A new archaeal β-glycosidase from Sulfolobus solfataricus: Seeding a novel retaining β-glycan-specific glycoside hydrolase family along with the human non-lysosomal glucosylceramidase GBA

Article abstract of DOI:10.1074/jbc.M109.086470

Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell.Onthe basis of their amino acid sequences they are classified in families and clans that show conserved catalytic mechanism, structure, and active site residues, but may vary in substrate specificity. We report here the identification and the detailed molecular characterization of a novel glycoside hydrolase encoded from the gene sso1353 of the hyperthermophilic archaeon Sulfolobus solfataricus. This enzyme hydrolyzes aryl β-gluco- and β-xylosides and the observation of transxylosylation reactions products demonstrates that SSO1353 operates via a retaining reaction mechanism. The catalytic nucleophile (Glu-335) was identified through trapping of the 2-deoxy-2-fluoroglucosyl enzyme intermediate and subsequent peptide mapping, while the general acid/base was identified as Asp-462 through detailed mechanistic analysis of a mutant at that position, including azide rescue experiments. SSO1353 has detectable homologs of unknown specificity among Archaea, Bacteria, and Eukarya and shows distant similarity to the non-lysosomal bile acid β-glucosidase GBA2 also known as glucocerebrosidase. On the basis of our findings we propose that SSO1353 and its homologs are classified in a new CAZy family, named GH116, which so far includes β-glucosidases (EC 3.2.1.21), β-xylosidases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as known enzyme activities.

Full text of DOI:10.1074/jbc.M109.086470

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 27, pp. 20691–20703, July 2, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
A New Archaeal ␤-Glycosidase from Sulfolobus solfataricus
SEEDING A NOVEL RETAINING ␤-GLYCAN-SPECIFIC GLYCOSIDE HYDROLASE FAMILY
ALONG WITH THE HUMAN NON-LYSOSOMAL GLUCOSYLCERAMIDASE GBA2*
Received for publication, November 18, 2009, and in revised form, April 28, 2010 Published, JBC Papers in Press, April 28, 2010, DOI 10.1074/jbc.M109.086470
Beatrice Cobucci-Ponzano‡, Vincenzo Aurilia‡, Gennaro Riccio‡, Bernard Henrissat§, Pedro M. Coutinho§,
Andrea Strazzulli‡, Anna Padula‡, Maria Michela Corsaro¶, Giuseppina Pieretti¶, Gabriella Pocsfalvi‡,
Immacolata Fiume‡, Raffaele Cannio‡, Mose` Rossi‡ʈ, and Marco Moracci‡1
From the ‡Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy, the §Architecture et Fonction des
Macromole´cules Biologiques, UMR6098, CNRS, Universite´s Aix-Marseille I & II, 163 Avenue de Luminy, 13288 Marseille, France, and
the ¶Dipartimento di Chimica Organica e Biochimica and the ʈDipartimento di Biologia Strutturale e Funzionale, Universita` di
Napoli Federico II, Complesso Universitario di Monte S. Angelo, Via Cinthia 4, 80126 Naples, Italy
Carbohydrate active enzymes (CAZymes) are a large class of
enzymes, which build and breakdown the complex carbohy-
drates of the cell. On the basis of their amino acid sequences they
are classified in families and clans that show conserved catalytic
mechanism, structure, and active site residues, but may vary in
substrate specificity. We report here the identification and the
detailed molecular characterization of a novel glycoside hydro-
lase encoded from the gene sso1353 of the hyperthermophilic
archaeon Sulfolobus solfataricus. This enzyme hydrolyzes aryl
␤-gluco- and ␤-xylosides and the observation of transxylosyla-
tion reactions products demonstrates that SSO1353 operates
via a retaining reaction mechanism. The catalytic nucleophile
(Glu-335) was identified through trapping of the 2-deoxy-2-
fluoroglucosyl enzyme intermediate and subsequent peptide
mapping, while the general acid/base was identified as Asp-462
through detailed mechanistic analysis of a mutant at that posi-
tion, including azide rescue experiments. SSO1353 has detecta-
ble homologs of unknown specificity among Archaea, Bacteria,
and Eukarya and shows distant similarity to the non-lysosomal
bile acid ␤-glucosidase GBA2 also known as glucocerebrosidase.
On the basis of our findings we propose that SSO1353 and its
homologs are classified in a new CAZy family, named GH116,
which so far includes ␤-glucosidases (EC 3.2.1.21), ␤-xylosi-
dases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as
known enzyme activities.
the mechanisms of the sugar code are not known in detail. They
include the control of the correct protein folding and activity
(2), the mediation of molecular recognition events regulating
cell-cell interactions (such as host-pathogen, cancer metastasis,
etc.), cell signal transduction (nucleo-cytoplasm communica-
tion, differentiation, immune response, etc.) (for reviews see
Refs. 1, 3, 4).
The ability of carbohydrates in functioning in intermolec-
ular interactions as encoders of biological information is
made possible by a large class of enzymes, collectively known
as carbohydrate-active enzymes (CAZymes),2 including glyco-
side hydrolases (GH), glycosyltransferases (GT), polysaccha-
ride lyases, carbohydrate esterases, and carbohydrate binding
modules, which are in charge of catalyzing the metabolism and
the correct shape of the sugars of the cell. CAZymes have been
classified on the basis of their amino acid sequences in families
sharing the same catalytic mechanism, structure, and active site
residues; in addition, families with similar three-dimensional
structure are further grouped in clans (5).
The number of CAZyme families is continuously increasing,
thanks to the advent of new DNA sequencing techniques and
subsequent sophisticated computer-aided sequence annota-
tion procedures combined with new biochemical characteriza-
tion. Interestingly, although the number of CAZy sequences
increased 14-fold in the last 8 years, the number of enzymatic
and structural characterization only doubled in the same time
span, with at present Ͻ10% of total proteins in the CAZy data-
base that have been characterized enzymatically (5). This con-
trast clearly shows that, in comparison with highly automated
sequencing techniques, enzymatic characterization of novel
Carbohydrates, whose structural diversity exceeds by far the
number of protein folds, are ubiquitous molecules that alone, or
in form of glycoconjugates, mediate many biological processes
(1). This extreme variety results from the diverse stereochem-
istry of the monosaccharide building blocks, from the enor-
mous number of intersugar linkages they can form and to the
fact that these molecules can decorate cell surface, large mac-
romolecules (sugars themselves, proteins, nucleic acids), or
small metabolites (lipids, antibiotics, etc). The breadth of the
biological functions of carbohydrates, from the classical ener-
getic and structural roles, is now well acknowledged although
2 The abbreviations used are: CAZyme, carbohydrate active enzyme; GBA1,
lysosomal acid ␤-glucosidase; GH, glycoside hydrolases; GT, glycosyltrans-
ferases; ORF, open reading frame; GBA2, non-lysosomal glucosylcerami-
dase; 4Np-Glc, 4-nitrophenyl-␤-D-glucopyranoside; 4Np-Xyl, 4-nitrophe-
nyl-␤-D-xylopyranoside; MU-Glc, methylumbellyferyl-␤-D-glucopyranoside;
MU-Xyl, methylumbellyferyl-␤-D-xylopyranoside; NB-DNJ, N-butyl-de-
oxynojirimycin; CBE, conduritol ␤-epoxide; TLC, thin layer chromatogra-
phy; Ss␤-gly, ␤-glycosidase from S. solfataricus; 4Np-Xyl2, 4NP-disaccha-
ride; 4NP-Xyl3, 4NP-trisaccharide; nano-ESI-MS/MS, nano-electrospray
ionization tandem mass spectrometry; EPS, exo-polysaccharides; 2,4DNp-
2F-Glc, 2,4-dinitrophenyl-␤-D-2-deoxy-2-fluoro-glucopyranoside; IDA,
information-dependent acquisition; CHAPS, 3-[(3-cholamidopropyl)di-
methylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted
laser desorption/ionization-time of flight.
* This work was supported by Project MoMa 1/014/06/0 of the Agenzia Spa-
ziale Italiana.
1 To whom correspondence should be addressed. Tel.: 390816132271; Fax:
390816132277; E-mail: m.moracci@ibp.cnr.it.
JULY 2, 2010•VOLUME 285•NUMBER 27
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Characterization of an Archaeal ␤-Glycosidase
CAZymes is a longer and laborious process representing the AGAGATGGACAATGCTTTTGGCGCTACCATCA-3Ј;
limiting step for the full exploitation of genome sequencing
efforts.
