α-galactosidase/sucrose kinase (AgaSK), a novel bifunctional enzyme from the human microbiome coupling galactosidase and kinase activities

Article abstract of DOI:10.1074/jbc.M111.286039

α-Galactosides are non-digestible carbohydrates widely distributed in plants. They are a potential source of energy in our daily food, and their assimilation by microbiota may play a role in obesity. In the intestinal tract, they are degraded by microbial glycosidases, which are often modular enzymes with catalytic domains linked to carbohydrate-binding modules. Here we introduce a bifunctional enzyme from the human intestinal bacterium Ruminococcus gnavus E1, α-galactosidase/sucrose kinase (AgaSK). Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide-binding motif. Its biochemical characterization showed that AgaSK is able to hydrolyze melibiose and raffinose to galactose and either glucose or sucrose, respectively, and to specifically phosphorylate sucrose on the C6 position of glucose in the presence of ATP. The production of sucrose-6-P directly from raffinose points toward a glycolytic pathway in bacteria, not described so far. The crystal structures of the galactosidase domain in the apo form and in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding and suggesting a cross-talk between the galactose and kinase domains.

Full text of DOI:10.1074/jbc.M111.286039

Enzymology:
α-Galactosidase/Sucrose Kinase (AgaSK), a
Novel Bifunctional Enzyme from the
Human Microbiome Coupling
Galactosidase and Kinase Activities
Laëtitia Bruel, Gerlind Sulzenbacher, Marine
Cervera Tison, Ange Pujol, Cendrine
Nicoletti, Josette Perrier, Anne Galinier,
David Ropartz, Michel Fons, Frédérique
Pompeo and Thierry Giardina
J. Biol. Chem. 2011, 286:40814-40823.
doi: 10.1074/jbc.M111.286039 originally published online September 19, 2011
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 47, pp. 40814–40823, November 25, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
␣-Galactosidase/Sucrose Kinase (AgaSK), a Novel
Bifunctional Enzyme from the Human Microbiome Coupling
□
S
Galactosidase and Kinase Activities*
Received for publication, August 1, 2011, and in revised form, September 3, 2011 Published, JBC Papers in Press, September 20, 2011, DOI 10.1074/jbc.M111.286039
Laëtitia Bruel‡§, Gerlind Sulzenbacher¶, Marine Cervera Tison‡, Ange Pujol‡, Cendrine Nicoletti‡,
Josette Perrier‡, Anne Galinier§, David Ropartzʈ, Michel Fons‡, Frédérique Pompeo§,
and Thierry Giardina‡1
From the ‡Faculté des Sciences et Techniques Saint-Jérôme, Université Paul Cézanne, ISM2/BiosCiences UMR CNRS 6263,
service 342, 13397 Marseille Cedex 20, the ¶Architecture et Fonction des Macromolécules Biologiques UMR CNRS 6098, Université
Aix-Marseille, Campus Luminy, Case 932, F-13288 Marseille Cedex 09, the §IMM/Laboratoire de Chimie Bactérienne, UPR CNRS
9043, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, and the ʈLaboratoire de Spectrométrie de Masse, Plate-forme
Biopolyme`res-Biologie Structurale, INRA UR1268 Biopolymères Interactions Assemblages, Rue de la Géraudière, B.P. 71627,
F-44316 Nantes cedex 3, France
Background: Raffinose, an abundant carbohydrate in plants, is degraded into galactose and sucrose by intestinal microbial
enzymes.
Results: AgaSK is a protein coupling galactosidase and sucrose kinase activity. The structure of the galactosidase domain sheds
light onto substrate recognition.
Conclusion: AgaSK produces sucrose-6-phosphate directly from raffinose.
Significance: Production of sucrose-6-phosphate directly from raffinose points toward a novel glycolytic pathway in
bacteria.
␣-Galactosides are non-digestible carbohydrates widely dis- necessaryforefficientsubstratebindingandsuggestingacross-talk
tributed in plants. They are a potential source of energy in our between the galactose and kinase domains.
daily food, and their assimilation by microbiota may play a role
in obesity. In the intestinal tract, they are degraded by microbial
glycosidases, which are often modular enzymes with catalytic
domains linked to carbohydrate-binding modules. Here we intro-
duce a bifunctional enzyme from the human intestinal bacterium
Ruminococcus gnavus E1, ␣-galactosidase/sucrose kinase (AgaSK).
Sequence analysis showed that AgaSK is composed of two
domains: one closely related to ␣-galactosidases from glycoside
hydrolase family GH36 and the other containing a nucleotide-
binding motif. Its biochemical characterization showed that
AgaSK is able to hydrolyze melibiose and raffinose to galactose and
either glucose or sucrose, respectively, and to specifically phospho-
rylate sucrose on the C6 position of glucose in the presence of ATP.
