ISSN 16076729, Doklady Biochemistry and Biophysics, 2013, Vol. 449, pp. 99–101. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © A.A. Trofimov, K.M. Polyakov, A.V. Tikhonov, E.Y. Bezsudnova, P.V. Dorovatovskii, V.M. Gumerov, N.V. Ravin, K.G. Skryabin, V.O. Popov, 2013,
published in Doklady Akademii Nauk, 2013, Vol. 449, No. 3, pp. 356–359.
BIOCHEMISTRY, BIOPHYSICS
AND MOLECULAR BIOLOGY
Structures of βGlycosidase from Acidilobus saccharovorans
in Complexes with Tris and Glycerol1
A. A. Trofimov, K. M. Polyakov, A. V. Tikhonov, E. Y. Bezsudnova, P. V. Dorovatovskii,
V. M. Gumerov, N. V. Ravin, Academician K. G. Skryabin,
and Corresponding Member of the RAS V. O. Popov
Received October 2, 2012
DOI: 10.1134/S1607672913020129
Glycosidases, enzymes catalyzing cleavage of O–
glycosidic bonds (EC 3.2.1), are widespread in nature
and are promising objects for biotechnologies and cre
ation of medicines [1, 2]. The accepted classification
of glycosidases based on the homology of amino acid
sequences (http://www.cazy.org/) considers more
than 110 glycosidase families. The object of this inves
tigation is β–glycosidase from the thermoacidophilic
archaeon Acidilobus saccharovorans (AsβGly, gene
ASAC1390 [3]), which belongs to the first glycosidase
family (GH1) according to the CAZy classification.
Glycosidases GH1 hydrolyze equatorial glycosidic
bonds, with the anomeric center configuration being
unchanged. The catalytic reaction is performed owing
to two glutamate residues; one of them is a proton
donor and the other is a nucleophile that attacks the
anomeric carbon atom. The basis of the glycosidase
GH1 tertiary structure is formed by the (β/α)8 barrel.
Among glycosidases with the structures deposited in
PDB (http://www.rcsb.org/), the enzymes from Sul
folobus solfataricus (SsβGlc1, PDB code 1uwq),
Thermosphaera aggregans (1qvb), and Pyrococcus furi
osus (3apg) are the most homological to AsβGly (71,
56, and 54% identities, respectively). In this work, the
3D structures of AsβGly in complexes with glycerol
and Tris are determined by Xray analysis.
To obtain AsβGly, the corresponding A. saccharo
vorans gene was cloned in the pQE60 vector and
expressed in the Escherichia coli strain DLT1270
(pRARE2). To isolate Asβ–Gly, cells were destroyed
in a French press, followed by the homogenate heating
(343 K, 10 min) and protein purification with the use
1 The article was translated by the authors.
Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences, Moscow, Russia
email: burayatina@gmail.com, kostya@eimb.ru,
alex_tikhonov@rambler.ru, eubez@yandex.ru,
paulgemini@mail.ru, netuns@gmail.com,
nravin@biengi.ac.ru, office@biengi.ac.ru,
vpopov@inbi.ras.ru
99
of hydrophobic chromatography (Toyepearl, Phenyl
650M; Source 15PHE) and gel filtration (Superdex
200 10/300). The activity was measured spectrophoto
metrically at the wavelength of 405 nm and 333 K
using onitrophenylβDgalactopyranoside as a sub
strate. To determine Ki of Tris, the enzyme activity was
measured in the standard conditions in the presence of
TrisHCl with the concentrations of 0–5 μM.
Crystallization of AsβGly was performed by the
hanging drop method in the following conditions:
(1) the protein solution contained 15 mg/ml AsβGly,
0.025 M NaCl, and 0.05 M Tris (pH 8.0); the precipi
tant solution contained 0.04 M cellobiose, 0.16 M mag
nesium acetate, 0.08 M sodium cacodylate (pH 6.5),
16% w/v Peg 8000, and 20% v/v glycerol; (2) the protein
solution contained 13 mg/ml AsβGly, 0.025 M NaCl,
and 0.025 M Hepes (pH 7.5); the precipitant solution
contained 1.0 M succinic acid (pH 7.0), 0.1 M Hepes
(pH 7.0), and 1% w/v Pegmonomethyl ether 2000.
3 μl drops contained equal volumes of precipitant and
protein solutions. Crystals grew for 12 days at 293 K.
Crystals of the complex AsβGly+Tris were obtained
in the conditions (1).
Diffraction data were collected on the beamline
K4.4 at the NRC Kurchatov Institute (Russia) using a
CCD detector at 100 K. Before data collection, a crys
tal from the conditions (2) was soaked in the cryopro
tectant solution containing 20% v/v glycerol, which
resulted in the formation of the complex AsβGly+Gol.
Diffraction data were processed in programs XDS and
XSCALE [4]. The structure of AsβGly was solved by
the molecular replacement method in the program
BALBES [5]. Refinement was performed in the pro
gram REFMAC [6, 7], taking into account the contri
bution of hydrogen atoms in scattering. The structure
AsβGly+Gol was refined anisotropically. Manual
model corrections based on electrondensity maps
were performed in the program COOT [8]. The data
collection and refinement statistics are given in the
table. Structures AsβGly+Gol and AsβGly+Tris
were deposited in the PDB database with codes 4ha4
and 4ha3, respectively.
100 TROFIMOV et al.