D462Grev, 5Ј-CAAAAGCATTGTCCATCTCTCCCTCCA-
TGA-3Ј. The genes containing the desired mutation were iden-
tified by direct sequencing and completely resequenced.
Expression and Purification of SSO1353 Wild Type and
Mutants—Escherichia coli BL21(DE3)Ril/pET1353 wild type
and mutants were grown in 2 liters of LB at 37 °C supplemented
with kanamycin (50 ␮g/ml) and chloramphenicol (30 ␮g/ml).
Gene expression was induced by the addition of 0.5 mM isopro-
pyl-1-thio-␤-D-galactopyranoside when the culture reached an
Here, we report the cloning, the heterologous expression and
the detailed enzymatic characterization of a novel GH from the
hyperthermophilic Archaeon S. solfataricus. This enzyme,
encoded by the ORF SSO1353 displays sequence similarity to
several unknown proteins from the three domains of life
(Archaea, Bacteria, and Eukarya) and, more distantly, to human
non-lysosomal glucosylceramidase, also known as ␤-glucosi-
dase 2 (GBA2) (6), an enzyme involved in an alternative cata-
bolic pathway of glucosylceramide (7).
A
600 of 1.0. Growth was allowed to proceed for 16 h, and cells
were harvested by centrifugation at 5,000 ϫ g. The resulting cell
Ϫ1
The enzymatic characterization of the product of gene
sso1353 allowed us to demonstrate the retaining reaction
mechanism followed by the enzyme, to evaluate its substrate
specificity toward ␤-linked aromatic glucosides and xylosides,
and to identify the catalytic amino acids in the active site. These
results allowed us propose the role possibly played in vivo by
this enzyme, which is expressed from a gene situated down-
stream of that coding for an endoglucanase in different Sulfolo-
bus species. Finally, by virtue of the established commonalities
within glycoside hydrolase families, the mechanistic data
obtained with SSO1353 can be extended to all members of the
newly created GH116 family including human GBA2.
pellet was thawed, resuspended in 3 ml g cells of 20 mM
sodium phosphate buffer, pH 7.4, 150 mM NaCl, 1% (v/v) Triton
X-100 and homogenized by French cell pressure treatment.
After centrifugation for 30 min at 10,000 ϫ g, the crude extract
was incubated with Benzonase (Novagen) for 1 h at room tem-
perature and then heat-fractionated for 30 min at 55 and 75 °C
and for 20 min at 85 °C. The supernatant obtained after heat
fractionations, equilibrated in 1 M ammonium sulfate, was
applied to a HiLoad 26/10 phenyl Sepharose high performance
(Amersham Biosciences), which had been equilibrated with 20
mM sodium phosphate buffer, pH 7.3, 1 M ammonium sulfate.
After washing with 2 column volumes with the loading buffer,
the protein was eluted with a linear gradient of water at a flow
rate of 3 ml minϪ1; the protein eluted in 100% water. Active
fractions were pooled, equilibrated in 20 mM sodium phosphate
buffer, pH 7.4, 150 mM NaCl and concentrated by ultrafiltration
on an Amicon YM30 membrane (cut off 30,000 Da). For the
wild-type enzyme, after concentration, the sample was loaded
onto a HiLoad 26/60 Superdex 200 prep grade column (Amer-
sham Biosciences). Active fractions were pooled and concen-
trated; protein concentration was determined with the method
of Bradford (8). The SSO1353 wild type and mutants were 95%
pure by SDS-PAGE and were stored at 4 °C.
EXPERIMENTAL PROCEDURES
Reagents—All commercially available substrates were pur-
chased from Sigma and Carbosynth. The GeneTailor Site-Di-
rected Mutagenesis system was from Invitrogen, and the syn-
thetic oligonucleotides were from PRIMM (Milan, Italy).
Plasmid Preparation—The SSO1353 ORF was cloned by
amplification of S. solfataricus, strain P2, chromosomal DNA
via PCR by using the following synthetic oligonucleotides:
1353Fw, 5Ј-ggaattccatatggttacatatactgataagg-3Ј, 1353Rv, 5Ј-ta-
catgccatggctagaataggaagctcc-3Ј, which introduce an NdeI and
NcoI sites at the 5Ј, just before the first ATG, and at the 3Ј-ends
of the ORF, respectively. The program was as follows: 5 min at
95 °C, 1,5 min at 50 °C, and 4 min at 72 °C; 30 cycles at 95 °C for
45 s, 50 °C for 1,5 min, and 72 °C for 4 min; final extension at
72 °C for 10 min. The resulting DNA fragment was cloned in
the pET29a plasmid (Novagen), obtaining the vector pET1353,
in which the SSO1353 ORF is under the control of the isopro-
pyl-1-thio-␤-D-galactopyranoside inducible T7 RNA polymer-
ase promoter that drives high expression levels in bacterial
hosts. The ORF obtained after amplification was controlled by
DNA sequencing.
Characterization of SSO1353 Wild Type and Mutants—
The molecular mass of native SSO1353 wild type was deter-
mined by gel filtration on a Superdex 200 HR 10/30 FPLC
column (Amersham Biosciences); molecular weight markers
were albumin (66,000), alcohol dehydrogenase (150,000), ␤-
amylase (200,000), and apoferritin (443,000).
The standard assay for SSO1353 activity was performed in
50 mM sodium citrate buffer at pH 5.5 at 65 °C on the indi-
cated substrates. Typically, in each assay we used 1–10 ␮g of
SSO1353 in the final volume of 1.0 ml. Kinetic constants of
SSO1353 wild type and D462G mutant on aryl glycosides 4Np-
Glc, 4Np-Xyl, and 2Np-Glc were measured at standard condi-
tions at 65 °C by using concentrations of substrate ranging
between 1 and 150 mM. The ⑀mM extinction coefficients at 405
nm for 2- and 4-nitrophenol under standard conditions and
Site-directed Mutagenesis—The mutants E335G, D406G,
D458G, D462G were prepared by site-directed mutagenesis
from the pET1353 plasmid, by following the instructions of the
manufacturer. The mutagenic oligonucleotides were the
following (mismatches are underlined): E335Gmut, 5Ј-ATGG-
ACAGTTCTGTGGTGCTCCGTAAATCGCA-3Ј; E335Grev,
5Ј-AGCACCACAGAACTGTCCATATTTAGGTAC-3Ј;
D406Gmut, 5Ј-TTCTCCTCCCAGATGGAAGGGTATGAA-
TCCGA-3Ј; D406Grev, 5Ј-CCTTCCATCTGGGAGGAGAA-
GTAGTACCAT-3Ј; D458Gmut, 5Ј-ATTCATGGAGGGAG-
AGATGGGCAATGCTTTTG-3Ј; D458Grev, 5Ј-CCATCTC-
Ϫ1
65 °C were 1.1 and 3.3 mM cmϪ1, respectively. One unit of
enzyme activity was defined as the amount of enzyme cata-
lyzing the hydrolysis of 1 ␮mol of substrate in 1 min at the
conditions described. The metal dependence of the wild-type
enzyme was evaluated using 5 mM 4Np-Xyl as substrate in the
presence of 1 mM EDTA and 5 mM MgCl2 or MnCl2, in standard
TCCCTCCATGAATGGTAAATTCC-3Ј; D462Gmut, 5Ј- conditions.
20692 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

Characterization of an Archaeal ␤-Glycosidase
The activity of the wild-type enzyme on different aryl glyco- least two experiments and were plotted and refined with the
program GraFit (10).
sides was tested in 50 mM sodium citrate buffer at pH 5.5 at
65 °C. Typically, in each assay we used 70 ␮g of SSO1353 in a
final volume of 0.2 ml. The reaction was started by adding the
enzyme, and it was stopped by adding 0.8 ml of 1 M iced sodium
carbonate. The optical density of the solution was measured at
420 nm at room temperature. The molar extinction coefficients
of 4-nitrophenol and 2-nitrophenol, measured at 420 nm, at
room temperature and in 1 M sodium carbonate buffer were
17.2 and 4.7 mMϪ1 cmϪ1, respectively. In all of the assays, spon-
taneous hydrolysis of the substrate was subtracted by using
appropriate blank mixtures without the enzyme.