The production of sucrose-6-P directly from raffinose points
toward a glycolytic pathway in bacteria, not described so far. The
crystal structures of the galactosidase domain in the apo form and
in complex with the product shed light onto the reaction and sub-
strate recognition mechanisms and highlight an oligomeric state
Recent studies have suggested that the human intestinal
microbiome plays an important role in the weight differ-
ences between human beings, namely that it could be signif-
icant in obesity (1–3). Indeed, obesity can be linked to an
alteration of the ratio of Bacteroidetes and Firmicutes in the
microbiome (4). Together, these bacteria represent over 90%
of the bacterial phylotypes in the intestinal tract (5). This
advocates a change in the fermentation pattern and a more
efficient energy extraction from foodstuff operated by Firmi-
cutes (6, 7). In particular, non-digestible carbohydrates can
be metabolized by bacteria residing in the intestinal tract,
and the imbalance of calorie consumption and expenditure
causing obesity can be attributed to variations of the intesti-
nal microbial ecology (8).
Among non-digestible carbohydrates, essentially originat-
ing from plant cell walls, ␣-galactosides are among the most
abundant oligosaccharides. For example, they represent
from 39 to 53 g/kg in soybean meals (9). They consist of
galactose units ␣-(1,6) linked to a mannopyranose backbone
(galactomannans) or ␣-(1,6) linked to glucose (melibiose) or
sucrose (raffinose) or raffinose (stachyose), with sucrose and
raffinose being the most abundant soluble carbohydrates
found in plant cell walls (9). However, there exists no
␣-(1,6)-galactosidase activity in the human intestine
mucosa, and ␣-galactosides are exclusively fermented by
microbial ␣-(1,6)-galactosidases (EC 3.2.1.22). Studies of the
bacterial metabolism of ␣-galactosides (10, 11) have shown
that melibiose and raffinose penetrate in the cell, where they
* This work was supported by the CNRS, the Agence Nationale de la
Recherche (ANR) contract “P-loop proteins,” and the “Ministère de
l’Enseignement Supérieur.”
The atomic coordinates and structure factors (codes 2YFN and 2YFO) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
The nucleotide sequences of the genes reported in this paper have been submit-
tedtotheEMBLNucleotideSequenceDatabase(ENA)withaccessionnumbers
FQ790377–FQ790383.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1–6.
1 To whom correspondence should be addressed. Tel.: 33-4-91-28-84-45; Fax:
33-4-91-28-84-40; E-mail: thierry.giardina@univ-cezanne.fr.
40814 JOURNAL OF BIOLOGICAL CHEMISTRY
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AgaSK, a Bifunctional Galactosidase/Sucrose Kinase
are hydrolyzed into galactose and glucose and into galactose nisms and highlight an oligomeric state necessary for efficient
and sucrose, respectively. Sucrose and galactose are subse- substrate binding and suggesting a cross-talk between the
quently phosphorylated by a kinase and utilized in the gly- galactose and kinase domains.
colytic pathways (12, 13).
EXPERIMENTAL PROCEDURES
According to the sequence-based classification of glycoside
hydrolases (GHs)2 (Carbohydrate-Active Enzymes Database,
Cecal Content Collection and Extraction of RNA—Animal
CAZy, classification) (14), ␣-galactosidases form eukaryotic experiments were performed according to the guidelines of the
organisms are mainly grouped into families GH27 and GH36, French Ethics Committee. Fisher axenic rats came from the
but those from microbial sources are also found in glycoside Animalerie Axe´nique de Micalis (ANAXEM) platform (INRA
hydrolase families GH4, GH57, GH97, and GH110. Iterative Jouy-en-Josas, France) and were reared in Trexler type isolators
database searches have shown that families GH27 and GH36, (La Calhène, Vélizy-Villacoublay, France). They were inocu-
together with families GH31 and GH66, share a common evo- lated with ϳ109 cells of the R. gnavus E1 strain (0.5 ml of fresh
lutionary origin with family GH13 (15). For that matter, fami- culture) by intragastric route. After 1 week, the animals were
lies GH27 and GH36, together with family GH31, form glyco- sacrificed, and the cecal contents were collected. Two hundred
side hydrolase clan D (16). Crystal structures of the clinically mg of fresh material were used for total RNA extraction accord-
important eukaryotic ␣-galactosidase and ␣-N-acetylgalac- ing to the protocol described by Doré et al. (32) and cleaned up
tosaminidase have been reported by the Garman group almost with the RNeasy mini kit (Qiagen). TURBOTM DNase
a decade ago (17, 18), and a wealth of supplementary structural (Ambionꢀ) was used for elimination of genomic DNA as rec-
information for other family GH27 members has become avail- ommended by the supplier.
