng, W., Li, S., Yang, H., Wang, J., Ehrlich, S.
3.5. Chemical rescue of the E416A mutant
[
The use of external nucleophilic anions such as azide has been
shown to enhance the rate of enzymes devoided of their catalytic
residues. Indeed, the replacement of the nucleophile or the
acid/base catalyst with smaller residues, such as glycine or alanine,
is generally sufficient to create a cavity in which exogeneous small
nucleophile compounds can penetrate and attack the anomeric
center of the substrate or the covalent glycosyl-enzyme intermedi-
ate, depending on the catalytic residue replaced, to form a glycosyl
azide product. Since the products of these reactions are
stereospecific a- or b-glycosyl azides, this strategy allows not only
identification of the catalytic residues but also to determine if the
mutated residue is the nucleophile or the general acid/base
catalyst (Fig. 5A). Using 2-NP-b-D-Xylp as substrate, a 16-fold
D., Nielsen, R., Pederson, O., Kristiansen, K. and Wang, J. (2012)
A
metagenome-wide association study of gut microbiota in type 2 diabetes.
Nature 490, 55–60.
[
3] Karlsson, F.H., Tremaroli, V., Nookaew, I., Bergström, G., Behre, C.J., Fagerberg,
B., Nielsen, J. and Bäckhed, F. (2013) Gut metagenome in European women
with normal, impaired and diabetic glucose control. Nature 498, 99–103.
[4] Karlsson, F.H., Fåk, F., Nookaew, I., Tremaroli, V., Fagerberg, B., Petranovic, D.,
Bäckhed, F. and Nielsen, J. (2012) Symptomatic atherosclerosis is associated
with an alterated gut metagenome. Nat. Commun. 3, 1245.
[
5] Hidaka, H., Tashiro, Y. and Eida, T. (1991) Proliferation of bifidobacteria by
oligosaccharides and their useful effect on human health. Bifidobact.
Microflora 10, 65–79.
6] Hsu, C.K., Liao, J.W., Chung, Y.C., Hsieh, C.P. and Chan, Y.C. (2004)
Xylooligosaccharides and fructooligosaccharides affect the intestinal
microbiota and precancerous colonic lesion development in rats. J. Nutr.
[
hydrolysis rate increase was obtained for the E416A mutant, in
the presence of 1 M azide. This chemical rescue was not as
significant as previously reported for other glycosidases, for which
134, 1523–1528.
2
3
[7] Campbell, J.M., Fahey, J. and Wolf, B.W. (1997) Selected indigestible
oligosaccharides affect large bowel mass, cecal and fecal short-chain fatty
acids, pH and microflora in rats. J. Nutr. 127, 130–136.
[8] Ebringerova, A., Hromadkova, Z., Alfodi, J. and Hribalova, V. (1998) The
immunologically active xylan from ultrasound-treated corncobs:
extractability, structure and properties. Carbohydr. Polym. 37, 231–239.
rate increase up to 10 –10 -fold were obtained [23,30,31].
Nevertheless, LC–ELSD analysis of the reaction product revealed
the formation of a new single product in the presence of azide,
instead of D-xylose (Fig. 5B). Reaction product characterization by
LC–MS analysis revealed, for this newly-formed sole peak, a mass
[
9] Ebringerova, A., Kardosova, A., Hromadkova, Z., Malovikova, A. and Hribalova,
V. (2002) Immunomodulatory activity of acidic xylans in relation to their
structural and molecular properties. Int. J. Biol. Macromol. 30, 1–6.
ꢁ
of 217 corresponding to [M+N
3
]
D
-xylopyranosyl azide adduct.
-xylopyranosyl
azide was analyzed by H NMR (Fig. 5C). The complete disappearance
of the H-1 signal of 2-NP-b- -Xylp at 5.06 ppm (d, 7.1 Hz) was
Furthermore, the reaction mixture containing the
D
[
[
10] Santos, A., San Mauro, M. and Diaz, D.M. (2006) Prebiotics and their longterm
influence on the microbial populations of the mouse bowel. Food Microbiol.
23, 498–503.
11] Gibson, G.R., Cummings, J.H. and McFarlane, G.T. (1991) Growth and activities
of sulphate-reducing bacteria in gut contents of healthy subjects and patients
with ulcerative colitis. FEMS Microbiol. Ecol. 86, 103–112.