Inhibition of SSO1353 Wild Type—The effect of the inhibi-
tor 2,4-dinitrophenyl-␤-D-2-deoxy-2-fluoro-glucopyranoside
(2,4DNp-2F-Glc) (Sigma) was analyzed as previously per-
formed by Shaikh et al. (11). Briefly, wild-type SSO1353 (0.1
␮g/␮l) was incubated at 45 °C in mixtures containing 0.3, 3.0,
7.0, and 18.0 mM concentrations of inhibitor and 50 mM sodium
citrate buffer, pH 5.5. An identical mixture containing all the
reagents with the exception of the enzyme was prepared as
control. At time intervals, aliquots from the two mixtures were
withdrawn and used to measure the enzymatic activity and as
blank, respectively. Assays were performed on 60 mM 2Np-Glc
in standard conditions. Initial rates at each time point were
elaborated as described in Ref. 11 to measure the inactivation
parameters Ki and ki with GraFit (10).
The chemically rescued activity of the D462G mutant was
measured in 50 mM sodium citrate buffer at pH 5.5 on 40 mM
2Np-Glc at 65 °C as described above; where indicated, the assay
mixture was supplemented with the indicated concentrations
of sodium azide as external nucleophile. In all of the assays,
spontaneous hydrolysis of the substrate was subtracted by
using appropriate blank mixtures without the enzyme. Aliquots
of the reaction mixtures were analyzed on a silica gel 60 F254
TLC by using ethyl acetate/methanol/water (70:20:10 v/v) as
eluant and were detected by exposure to 4% ␣-naphthol in 10%
sulfuric acid in ethanol followed by charring. The ␤-D-glucosyl
azide isolated from the enzymatic reaction mixture of the
To determine the effect of the inhibitors N-butyldeoxynojiri-
mycin (NB-DNJ) and conduritol ␤-epoxide (CBE), SSO1353
(11 ␮g) was incubated in the presence of increasing concentra-
tions of the inhibitors (0.1–5 mM for NB-DNJ and 0.1–2 mM for
CBE) in 50 mM sodium citrate buffer, pH 5.5, for 30 min at
45 °C, in a final volume of 80 ␮l. Identical mixtures containing
all the reagents with the exception of the inhibitor were used to
determine the 100% of activity. After incubation, the samples
were diluted 3-fold in 5 mM MU-Glc, 50 mM sodium citrate
buffer, pH 5.5 in a final volume of 0.25 ml, and assayed as
described above.
1
D462G mutant was identified by H and 13C NMR spectros-
copy. NMR: ␦ 4.75 H-1 (d, 3JH1,H2 ϭ 8.8 Hz), ␦ 91.4 C-1; ␦ 3.25
H-2 (t), ␦ 74.0 C-2; ␦ 3.51 H-3 (t), ␦ 76.9 C-3; ␦ 3.41 H-4 (t), ␦
70.5 C-4; ␦ 3.53 H-5 (m), ␦ 79.0 C-5; ␦ 3.73 H-6a (dd), ␦ 3.90
H-6b (dd), 61.6 C-6.
Transglycosylation Reactions—Wild-type SSO1353 (7–148
␮g), was incubated in 50 mM sodium citrate buffer pH 5.5 with
5 mM 4Np-Xyl or 4Np-Glc, or both, at 5 mM each, at 65 °C for
16 h in a final volume of 0.2–1 ml. Identical mixtures containing
all the reagents but the enzyme were prepared as control.
Where specified, after incubation, 50 ␮g of the ␤-glycosidase
from S. solfataricus (Ss␤-gly) were added to aliquots (90 ␮l) of
the reaction mixtures and further incubated 2 h at 65 °C. All the
reactions were examined by TLC by using ethyl acetate/meth-
anol/water (70:20:10 v/v) as eluant as described above.
Identification of the Reaction Products—The 1H and 13C
NMR spectra were recorded in D2O at 600 MHz with a spec-
trometer equipped with a cryo probe, in the FT mode at 303 K.
1H chemical shifts are expressed in ␦ relative to HOD signal
(4.72 ppm).
The activity of the wild-type enzyme in the presence of
0.5–2.0 mg mlϪ1 Triton X-100 or CHAPS was determined on
20 mM 4Np-Glc as described above. The activity of the wild
type on ␤-D-oligosaccharides of glucose (G3 to G5) (5 mM) and
xylose (X2 to X5) (2.5 mM) was tested in 50 mM sodium citrate
buffer at pH 5.5, by using 12–63 ␮g of enzyme, at 65 °C in a final
volume of 0.2 ml. Aliquots of the reaction mixtures were ana-
lyzed by TLC by using ethyl acetate/acetic acid/isopropanol/
formic acid/water (50:20:10:2:30 v/v) (for G3-G5) or acetone/
isopropylic alcohol/water (60:30:15 v/v) (for X2–X5) as the
eluant and were detected as described above. The activity of the
wild type on glucocerebroside (Matreya), octyl-␤-D-glucopy-
ranoside (Sigma), and gangliosides, measured at the same con-
ditions was analyzed by TLC by using chloroform/methanol/
CaCl2 15 mM (60:40:9 v/v).
The linkage analysis of the oligosaccharide was obtained
by methylation according to Ciucanu procedure, as already
reported (12). The sample obtained was injected into GC-MS
and partially methylated alditol acetates were recognized from
their EI-MS spectra and by comparison with pure synthetic
standards. Partially methylated alditol acetates were analyzed
on an Agilent Technologies gas chromatograph 6850A
equipped with a mass selective detector 5973N and a Zebron
ZB-5 capillary column (Phenomenex, 30 m ϫ 0.25 mm i.d., flow
rate 1 ml/min, He as carrier gas). The temperature program
was: 90 °C for 1 min, 90 °C 3 140 °C at 25 °C minϪ1, 140 °C 3
200 °C at 5 °C minϪ1, 200 °C 3 280 °C at 10 °C minϪ1, 280 °C
for 10 min.
The steady-state kinetic constants of the wild type on MU-
Glc and MU-Xyl were calculated by following a slight modified
method already described (9). Briefly, the enzyme activity was
determined fluorimetrically with MU-Glc (0.1–6 mM) and
MU-Xyl (0.025–5 mM) as substrates in 50 mM sodium citrate
buffer at pH 5.5, by using 1 ␮g of the enzyme. Assays (0.25 ml
final volume) were conducted for 1–5 min at 65 °C, and the
reaction was stopped with 0.5 ml of 0.1 M glycine-NaOH buffer,
pH 10.3. The formation of the methylumbelliferone was meas-
ured by emission at 450 nm with excitation at 384 nm. In all of
the assays, spontaneous hydrolysis of the substrate was sub-
tracted by using appropriate blank mixtures without the
The reaction mixture was purified by reverse phase chroma-
tography (Polar-RP 80A, Phenomenex, 4 ␮, 250 ϫ 10 mm) on
enzyme. All kinetic data were calculated as the average of at an Agilent HPLC instrument 1100 series, using H2O/CH3OH
JULY 2, 2010•VOLUME 285•NUMBER 27
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Characterization of an Archaeal ␤-Glycosidase
6/4 with 20 mM trifluoroacetic acid (final concentration) as elu- ratio at 37 °C for 30 min. Resulting peptide mixtures (5 ␮l) were
ant. The eluted products were first analyzed by positive ions loaded, purified, and concentrated on a monolithic trap column
reflectron MALDI-TOF mass spectrometry.
(200 ␮m inner diameter ϫ 5 mm, LCPackings, Sunnyvale, CA)
The trisaccharide ␤-Xyl-(134)-␤-Xyl-O-4-Np showed a at 25 ␮l/min flow rate and separated by nanoflow reverse-phase
ϩ
pseudomolecular ion (MϩNa) at m/z 425.94 (calculated m/z chromatography on a PS-DVB monolithic column (200 ␮m
426.10) and the methylation analysis indicated the presence of ter- inner diameter ϫ 5 cm, LCPackings) at 300 nl/min using an
minal xylopyranose and 4-substituted xylopyranose. NMR: ␦ 5.24 UltiMateTM 3000 HPLC (Dionex, Sunnyvale, CA). The follow-
H-1A (d, JH-1,H-2 ϭ7.3Hz), ␦101.0C-1A;3.66H-2A (t), ␦73.7C-2A; ing solvents and gradient conditions were used: solvent A: 2%
␦ 3.72 H-3A (t), ␦ 74.5 C-3A; ␦ 3.89 H-4A (m), ␦ 77.4 C-4A; ␦ 4.19 acetonitrile in 0.1% formic acid and 0.025% trifluoroacetic acid,
Ha-5A (dd), ␦ 3.62 Hb-5A (t), ␦ 64.3 C-5A; ␦ 4.49 H-1B (d, JH-1,H-2 ϭ solvent B: 98% acetonitrile in 0.1% formic acid and 0.025% tri-
7.3 Hz), ␦ 103.1 C-1B; 3.29 H-2B (t), ␦ 74.0 C-2A; ␦ 3.44 H-3B (t), ␦ fluoroacetic acid, gradient: 5–50% B in 40 min, 50–98% B in 6 s.