able later on. On the basis of the high level of sequence homol-
RT-PCR—cDNA synthesis was performed by reverse tran-
ogy between members of family GH27 and GH36, which scription (RT) of 100 ng of RNA primed with 50 ng of random
reflects intrinsically structural homology, Comfort et al. (19) primers. The reaction was carried out at 50 °C for 1 h with the
were able to infer the catalytic residues of Thermotoga maritima SuperScriptTM III reverse transcriptase (Invitrogen), as recom-
TmGalA, as well as to confirm their identity by elegant biochemi- mended by the supplier. The enzyme was inactivated by heating
cal studies. For family GH36, only the crystal structures of T. mari- for 15 min at 70 °C, and 1 ␮l of the mixture was used directly as
tima TmGalA (Protein Data Bank (PDB) 1zy9) and Lactobacillus a template for PCR amplifications. PCR reactions were per-
brevis LbR11 (PDB 3mi6) have been deposited within the Protein formed in a 50-␮l volume by using the Platinumꢀ Taq DNA
Data Bank by Structural Genomics Consortia, and the crystal polymerase (Invitrogen) under the following conditions: 94 °C
structure of Lactobacillus acidophilus NCFM LaMel36A (PDB for 2 min; 30 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for
2xn0) has been solved very recently (20).
1 min; and a final elongation step at 72 °C for 10 min. As a
Family GH36 members originate from bacteria, fungi, and control, additional reactions were performed by using as a tem-
plants and carry out hydrolysis with a net retention of the ano- plate an RT mixture without enzyme, an RT mixture without
meric configuration, which is why many enzymes from this RNA, and chromosomal DNA.
family have been shown to possess transglycosylation activity
Bioinformatic Analysis—Manual validation of the automatic
(21–27). For their ability to hydrolyze ␣-galactosides non-di- annotation of the R. gnavus E1 genome was performed using
gestible by humans and to synthesize diverse oligosaccharides, the MaGe (Magnifying Genomes) web-based interface from
GH36 ␣-galactosidases have potential applications for the pro- Genoscope. Putative transcriptional terminators were pre-
duction of prebiotics. These non-digestible oligosaccharides dicted in silico using the RNAfold program. Functional predic-
are beneficial for human health by their ability to stimulate tions were based on sequence comparisons with the help of the
selectively the growth of microbiota with advantageous physi- sequence databases Profils pour l’Identification Automatique
ological effects on the host (28–30).
du Métabolisme (PRIAM), Clusters of Orthologous Groups of
In this work, we have characterized from a functional point of protein (COG) (www.ncbi.nlm.nih.gov/COG), and Swiss-Prot
view a bifunctional enzyme, hereafter called ␣-galactosidase/ the domain/motif database InterProScan.
sucrose kinase (AgaSK), from Ruminococcus gnavus E1. R. gna-
agaSK Amplification and Cloning into pOPIN E Expression
vus is a strict anaerobic Gram-positive bacterium from the phy- Vector—The agaSK gene was cloned from the genomic DNA of
lum Firmicutes. It belongs to the most common 57 species R. gnavus E1 using DNA polymerase (PrimeSTAR HS DNA
present in 90% of individuals (31). The enzyme AgaSK is com- polymerase, TAKARA BIO Inc.). PCR reaction primers (for-
posed of a GH36 ␣-galactosidase and a kinase domain and is ward, 5Ј-AGGAGATATACCATGGCAATTATATACAAT-
able to release galactose from ␣-galactosides and to phospho- CCA-3Ј; reverse, 5Ј-GTGATGGTGATGTTTCTGTTCATA-
rylate sucrose provided either by raffinose hydrolysis or by the AACACTTCC-3Ј) were used for 30 cycles of denaturation (1
environmental medium. The crystal structures of the galacto- min, 98 °C), annealing (1.5 min, 48 °C), and extension (3 min,
sidase domain in the apo form and in complex with the product 60 °C) in a Mastercyclerꢀ gradient thermocycler (Eppendorf,
shed light onto the reaction and substrate recognition mecha- Hamburg, Germany). The PCR products amplified with the In-
Fusion primers were combined with the expression vector
pOPIN E, kindly provided by Dr. Ray Owens (Oxford Univer-
2 The abbreviations used are: GH, glycoside hydrolase; AgaSK, ␣-galactosid-
ase/sucrose kinase; PNPG, p-nitrophenyl-␣-D-galactopyranoside; ABC,
ATP-binding cassette transporter; Q-TOF, quadrupole time-of-flight; Gal-
DNJ, D-galacto-1-deoxynojirimycin; PTS, phosphotransferase system.
sity), following the manufacturer’s instructions (In-FusionTM
,
Clontech). The recombinant vectors were checked by double-
stranded DNA sequencing (Genome Express, Meylan, France).