[12] Babidge, W., Millard, S. and Roediger, W. (1998) Sulfides impair short chain
fatty acid beta-oxidation at acyl-CoA dehydrogenase level in colonocytes:
implications for ulcerative colitis. Mol. Cell. Biochem. 181, 117–124.
13] Pitcher, M.C.L. and Cummings, J.H. (1996) Hydrogen sulphide: a bacterial toxin
in ulcerative colitis? Gut 39, 1–4.
1
D
accompanied by parallel changes of the 2-NP aglycone signals into
free 2-NP signals (signals between 6.5 and 7.9 ppm) and the
appearance of one alone new anomeric signal at 4.43 ppm (d,
8
.5 Hz) attributed to the H-1 of the b-
-b- -Xylp) [23].
All together these data, including the kinetic behavior displayed
D-xylopyranosyl azide
(N
3
D
[
4
16
[
14] Loubinoux, J., Bronowicki, J.P., Pereira, I.A.C., Mougenel, J.L. and Le Faou, A.E.
by the Glu
mutants as well as the pH dependency of E416A
(
2002) Sulfate-reducing bacteria in human feces and their association with
activity and its chemical rescue, provide definitive evidence that
inflammatory bowel diseases. FEMS Microbiol. Ecol. 40, 107–112.
this GH120 b-xylosidase uses a retaining mechanism of glycoside
[15] Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P.M. and Henrissat, B.
(2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic
Acids Res. 42, D490–D495.
16] Cecchini, A.D., Laville, E., Laguerre, S., Robe, P., Leclerc, M., Doré, J., Henrissat,
B., Remaud-Siméon, M., Monsan, P. and Potocki-Véronèse, G. (2013)
Functional metagenomic reveals novel pathways of prebiotic break down by
human gut bacteria. PLoS One 8, e72766.
4
16
hydrolysis, with Glu
(BaXylB numbering) playing the role of
393
the acid/base catalyst and Asp that of the nucleophile, assisted
to stabilize the enzyme-substrate covalent intermediate.
[
[
[
3
64
by Glu
Acknowledgements
17] Lagaert, S., Pollet, A., Delcour, A.J., Lavigne, R., Courtin, M.C. and Volckaert, G.
(
2011) Characterization of two b-xylosidases from Bifidobacterium adolescentis
This work was funded by the Institut National de la Recherche
Agronomique (INRA). We thank Nelly Monties for technical assis-
tance on LC–ELSD and LC–MS equipments provided by the ICEO
facility dedicated to enzyme screening and discovery, part of the
Integrated Screening Platform of Toulouse (PICT, IBiSA). We cor-
dially thank David Guieysse and Alvaro Lafraya for their helpful
expertise in LC–MS. MetaToul (Metabolomics & Fluxomics Faci-
tilies, Toulouse, France, www.metatoul.fr) and its staff members
and their contribution to the hydrolysis of prebiotic xylooligosaccharides.
Appl. Microbiol. Biotechnol. 92, 1179–1185.
18] Lagaert, S., Van Campenhout, S., Pollet, A., Bourgois, T.M., Delcour, J.A., Courtin,
C.M. and Volckaert, G. (2007) Recombinant expression and characterization of
a
reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium
adolescentis. Appl. Environ. Microbiol. 73, 5374–5377.
[
19] Shao, W., Xue, Y., Wu, A., Kataeva, I., Pei, J., Wu, H. and Wiegel, J. (2011)
Characterization of a novel b-xylosidase, XylC, from Thermoanaerobacterium
saccharolyticum JW/SL-YS485. Appl. Environ. Microbiol. 77, 719–726.