76.9 C-3B; ␦ 3.62 H-4B (m), ␦ 70.4 C-4B; ␦ 3.99 Ha-5B (dd), ␦ 3.32 Eluting peptides were directly analyzed by nano-ESI-MS in pos-
Hb-5B (t), ␦ 66.5 C-5B.
itive ion mode using information-dependent acquisition (IDA).
The trisaccharide ␤-Xyl-(134)-␤-Xyl-(134)-␤-Xyl-O- The two most abundant multiply charged ions were automati-
4-Np showed a pseudomolecular ion in a MALDI-TOF-MS cally selected and subjected for collision induced dissociation
ϩ
(MϩNa) at m/z 557.82 (calculated m/z 558.15); methyla- experiments. Nitrogen was used as collision gas. Tandem mass
tion analysis: terminal xylopyranose and 4-substituted spectra were analyzed by manual inspection and by the use of
xylopyranose.
Mascot Server (version 2.2). Peak lists for Mascot containing all
NMR: ␦ 5.25 H-1A (d, JH-1,H-2 ϭ 7.3 Hz), ␦ 101.2 C-1A; 3.66 acquired MS/MS spectra were generated by Analyst QS 2.0
H-2A (t), ␦ 73.8 C-2A; ␦ 3.72 H-3A (t), ␦ 74.6 C-3A; ␦ 3.90 H-4A software using the default parameters. Mascot was set up to
(m), ␦ 77.3 C-4A; ␦ 4.19 Ha-5A (dd), ␦ 3.62 Hb-5A (t), ␦ 64.3 C-5A; search database containing a single protein (SSO1353) se-
␦ 4.51 H-1B (d, JH-1,H-2 ϭ 7.3 Hz), ␦ 103.0 C-1B; 3.31 H-2B (t), ␦ quence extracted from NCBInr and was run with a fragment
74.0 C-2A; ␦ 3.56 H-3B (t), ␦ 75.0 C-3B; ␦ 3.80 H-4B (m), ␦ 77.6 ion mass tolerance of 0.1 Da and a parent ion tolerance of 50
C-4B; ␦ 4.12 Ha-5B (dd), ␦ 3.39 Hb-5B (t); ␦ 64.3 C-5B; ␦ 4.47 ppm. MS/MS ion score cut-off was set to 10. No enzyme was
H-1C (d, JH-1,H-2 ϭ 7.3 Hz), ␦ 103.1 C-1C; ␦ 3.26 H-2C (t), ␦ 74.1 specified. 2F-Glc was defined as variable modification in Mas-
C-2C; ␦ 3.43 H-3C (t), ␦ 76.9 C-3C; ␦ 3.63 H-4C (m), ␦ 70.4 C-4C; cot searches. Three independent inhibition experiments were
␦ 3.97 Ha-5C (dd), ␦ 3.31 Hb-5C (t); ␦ 66.5 C-5C.
performed and on each resulting samples two analytical mea-
Nano-ESI-MS of Intact Protein Samples—Samples were surements were run.
analyzed using a triple quadrupole time of flight instrument
Definition and Analysis of a New Glycoside Hydrolase Family—
(QSTAR Elite, Applied Biosystems, Foster City, CA/Toronto, Gapped BLAST searches (13) were performed against the
Canada) equipped with a nanoflow electrospray ion source. non-redundant protein set at the NCBI and against classified
Pulled silica capillary (170 ␮m outer diameter/100 ␮m inner and unclassified sequences present in carbohydrate-active
diameter, tip 30 ␮m inner diameter) was used as nanoflow tip. enzymes database (CAZy) (5). A total of 90 sequences were
For the analysis of intact proteins, 4 ␮g of samples were purified used to define new family. This family includes proteins from
using ZipTip C4 (Millipore, Billerica, MA). Proteins were archaeal, bacterial, and eukaryotic origin, several already col-
eluted by 50% acetonitrile and 0.1% formic acid. Purified pro- lected in CAZy by similarity to human non-lysosomal bile acid
teins (10 ␮M) were loaded into the ion source at 300 nl/min flow ␤-glucosidase 2 (Gba2), but not yet assigned to a family. This
rate using a syringe pump. Single-stage ESI mass spectra were family was designated as glycoside hydrolase family 116 and will
acquired in the range of m/z 300–2000. For protein molecular be released in CAZy. The sequences were aligned with Muscle
mass determination three independent measurements were 3.7 (14), and the resulting alignments were subsequently
performed. The expected mass error on the average molecular manipulated and analyzed with an in-house modified version of
mass of intact proteins was about Ϯ0.01%. For data acquisition Jalview (15).3 Estimated sequence distances were determined
and Bayesian protein reconstruction the Analyst QS 2.0 soft- by maximum likelihood using LG distances (16) and con-
ware (Applied Biosystems, Foster City, CA/Toronto, Canada) structed a distance tree using the Ward hierarchical clustering
was used.
method (17).
Nano-HPLC-ESI-MS/MS Experiments—Wild-type SSO1353
(22 ␮g, 0.3 nmol) was incubated with 2.9 mM 2,4-dinitrophenyl-
␤-D-2-deoxy-2-fluoro-glucopyranoside (2,4DNp-2F-Glc) (Sigma)
at 1:1000 enzyme/inhibitor ratio in 50 mM sodium citrate
buffer, pH 5.5 at 45 °C. An identical mixture containing all the
reagents with the exception of the inhibitor was prepared as
control. At time intervals, aliquots from the two mixtures were
withdrawn and assayed on 60 mM 2Np-Glc in standard
conditions.
RESULTS
Isolation of ORF SSO1353—The inspection of the genomic
sequence of the archaeon S. solfataricus, strain P2, revealed an
ORF downstream of the gene sso1354 encoding for an endoglu-
canase (Fig. S1) (18). Sso1353 is presently annotated as an hypo-
thetical protein while the other ORFs in this cluster, sso1351,
sso1352, and sso1355, are a putative permease, a transcriptional
regulator, and a carboxypeptidase, respectively. ORFs sso1354
and sso1353 are transcribed in the same direction and are sep-
arated by 57 bp in which the latter ORF is preceded by a putative
Samples (0.086 ␮g/␮l) were enzymatically digested in the
acidified inhibition buffer (formic acid to 5% (v/v) final concen-
tration, pH 2) using pepsin from porcine stomach mucosa
(3,260 units/mg, Sigma-Aldrich) at 1:20 enzyme to substrate 3 P. M. Coutinho and B. Henrissat, unpublished data.
20694 JOURNAL OF BIOLOGICAL CHEMISTRY
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Characterization of an Archaeal ␤-Glycosidase
promoter formed by an AT-rich box A (centered at Ϫ30 nt abinoside; 4Np-␤-L-Fuc, ␤-D-oligosaccharides of glucose and
from ATG) and a TFB-responsive element (centered at Ϫ38 nt) xylose (di-, tri-, tetra- and pentaose), and glucocerebroside,
(not shown). Northern blot analysis showed that sso1353 is octyl-␤-D-glucopyranoside and gangliosides even after pro-
expressed as an isolated gene (not shown) and the absence of longed incubations. Steady-state kinetic parameters of
a clear Shine-Dalgarno-like motif in the intergenic region SSO1353 were measured for the glycosides that were substrates
suggests that sso1353 gene is translated as a leaderless gene (19). of the enzyme (Table 1). SSO1353 showed similar kinetic con-
Initial gapped BLAST searches (13) revealed that SSO1353 is stants for the substrates tested: the highest specificity constant
similar to proteins of unknown function from Archaea, Bacte- for MU-glycosides results from a reduced Km. Therefore, this
ria, and Eukarya and, to a lesser extent, to eukaryotic non-lyso- kinetic characterization showed that SSO1353 is a ␤-D-glycosi-
somal bile acid ␤-glucosidases. The higher sequence identity dase specific for gluco- and xylosides (EC 3.2.1.21/37) showing
scores (Ͼ31%) were with archaeal proteins with the highest increased affinity for substrates having hydrophobic leaving
(86%) with loci sso1948 from S. solfataricus, strain P2, and groups. Finally, interestingly, the activity of SSO1353 on MU-
M1425_0924 and M1627_099 from S. islandicus, strains Glc was inhibited by both N-butyl-deoxynojirimycin (NB-DNJ)
M.14.25 and L.S.2.15, respectively. Interestingly, these highly and conduritol ␤-epoxide (CBE). The former showed an IC50 of
similar genes also lie downstream to a locus encoding for an 1.5 mM while 2 mM CBE gave 93% inhibition after 30 min of
endoglucanase (SSO1949 in S. solfataricus (20)), suggesting incubation. The sensitivity to this irreversible inhibitor differ-
that gene duplication occurred in these organisms. Among entiates SSO1353 from human non-lysosomal glucosylcerami-
non-lysosomal bile acid ␤-glucosidases, the scores were much dase, which is insensitive to CBE (see below) (6).