Datacollection and refinement statistics for AsβGly. Values in parentheses are for the highest resolution shell
Study object AsβGly + Tris AsβGly + Gol
Space group P42212 P42212
Unitcell parameters a = b = 84.18, c = 166.22 Å; a = b = 84.12, c = 166.27 Å;
α = β = γ = 90° α = β = γ = 90°
Xray source Kurchatov SNC Kurchatov SNC
Wavelength, Å 0.9779 0.9815
Temperature, K 100 100
Resolution, Å 8.0–1.46 (1.55–1.46) 4.0–1.37 (1.45–1.37)
Total no. of reflections 1076686 (123508) 735518 (73860)
No. of unique reflections 102050 (15340) 124046 (18722)
Completeness, % 98.0 (91.1) 98.8 (96.5)
Rmeas * 0.063 (0.519) 0.063 (0.810)
Mean I/σ(I) 26.8 (4.5) 17.2 (2.1)
B factor from Wilson plot, Å2 20.3 20.1
Rwork 0.143 0.131
Rfree 0.159 0.163
RMSD of bond lengths, Å 0.015 0.015
RMSD of bond angles, degrees 1.531 1.559
No. of protein residues 489 489
No. of water molecules 516 543
N
*Rmeas = Σhkl [  Σi |Ii (hkl) – 〈I(hkl)〉|]/Σhkli (Ii (hkl)), where N is the multiplicity of the observed reflection hkl.
1/2
(N – 1)

There is one polypeptide chain of AsβGly in the
asymmetric unit cell. In crystal AsβGly, a tetramer
similar to the tetramer of SsβGlc1 forms. The sub
302
104 301
93
Fig. 1. Superposition of the subunit structures of AsβGly
and SsβGlc1 (1uwr) using equivalent Cα atoms. The
model of AsβGly is colored black; the SsβGlc1 model is
gray. The maximum differences in the structures were
found in the regions 93–104 and 301–302 (numbering for
AsβGly).
units of AsβGly in the tetramer are related by the
crystallographic fourfold axis.
The structure of the AsβGly subunit (Asβ
Gly+Gol) can be superposed with the structure of
SsβGlc1 (1uwr) with an rms deviation of 0.54 Å using
equivalent Cα atoms of 474 residues. The only essen
tial differences between AsβGly and SsβGlc1 are
found in the sequence regions 301–302 and 93–104
(AsβGly) (Fig. 1). Residues 301–302 of AsβGly ter
minate a short βsheet, whereas in SsβGlc1 the sim
ilar βsheet is terminated by a loop of six residues
(299–304). The differences in the second region
appear because the residues 99–103 of AsβGly form
a βsheet with the residues 111–106, whereas the sim
ilar residues in SsβGlc1 (92–109) form a big loop.
The catalytic residues of AsβGly are Glu208 (proton
donor) and Glu385 (nucleophile). The active site
structures of AsβGly and SsβGlc1 are the same; the
distance between the catalytic residues of AsβGly is
3.73 Å (AsβGly+Gol).
In the structure AsβGly+Gol, glycerol is bound in
the active site in two possible conformations. Atoms of
the glycerol molecule occupy almost the same posi
tions as the atoms of substratelike inhibitors in the
structures of the SsβGlc1 complexes (1uws, 1uwu)
(Fig. 2), the O3 glycerol atom forming a hydrogen
bond with the OE2 atom of the nucleophile Glu385
(2.67 Å). In this case, the glycerol molecule mimics the
substrate, but it can form only eight hydrogen bonds
with the side chains of AsβGly residues, and hence it

DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 449 2013

 STRUCTURES OF ΒGLYCOSIDASE FROM ACIDILOBUS SACCHAROVORANS
Asn207
His152
Trp431
Glu208
Glu385
Gln19
Glu430
Fig. 2. Active sites in the structures of AsβGly+Gol and
the SsβGlc1 complex with Dglucohydroximolactam
(1uwu). The structures are superposed using equivalent
Cα atoms. The AsβGly+Gol model is colored black;
SsβGlc1 is gray. The glycerol molecule has two conforma
tions. The hydrogen bonds shown and the residues labeled
are for AsβGly+Gol.
should be a weaker inhibitor compared to such com
pounds as Dglucohydroximolactam and Dgalacto
hydroximolactam [9], which are like the substrate in
their nature and form more hydrogen bonds in the gly
cosidase active site.
Tris is a rather strong competitive inhibitor of Asβ
Gly. The Ki value for AsβGly inhibition by Tris is
1.7 μM, which is close to the Ki values for SsβGlc1
inhibition by Dgluco and Dgalactohydroximolac
tams (1.04 and 1.08 μM, [9]). In the structure Asβ
Gly+Tris, Tris is bound in the active site in two possi
ble conformations (Fig. 3). Neither cellobiose nor
glycerol, which were present in the crystallization con
ditions, was detected on electrondensity maps in the
AsβGly active site. The O1 atom of Tris forms hydro
gen bonds with the nucleophile Glu385 (2.65 Å) and
the proton donor Glu208 (3.18 Å). The side chain of
Glu208 has two conformations; the mobility of this
residue might be important for the catalysis.
In the active site of AsβGly, Tris can form 10
hydrogen bonds with the side chains of the residues,
whereas Dglucohydroximolactam forms 14 hydro
gen bonds with the protein residues in the structure of
the SsβGlc1 complex (1uwu), which is in agreement
with the Ki values for these inhibitors.
The binding of Tris in the active site of AsβGly is
an example of the relatively poorly investigated effec
tive inhibition of glycosidases by compounds that sig
nificantly differ in their nature from the substrate—
sugar.
DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 449
101
Asn207
His152
Trp431
Glu208
Glu385
Gln19
Glu430 Trp423
Fig. 3. Binding of Tris in the active site of AsβGly. The Tris
molecule and Glu208 both have two conformations.
Hydrogen bonds are indicated by dashed lines.
This work was supported by the Ministry of Educa
tion and Science of the Russian Federation (state con
tract no. 16.512.11.2234).
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