20] Huang, C.H., Sun, Y., Ko, T.P., Chen, C.C., Zheng, Y., Chan, H.C., Pang, X., Wiegel,
J., Shao, W. and Guo, R.T. (2012) The substrate/product binding modes of a
[
(
Lindsay Peyriga, Edern Cahoreau and Jean-Charles Portais) are
novel
GH120
b-xylosidase
(XylC)
from
Thermoanaerobacterium
gratefully acknowledged for the technical support and access to
NMR facility. MetaToul is part of the national infrastructure
MetaboHUB-ANR-11-INBS-0010 (The French National infrastruc-
ture for metabolomics and fluxomics, www.metabohub.fr). Meta-
Toul is supported by grants from the Région Midi-Pyrénées, the
European Regional Development Fund, the SICOVAL, the Infras-
tructures en Biologie Sante et Agronomie (IBiSa, France), the Centre
National de la Recherche Scientifique (CNRS) and INRA. The
authors also acknowledge support from the Mass spectrometry
facility of the Chemistry Nanobio Platform-ICMG (FR 2607),
Grenoble.
saccharolyticum JW/SL-YS485. Biochem. J. 448, 401–407.
[21] Ziser, L., Setyawati, I. and Withers, S.G. (1995) Syntheses and testing of
substrates and mechanism-based inactivators for xylanases. Carbohydr. Res.
274, 137–153.
[
22] Gottlieb, H.E., Kotlyar, V. and Nudelman, A. (1997) NMR chemical shifts of
common laboratory solvents as trace impurities. J. Org. Chem. 62, 7512–7515.
23] Bravman, T., Belakhov, V., Solomon, D., Shoham, G., Henrissat, B., Baasov, T.
and Shoham, Y. (2003) Identification of the catalytic residues in family 52
glycoside hydrolase, a b-xylosidase from Geobacillus stearothermophilus T-6. J.
Biol. Chem. 278, 26742–26749.
[
[
24] MacLeod, A.M., Tull, D., Rupitz, K., Warren, A.J. and Withers, S.G. (1996)
Mechanistic consequences of mutation of active site carboxylates in
retaining b-1,4-glycanase from Cellulomonas fimi. Biochemistry 35, 13165–
13172.
a

3
106
D.A. Cecchini et al. / FEBS Letters 589 (2015) 3098–3106
[
[
[
25] Ly, H.D. and Withers, S.G. (1999) Mutagenesis of glycosidases. Annu. Rev.
[31] MacLeod, A.M., Lindhorst, T., Withers, S.G. and Warren, R.A.J. (1994) The acid/
base catalyst in the exoglucanase/xylanase from Cellulomonas fimi is glutamic
acid 127: evidence from detailed kinetic studies of mutants. Biochemistry 33,
6371–6376.
[32] Heightman, T.D. and Vasella, A.T. (1999) Recent insights into inhibition,
structure, and mechanism of configuration-retaining glycosidases. Angew.
Chem. Int. Ed. 38, 750–770.
Biochem. 68, 487–522.
26] Zechel, L.D. and Withers, S.G. (2000) Glycosidase mechanisms: anatomy of a
finely tuned catalyst. Acc. Chem. Res. 33, 11–18.
27] Shallom, D., Belakhov, V., Solomon, D., Gilead-Gropper, S., Baasov, T., Shoham,
G. and Shoham, Y. (2002) The identification of the acid-base catalyst of
a
-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51
glycoside hydrolase. FEBS Lett. 514, 163–167.
[33] Nerinckx, W., Desmet, T., Piens, K. and Claeyssens, M. (2005) An elaboration on
the syn-anti proton donor concept of glycoside hydrolases: electrostatic
stabilisation of the transition state as a general strategy. FEBS Lett. 579, 302–
312.
[
28] Vocadlo, J.D., Wicki, J., Rupitz, K. and Withers, S.G. (2002) Mechanism of
Thermoanaerobacterium saccharolyticum b-xylosidase: kinetic studies.
Biochemistry 41, 9727–9735.
[
[
29] Kempton, J.B. and Whiters, S.G. (1992) Mechanism of Agrobacterium
b-glucosidase: kinetic studies. Biochemistry 31, 9961–9969.
30] Wang, Q., Trimbur, D., Graham, R., Warren, R.A.J. and Withers, S.G. (1995)
Identification of the acid/base catalyst in Agrobacterium faecalis b-glucosidase
by kinetic analysis of mutants. Biochemistry 34, 14554–14562.
[34] Sinnott, L.M. and Souchard, J.L.I. (1973) Effect of aglycone nature and
a-deuterium substitution on the hydrolysis of aryl galactosides. Biochem.
J. 133, 89–98.