lower, the best ones being with human and Ciona intestinalis
SSO1353 Has Transglycosylation Activity and Follows a
enzymes (19% identity). Sequences from the NCBI were Retaining Reaction Mechanism—The products of the reaction
searched to complement the set of unclassified glycoside mixtures containing SSO1353 and 4Np-Xyl were examined by
hydrolase sequences already present in CAZy previously col- thin layer chromatography (TLC) (Fig. 3). Interestingly, the
lected based on the bile acid-glycosidases, and integrated to products include not only xylose, but also oligosaccharides with
create a new family, that we have designated as Glycoside a higher degree of polymerization than the 4Np-Xyl substrate,
Hydrolase family 116 (GH116). Once the conserved catalytic indicating that the enzyme performed transglycosylation reac-
regions were aligned, a distances tree was obtained (Fig. 1).
tions (Fig. 3A, lane 4). The transglycosylation products could be
To ascertain if the sso1353 encodes for a novel glycoside completely hydrolyzed (Fig. 3A, lane 5) by the addition of lim-
hydrolase, the corresponding gene was cloned by PCR from the iting amounts of ␤-glycosidase from S. solfataricus (Ss␤-gly),
genomic DNA of S. solfataricus, strain P2. The primer at the 5Ј which shows broad substrate specificity for ␤-D-glycosides (22),
of the gene was designed starting from the first Met. Attempts indicating that SSO1353 catalyzed the formation of ␤-D-xylo-
to express SSO1353 fused to glutathione S-transferase (at the oligosaccharides. Remarkably, the enzyme catalyzed transgly-
N terminus) or to a His tag (at the C terminus) were unsuccess- cosylation reactions also by using 4Np-Glc (5 mM) as a substrate
ful; therefore, the gene was cloned in pET29a without any puri- and an increased number of transglycosylation products were
fication tag, obtaining the plasmid vector pET1353. The result- found when both substrates were included in the reaction mix-
ing recombinant SSO1353 protein was successfully expressed ture: at least five compounds are easily observable by TLC (Fig.
in the soluble fraction and purified to homogeneity by perform- 3B, lane 8).
ing three subsequent heating steps followed by a hydrophobic
To determine the stereo- and regioselectivity of the trans-
chromatography and a gel filtration (Fig. 2). After the last puri- glycosylation activity of SSO1353 in the presence of 4Np-Xyl
fication step we obtained about 1.5 mg of pure protein per liter we scaled up the reaction to purify the products. The reverse
of E. coli culture.
phase HPLC purification revealed the presence of two trans-
Properties of SSO1353—A gel filtration run with the suitable glycosylation products corresponding to 4NP-disaccharide
molecularweightstandardsrevealedthatSSO1353wasamono- (4Np-Xyl2) and 4NP-trisaccharide (4NP-Xyl3). Each product
mer of about 76 kDa in native conditions (not shown). The was identified by MALDI-TOF mass spectrometry, methyla-
purified enzyme was optimally active at pH 5.5 (50 mM sodium tion analysis, 1H and 13C-NMR spectroscopy. The positive ions
citrate) when assayed on 5 mM 4-nitrophenyl-␤-D-gluco- and MALDI-TOF mass spectrum of 4Np-Xyl2 showed a pseudo-
ϩ
-xylopyranoside (4Np-Glc, 4NP-Xyl) substrates at 65 °C, with molecular ion (MϩNa) at m/z 425.84, which accounted for
specific activities of 0.3 and 0.8 units mgϪ1, respectively. The the presence of a 4-phenyl glycoside of xylose disaccharide.
1
activity on 4Np-Xyl was not affected by Triton X-100 (0.5–2.0
The H-NMR spectrum showed two anomeric doublets in
mg mlϪ1), CHAPS (0.5–2.0 mg mlϪ1), Mn2ϩ, Mg2ϩ (5 mM), and the ratio of 1:1 of xylose at 5.24 ppm (H-1 of ␤ anomer A, 1,2JH,H
1,2
1 mM EDTA.
7.3 Hz) and 4.49 ppm (H-1 of ␤ anomer B
J
7.3 Hz),
H,H
To determine the substrate specificity of this enzyme, the respectively, together with signals of protons geminal to
hydrolytic activity of SSO1353 toward various substrates was hydroxyl groups in the region between 3.0 and 4.0 ppm. More-
investigated at 65 °C in 50 mM sodium citrate, pH 5.5. In addi- over signals having the same intensity as anomeric signals and
tion to 4Np-Glc and -Xyl, also 4Np-Gal, 2Np-Glc, -Gal, -Xyl, attributable to the 4-nitrophenyl moiety, occurred at 7.25, and
methylumbellyferyl-␤-D-glucopyranoside (MU-Glc), and MU- 8.28 ppm. The chemical shifts of these signals were in agree-
Xyl were substrates of the enzyme. Instead, no activity was ment with those of the disaccharide ␤-Xyl-(134)-␤-Xyl-O-4-
observed on 4Np-Man and -GlcNAc; X-Glc and X-Gal; 2Np- Np, as obtained by analysis of two-dimensional H and 13C
1
cellobioside; 4Np-␣-D-Gal,-Glc, and, -Man; 4Np-␣-L-Fuc, -ar- NMR spectroscopy. In particular the (134) linkage was
JULY 2, 2010•VOLUME 285•NUMBER 27
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Characterization of an Archaeal ␤-Glycosidase
20696 JOURNAL OF BIOLOGICAL CHEMISTRY
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Characterization of an Archaeal ␤-Glycosidase
FIGURE 2. SDS-PAGE analysis of SSO1353. Lane 1, molecular weight mark-
ers; lane 2, E. coli BL21(DE3)Ril/pET1353 soluble protein extract (45 ␮g); lanes
3–5, protein extract after heat treatment at 55, 75, and 85 °C, respectively (70,
120 and 84 ␮g, respectively); lane 6, typical sample after hydrophobic chro-
matography (21 ␮g); lane 7, sample after gel filtration (3 ␮g).
TABLE 1
Steady-state kinetic constants of SSO1353
Substrate
kcat
s
Km
mM
k
s
cat/Km
Ϫ1
Ϫ1
Ϫ1 mM
2Np-Glc
4Np-Glc
4Np-Xyl
MU-Glc
MU-Xyl
4.7 Ϯ 0.3
4.9 Ϯ 0.5
4.3 Ϯ 0.4
1.2 Ϯ 0.1
0.8 Ϯ 0.1
13 Ϯ 2
54 Ϯ 12
25 Ϯ 8
0.37
0.09
0.17
0.47
0.71
2.6 Ϯ 0.5
1.2 Ϯ 0.3
deduced from the C-4 glycosylation shift at 77.4 ppm of xylose
unit A respect to the value of 70.4 ppm for an unsubstituted
xylopyranoside (23). Further support to this structure derived
from the methylation analysis, which showed the presence of
1,4,5-tri-O-acetyl-2,3-di-O-methyl xylitol, corresponding to
4-substituted xylopyranose unit, and of 1,5-di-O-acetyl-2,3,4-
tri-O-methyl xylitol, corresponding to a terminal non-reducing
end xylopyranose unit.
FIGURE 3. Reaction products of SSO1353. Thin layer chromatography of the
transxylosylation reactions with 4Np-Xyl. (A) In each lane were loaded 20 ␮l of
the following reaction mixtures: lane 1, xylose standard 25 mM; lane 2, blank
mixture containing 50 mM sodium citrate buffer pH 5.5, 4Np-Xyl 5 mM; lane 3,
SSO1353 after TP85 °C (30 ␮g); lane 4, reaction mixture same as lane 2 with
added SSO1353 after TP85 °C (150 ␮g); lane 5, sample loaded in lane 4 with
added Ss␤-gly (50 ␮g); lane 6, Ss␤-gly (25 ␮g); lane 7, sample loaded in lane 2
with added Ss␤-gly (25 ␮g). Transxylosylation reactions with 4Np-Xyl and
4Np-Glc. (B) In each lane were loaded 20 ␮l of the following reaction mixtures:
lane 1, 4Np-Xyl 5 mM; lane 2, xylose standard 25 mM; lane 3, blank mixture
containing 50 mM sodium citrate buffer, pH 5.5, 4Np-Xyl 5 mM; lane 4, reaction
mixture same as lane 3 with added pure SSO1353 (7 ␮g); lane 5, blank mixture
containing 50 mM sodium citrate buffer, pH 5.5, 4Np-Glc 5 mM; lane 6, same as
lane 5 with added pure SSO1353 (7 ␮g); lane 7, blank mixture containing 50
mM sodium citrate buffer, pH 5.5, 4Np-Xyl 5 mM and 4Np-Glc 5 mM; lane 8,
same as lane 7 with added pure SSO1353 (7 ␮g); lane 9, glucose standard 25
mM; lane 10, 4Np-Glc 5 mM.
The positive ions MALDI-TOF mass spectrum of 4NP-Xyl3
ϩ
showed a pseudomolecular ion (MϩNa) at m/z 557.52, which
accounted for the presence of a nitro-phenyl glycoside of xylose
trisaccharide. The 1H NMR spectrum showed the presence of
three anomeric proton signals at 5.25, 4.51, and 4.47 ppm,
respectively. The beta anomeric configuration for all the xylose
1,2
units was inferred from the 7.3 Hz value of
J
coupling
H,H
constants. The methylation analysis revealed the same resi-
dues as for 4Np-Xyl2, suggesting a linear trisaccharide struc-
ture ␤-Xyl-(134)-␤-Xyl-(134)-␤-Xyl-O-4-Np for 4NP-
Xyl3. The complete assignment of the 1H and 13C values (see
“Experimental Procedures”) confirmed the above structure.
These results unequivocally demonstrate that SSO1353 pro- ate (glycosylation step) (Fig. S2). In the second step (de-glyco-
moted the transglycosylation reaction by following a retaining sylation step), the group previously acting as an acid now works
reaction mechanism.
as a base catalyst deprotonating the water and resolving the
Identification of the Catalytic Residues of SSO1353 by Site- glycosyl enzyme intermediate. Both steps proceed via transition
directed Mutagenesis—Retaining glycosidases generally utilize states with substantial oxocarbenium ion character (24).
a double displacement mechanism catalyzed by two enzymatic
The identification of key active site residues in a glycoside
carboxylates and in which a glycosyl intermediate is formed and hydrolase is crucial to determine the catalytic machinery for the
hydrolyzed. In the first step of the reaction, one of the carbox- classification of this class of enzymes (5, 25, 26). These residues
ylic acids functions as a general acid catalyst protonating the can be identified by using several different techniques, site-
glycosidic oxygen while the nucleophile residue attacks the directed mutagenesis followed by kinetic analysis of the
sugar anomeric center to form the glycosyl enzyme intermedi- mutants being one of the approaches most used. Briefly, con-
FIGURE 1. Phylogenetic tree of family GH116 using Ward hierarchical clustering distances. The leaves of the tree indicate the organism genus and species
information (e.g. Hom_sapie corresponds to Homo sapiens), the gene or Locus name, the EC activities if characterized experimentally, and a database accession
number. Tree branches were colored according to identified significant subgroups using Dendroscope 2.3 (21).
JULY 2, 2010•VOLUME 285•NUMBER 27
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Characterization of an Archaeal ␤-Glycosidase
served aspartic/glutamic acid residues identified by sequence
analysis are mutated with non-nucleophilic amino acids; the
reduction or even abolition of the enzymatic activity is a strong
indication that the mutation removed catalytic residues. The
activity of the mutants can be chemically rescued in the pres-
ence of external nucleophiles such as sodium azide. The char-
acterization of the anomeric configuration of the glycosyl-azide
products allows the assignment of the mutated residue as the
nucleophile or the acid/base of the reaction (Fig. S2B) (27).
We aligned the amino acid sequence of SSO1353 to eight
other hypothetical proteins identified by BLAST analysis with
an identity Ն22%. The multi-alignment led to the identification
of 15 Asp/Glu residues highly conserved (Fig. S3); among these,
Glu-335, Asp-406, and Asp-426 were invariant and, together
with Asp-458, were mutated by site-directed mutagenesis
obtaining the mutants E335G, D406G, D458G, and D462G.
These SSO1353 mutants were expressed and purified as
described above. During this procedure the proteins showed
identical behavior, suggesting that the mutations did not
affected the stability of the enzymes. These purification steps
yielded proteins with similar concentrations and purification
degrees (Fig. S4). The mutants assayed at 65 °C on 2Np-Glc 40
mM in 50 mM sodium citrate buffer pH 5.5 were completely
inactive indicating that the mutations affected the catalytic
machinery of SSO1353. When 1 M sodium azide was included in
the assay on 40 mM 2Np-Glc we observed the reactivation of the
D462G mutant, which showed a specific activity of 0.5 units
mgϪ1, which is about 7-fold lower than that of the wild type (3.6
units mgϪ1) assayed in the same conditions. Instead, the exter-
nal ion did not modify the specific activity of the wild type and
did not reactivate the E335G, D406G, and D458G mutants.
The mutant D462G was assayed at standard conditions on 40
mM 2Np-Glc in the presence of increasing concentrations of
sodium azide: the maximal activity was observed at 0.5 M
sodium azide (Fig. 4A). At these conditions the kinetic con-
stants were kcat of 0.64 Ϯ 0.1 sϪ1, Km of 16.2 Ϯ 6 mM, and
FIGURE 4. Chemical rescue of the activity of SSO1353 mutants.
(A) Dependence of activity of D462G mutant on different concentrations of
sodium azide. (B) TLC analysis of the reaction mixtures of SSO1353 wild type
and mutants in the absence (lanes 1–6) and in the presence of 0.1 M sodium
azide (lanes 8–13). Standard assays were performed overnight at 65 °C on 40
mM 2NP-Glc by using 11 ␮g of enzyme. Lane 1, blank with no enzyme; lane 2,
wild type; lane 3, E335G mutant; lane 4, D462G; lane 5, D458G; lane 6, D406G;
lane 7, standards (2NP-Glc and Glc); lane 8, blank with no enzyme; lane 9, wild
type; lane 10, E335G; lane 11, D462G; lane 12, D458G; lane 13, D406G; lane 14,
␤-glucosyl-azide standard.
k
cat/Km of 0.04 sϪ1 mMϪ1, showing that D462G maintained a
similar affinity for the substrate, but a specificity constant
10-fold lower than the wild type. Reaction mixtures prepared at
these conditions and containing the wild type and the mutants
were analyzed by TLC after prolonged incubation. The D462G
produced a novel compound, which was observed only in trace
amounts with the other mutants. Instead, the wild type com-
pletely converted the substrate producing transglycosylation group of these inhibitors includes activated 2-deoxy-2-fluoro-
products (Fig. 4B). D462G reaction mixtures in preparative glycosides; the presence of fluorine substituent at C2 slows both
scale allowed the isolation and structural characterization of the glycosylation and the deglycosylation steps of the reaction
this product that was unequivocally identified as ␤-glucosyl by destabilizing the transitions states. The incorporation of
azide. The reactivation in the presence of the external ion and good leaving groups (as 2,4-dinitrophenol or fluoride) acceler-
the anomeric configuration of this product strongly indicate ates the glycosylation step relative to the deglycosylation step of
that Asp-462 is the acid/base of the reaction.
the reaction with the effect that the incubation of the enzyme
Identification of the Catalytic Nucleophile of SSO1353—To with its corresponding 2-deoxy-2-fluoro-glycosides results in a
identify the nucleophile of the reaction we used the mecha- time dependent inactivation with the accumulation of the 2-de-
nism-based inhibition approach in combination with nano- oxy-2-fluoro-glycoside enzyme intermediate. Consequently,
electrospray ionization tandem mass spectrometry (nano-ESI- the nucleophile of the reaction labeled with the inhibitor can be
MS/MS) analysis. Mechanism-based inhibitors are ligands that identified by mass spectrometry (29).
bind to the active site by competing with the substrate of retain-
Time-dependent inactivation of SSO1353 was observed
ing glycosidases and require mechanism-based activation to upon incubation of the enzyme with 2,4DNp-2F-Glc (Fig. 5).
react covalently with the enzyme (for a review see Ref. 28). One Inhibition was incomplete after 4 h of incubation (about 40%)
20698 JOURNAL OF BIOLOGICAL CHEMISTRY
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Characterization of an Archaeal ␤-Glycosidase
the following inactivation parame-
Ϫ4 Ϫ1
ters: ki ϭ (6.9 Ϯ 1.3) ϫ 10
s
;
Ki ϭ 5.5 Ϯ 2.7 mM; ki/Ki ϭ 1.2 ϫ
10Ϫ4 sϪ1 mM
.
Ϫ1
SSO1353 samples incubated in
the absence and the presence of
2.9 mM 2,4DNp-2F-Glc for 2 h
were analyzed by single-stage nano-
ESI-MS to monitor alteration in
the molecular mass of the protein.
Nano-ESI mass spectra of intact
proteins yield series of multiply
charged molecular ion peaks with
40–100 positive charges under the
experimental condition applied.
Molecular mass of SSO1353 in the
absence of inhibitor was measured
to be 75914 Ϯ 6 Da, which is com-
parable to the theoretical average
molecular mass (75907.7 Da) within
experimental error (0.009%) (Fig.
S5A). After inhibition, molecular
ion peaks shift toward higher m/z
values leading a molecular mass of
76077 Ϯ 5 Da and accounting for
163 Ϯ 5.5 Da difference between the
two species (Fig. S5B). To gain fur-
ther evidence and a more detailed
structural insight into the site-di-
rected inhibition, samples were pro-
teolytically digested by pepsin and
the resulting peptide mixtures
were analyzed by nano-HPLC-ESI-
MS/MS in IDA mode. Based on
MS/MS sequence data, 91 and 87%
protein sequence coverage were
respectively obtained in the absence
and in the presence of inhibitor
(Table S1A and Table S1B respec-
tively). Interestingly, in the inhib-
ited sample five peptides compris-
ing residues 332–345, 332–343,
332–347, 332–348, and 332–349
showed considerable decrease in
intensity, and in the same time,
six new peptide molecular ions
appeared in the corresponding
scans (Table 2). The peptide molec-
ular ion pairs corresponding to the
normal and the modified sequences
eluted at the same retention time
and thus they were detected in the
same survey scan in the sample con-
FIGURE 5. Time-dependent inactivation of SSO1353 using 2,4DNp-2F-Glc inhibitor. A plot of % residual
activity versus time at four 2,4DNp-2F-Glc concentrations (F 0.3 mM, Ⅺ 3 mM, E 7 mM, and f 18 mM) is reported
in A with the plot of rate versus time shown as inset. ki, obs were obtained from fitting the curves in A to a single
exponential decay with offset because time-dependent inactivation did not decay to zero, and plotted versus
inhibitor concentration (B), to determine Ki and ki. The reciprocal plot is shown as inset in B.
even at the highest concentration of inhibitor used (18 mM). taining 2,4DNp-2F-Glc. Therefore, they are likely due to in-
This is not surprising as the catalytic competence of GH inac- source ion fragmentation process indicating a relatively labile
tivated by mechanism-based inhibitors, occurring via turnover bond between amino acid and inhibitor. These peptides
of the intermediate via hydrolysis or transglycosylation, has showed an increase of 164.05 Da in molecular mass which cor-
been well documented (28). At these conditions we obtained responds well to the difference between unmodified and 2F-Glc
JULY 2, 2010•VOLUME 285•NUMBER 27
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Characterization of an Archaeal ␤-Glycosidase
TABLE 2
Peptide mapping of SSO1353
Characteristic peptide molecular ions containing residue E at position 335 observed during nano-HPLC-ESI-MS/MS IDA analyses of SSO1353 incubated in the absence and
in the presence of 2,4DNp-2F-Glc inhibitor and digested by pepsin. Peptide sequences (both unmodified and modified) were elucidated by the interpretation of nano-ESI-
MS/MS spectra acquired on the doubly charged (z ϭ 2) precursor ions (Fig. 4). Modification corresponds to the covalent attachment of 2F-Glc ligand at E335 (indicated in
the sequence as E*).
SSO1353
m/z
SSO1353 inhibited
Unmodified and modified
From-To
Mw (calc.)
peptide sequences
Rt
Intensity
Rt
m/z
Intensity
min
cps
min
cps
332–345
332–343
332–347
332–348
332–349
332–340
AIYEAPQNCPYLGT
AIYE*APQNCPYLGT
AIYEAPQNCPYL
1538.708
1703.188
1380.638
1544.689
1708.813
1873.293
1779.85
20.7
770.36
n.d.a
691.33
n.d.
26
21.2
770.36
852.39
691.33
773.35
855.42
937.44
890.93
972.96
942.44
1024.46
n.d.
10
–
–
34
21.8
–
30
–
22.0
23.2
23.7
24.6
25.2
17
AIYE*APQNCPYL
44
AIYEAPQNCPYLGTIG
AIYE*APQNCPYLGTIG
AIYEAPQNCPYLGTIGA
AIYE*APQNCPYLGTIGA
AIYEAPQNCPYLGTIGAC
AIYE*APQNCPYLGTIGAC
AIYEAPQNC
23.2
–
855.42
n.d.
42
–
16
37
23.5
–
890.94
n.d.
280
–
63
1943.898
1882.859
2046.07
240
106
384
–
24.8
–
942.46
n.d.
320
–
1007.447
1171.495
–
n.d.
–
AIYE*APQNC
–
n.d.
–
586.76
94
a n.d., not determined.
modified peptides, and indicated that the ligand was likely (EC 3.2.1.45) activity. As for the other GH families, the retain-
bound to one of the amino acids present in peptide 332–349. To
confirm the site of modification, nano-ESI-MS/MS spectra of
the unmodified/modified peptide pairs were analyzed (Table 2,
Fig. 6). MS/MS spectra show a very similar fragmentation pat-
tern yielding characteristic b-type N-terminal fragment ions at
the low m/z range. Based on these ions, and in particular, on the
appearance of bn* (nՆ3) modified fragment ions at m/z 641.28
(b3*), 712.32 (b4*) and 809.37 (b5*) in the inhibited sample,
modification was unequivocally localized on amino acid Glu-
335. Therefore, it was concluded that Glu-335 is the nucleo-
phile of the reaction of SSO1353. Though we had no direct
evidence from the chemical rescue experiment, we deduce that
the invariant residue Asp-462 is the acid/base of the reaction.
ing reaction mechanism and the catalytic role for the acid/base
and the nucleophile, experimentally determined here, can be
easily extended to all the enzymes belonging to this new family.
Interestingly, all the archaeal putative enzymes belonging to
this new family are from Crenarchaea, and the vast majority
originates from the genus Sulfolobus. A PSI-BLAST search con-
ducted using SSO1353 as the query sequence retrieved (with
low scores) uncharacterized bacterial glycosidases belonging to
families GH15, GH63, and GH78. The latter families include
mainly glucoamylases, ␣-glucosidases, and ␣-L-rhamnosidases,
respectively, and are characterized by an (␣/␣)6 fold. Although
SSO1353 is inactive on ␣-glycosides, this perhaps hints at struc-
tural similarities with enzymes from family GH116. Similar
structural similarity between (␣/␣)6 fold glycoside hydrolase
families degrading both ␣ and ␤ glycosidic bonds have already
been described (30).
The phylogenetic analysis (Fig. 1) shows that sequences from
the new family GH116 can be subdivided into two major
groups, one containing sequences from Archaea and another
one composed mostly of sequences from Cyanobacteria and
Eukaryotes. The archaeal subgroup can be further subdivided
into at least two subgroups, in which, interestingly, all the
archaeal homologs of SSO1353 are present as multiple copies in
the genomes of Caldivirga maquilingensis, S. tokodaii, S. solfa-
taricus, and in the six strains of S. islandicus. The sso1353
homologs with identity Ͼ80%, lie downstream of genes encod-
ing endoglucanases, and, interestingly, in S. solfataricus, this
gene arrangement occurs twice. Presumably, the ␤-glycosidase
activity of SSO1353 is involved, in combination with the
secreted endoglucanase, in the degradation of exogenous glu-
cans used as carbon energy source or, possibly, of the exo-
polysaccharides (EPS) that are produced by S. solfataricus itself
(31–33). Other sso1353 homologs, with identities in the range
21–33% exemplified by sso2674 and sso3039 in S. solfataricus,
flank a putative peptidase or a putative gluconolactonase,
respectively. These two other subgroups of enzymes similar to
SSO1353 are present also in C. maquilingensis, S. tokodaii, and
DISCUSSION
We report here the molecular cloning, the expression in
E. coli and the functional characterization of the product of the
gene sso1353 from the hyperthermophilic archaeon S. solfa-
taricus. The molecular characterization revealed the specificity
of the enzyme for gluco- and -xylosides ␤-bound to hydropho-
bic groups that are hydrolyzed by following a retaining reaction
mechanism. In addition, site-directed mutagenesis of con-
served glutamic/aspartic amino acids and the chemical rescue
of the ␤-glycosidase activity of the mutants, combined with the
use of mechanism based inhibitors and mass spectrometric
analysis, allowed us to identify Asp-462 and Glu-335 as the
acid/base and the nucleophile of the reaction, respectively.
Mutagenic studies also suggested that Asp-406 and Asp-458
residues play a role in catalysis, but elucidation of their function
requires further investigations. Amino acid sequence analysis
showed that SSO1353 shared identity with other hypothetical
proteins and, remarkably, with eukaryotic non-lysosomal bile
acid ␤-glucosidases.
So far, SSO1353 was not assigned to a defined glycoside
hydrolase family in the carbohydrate active enzyme database.
On the basis of our findings we propose that SSO1353 and its
homologs define a new sequence-based family, namely GH116,
which presently includes enzymes with ␤-glucosidases (EC
3.2.1.21), ␤-xylosidases (EC 3.2.1.37), or glucocerebrosidases S. islandicus showing a remarkable identity (Ͼ80%) within each
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Characterization of an Archaeal ␤-Glycosidase
JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 20701

Characterization of an Archaeal ␤-Glycosidase
is inhibited by hydrophobic
deoxynojirimycin (DNJ), and it is
relatively insensitive to CBE (6, 34,
37). In humans, no known patholo-
gies related to defects of GBA2 have
been reported so far while only in
certain mice strains treatments with
NB-DNJ or gba2 gene knock-outs
led to impaired spermatogenesis
(38). However, such deleterious
effects were not observed in other
organisms including humans (6, 39,
40). These studies demonstrate the
importance of understanding at the
molecular level the reaction mecha-
nism and the catalytic machinery of
carbohydrate active enzymes for the
development of specific inhibitors
for bio-medical applications. The
experimental identification of the
catalytic amino acids of SSO1353
reported here, allows to easily iden-
tifying the catalytic machinery of
human GBA2 despite the low
sequence identity (18%) between
the two enzymes. In GBA2 the
nucleophile and the acid/base of the
FIGURE 7. Multi-alignment of SSO1353 with glucosylceramidases. Invariant residues are indicated with “*”;
increased level of conservation is indicated with “:” and “.” The residues corresponding to the nucleophile
Glu-335 and acid/base Asp-462 of SSO1353 are boxed. Pan is XP_001167952.1 from Pan troglodytes; Homo is
NP_065995.1 from Homo sapiens; Ciona is XP_002127036.1 from Ciona intestinalis; Sulfolobus is SSO1353 from
S. solfataricus P2.
subgroup. The observation that the archaeal ␤-glycosidases
reaction are Glu-528 and Asp-678, respectively, which, as
observed in a multi-alignment of putative glucocerebrosidases
from mammals, plants, and tunicates belonging to this new GH
family, are located in two conserved motifs (Fig. 7). In particu-
lar, amino acids with hydrophobic side chains are almost invari-
ant in the position preceding the catalytic glutamic and aspartic
acids in the enzymes belonging to the new family GH116
(Fig. S3 and Fig. 7). Our findings can now allow the planning of
more detailed site-directed mutagenesis studies to better
understand the molecular bases of the substrate recognition of
GBA2.
from this novel GH family can be subgrouped according to their
identity suggests they are present in multiple copies for func-
tional purposes, possibly, for the degradation/modification of
different substrates. A more detailed characterization of these
enzymes is needed to understand their function in vivo.
The other major subdivision of the family is prone to be sub-
divided into several subgroups, one containing sequences from
Cyanobacteria, the other having plant, animal, and mixed bac-
terial subdivisions. One of the members of the animal subgroup
in this newly proposed family is human non-lysosomal glu-
cosylceramidase or ␤-glucosidase 2 (GBA2). This enzyme, pre-
viously described as bile acid ␤-glucosidase (34), is involved in
the catabolism of glucosylceramide, which is then converted to
sphingomyelin (6). Glucocerebrosidases are important en-
zymes involved in the metabolism of gangliosides and globo-
sides. Deficiency of this enzymatic activity is the cause of the
most common lysosomal storage disorder named Gaucher dis-
ease (35) resulting from a defect in the lysosomal acid ␤-gluco-
sidase (GBA1) belonging to GH30. This deficiency leads to the
accumulation of glycosylceramides in certain organs, typically
spleen, kidney, lungs, brain, and bone marrow (36). The finding
that other cell types of Gaucher patients did not show accumu-
lation of glycosylceramides suggested the existence of an alter-
SSO1353 has substrate specificity and inhibitor sensitivity
slightly different from those of GBA2. In fact, the archaeal
enzyme can hydrolyze both aryl ␤-gluco and ␤-xylosides and
it is inhibited with mM affinity by both NB-DNJ and CBE.
Instead, GBA2 is inactive on MU-Xyl and it is relatively
insensitive to CBE (6). These differences presumably reflect
the different function of the two enzymes in vivo: the wider
substrate specificity of the archaeal enzyme might allow to
degrade a variety of substrates ensuring an efficient availabil-
ity of sugars as energy source while GBA2 is involved in a
well defined catabolic pathway. The purification of GBA2 is
made difficult by its instability to detergents precluding its
native catabolic pathway that later was demonstrated to be cat- production in abundant and homogeneous form (6). Instead,
alyzed by GBA2 (6). This enzyme is ubiquitously expressed and robust GBA2 homologs from hyperthermophilic Archaea
it is associated to the cell surface. GBA2 is inactive on MU-Xyl, can be more easily expressed and purified from conventional
FIGURE 6. Identification of the catalytic nucleophile site of SSO1353 by nano-HPLC-ESI mass spectrometry. (A) Nano-ESI mass spectrum of peptides
eluted at 24.3–24.9 min of SSO1353 incubated with 2,4DNp-2F-Glc for 2 h. Doubly charged peptide ions at m/z 942.44 and 1024.46 correspond to the
unmodified and the modified peptide 339–349, respectively. Tandem mass spectra on the unmodified (B) and modified peptides 339–349 (C) reveal modifi-
cation on E4 residue.
20702 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 27•JULY 2, 2010

Characterization of an Archaeal ␤-Glycosidase
7, 47–54
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JULY 2, 2010•VOLUME 285•NUMBER 27
JOURNAL OF BIOLOGICAL CHEMISTRY 20703

Enzymology:
A New Archaeal β-Glycosidase from
Sulfolobus solfataricus: SEEDING A
NOVEL RETAINING β
-GLYCAN-SPECIFIC GLYCOSIDE
HYDROLASE FAMILY ALONG WITH
THE HUMAN NON-LYSOSOMAL
GLUCOSYLCERAMIDASE GBA2
Beatrice Cobucci-Ponzano, Vincenzo Aurilia,
Gennaro Riccio, Bernard Henrissat, Pedro M.
Coutinho, Andrea Strazzulli, Anna Padula,
Maria Michela Corsaro, Giuseppina Pieretti,
Gabriella Pocsfalvi, Immacolata Fiume,
Raffaele Cannio, Mosè Rossi and Marco
Moracci
J. Biol. Chem. 2010, 285:20691-20703.
doi: 10.1074/jbc.M109.086470 originally published online April 28, 2010
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