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
JOURNAL OF BIOLOGICAL CHEMISTRY 40815

AgaSK, a Bifunctional Galactosidase/Sucrose Kinase
The In-Fusion system led to the production of AgaSK with a relevant mother liquor supplemented with 30% glycerol and
His6 tag at the C terminus.
were flash-frozen in liquid nitrogen. X-ray data for AgaSK-tru,
Expression and Purification of AgaSK—Recombinant AgaSK extending to 2.0 Å resolution, were measured at beam line
was synthesized from Escherichia coli BL21 (DE3) grown in Proxima1 (SOLEIL, Gif-sur-Yvette, France), and a data set at
Luria broth, containing 50 mgꢁliterϪ1 ampicillin, for 4 h. Bacte- 1.45 Å resolution for AgaSK-tru and a data set at 1.35 Å reso-
rial lysis was carried out with a cell disruptor (Constant Systems lution for AgaSK-tru in complex with galactose were subse-
Ltd.) in the binding buffer for affinity chromatography (50 mM quently measured at beam lines ID14-1 and ID29, respectively
sodium phosphate buffer, pH 8, 40 mM imidazole) at 1.37 kbar. (European Synchrotron Radiation Facility (ESRF), Grenoble,
After a centrifugation run at 33,000 ϫ g, the soluble fraction was France). Oscillation images of native data sets were integrated
loaded onto a nickel-nitrilotriacetic acid column, and the with Mosflm (35), and data for the complex of AgaSK-tru with
recombinant protein was eluted with 400 mM imidazole in galactose were processed with XDS (36). All scaling was per-
sodium phosphate buffer, pH 8. Fractions were assayed for formed with SCALA (37).
␣-galactosidase activity, pooled, and dialyzed against McIl-
vaine’s buffer, pH 6. The protein was purified to 95% homoge- (38) using the structure of the ␣-galactosidase LbR11 from
neity, with an overall yield of 20 mg per liter of culture. L. brevis (PDB 3mi6) as the search model. The resulting model
The structure of AgaSK-tru was determined with PHASER
␣-Galactosidase Activity Measurement and Analysis of was rebuilt in an automatic fashion with the program ARP/
Mono- and Disaccharides—␣-Galactosidase activity was wARP (39) and completed manually using the program COOT
assayed by monitoring the absorbance at 410 nm of p-nitrophe- (40). The resulting model was refined with REFMAC (41). For
nol released from 2 mM p-nitrophenyl-␣-D-galactopyranoside the structure of the enzyme-galactose complex, the final native
(PNPG) in McIlvaine’s buffer, pH 6, at 37 °C, with 9.8 nM of model was subjected to rigid body refinement and further
enzyme. One unit of ␣-galactosidase activity was defined as the refined with REFMAC (41).
amount of enzyme required to liberate one ␮mol of p-nitrophe-
The final protein structures encompass residues Ala-2 to
nol per minute at 37 °C. Hydrolysis products formed by recom- Leu-720 and, in addition to galactose in the complex structure,
binant AgaSK with 2 mM melibiose or raffinose in McIlvaine’s also contain several glycerol and polyethylene glycol molecules
buffer, pH 6, at 37 °C for 30 min were detected by thin-layer and a number of sodium and phosphate ions, arising form the
chromatography (TLC, Silica gel 60, Merck) and separated by purification, crystallization, and cryo buffers. The root mean
high performance anion exchange chromatography coupled square deviation between the apo- and sugar-bound forms is
with pulsed amperometric detection (Dionex, Sunnyvale, CA). 0.07 Å for 720 Calpha atoms. The stereochemistry of the model
TLC plates were developed with n-propyl alcohol/water (8:2; was analyzed with MolProbity (42), and only one residue, Ser-
v/v), and the sugars were detected by heating at 140 °C for 5 min 225, was found in the disallowed regions of the Ramachandran
after spraying with 0.1% orcinol dissolved in 5% sulfuric acid plot. This residue is part of a loop region of the ␤-supersand-
(H2SO4).
wich domain contacting the central (␤/␣)8 barrel. The PISA
The release of products from raffinose and melibiose by the server (43) was used for quaternary structure analysis, and fig-
action of AgaSK was analyzed by HPLC coupled with pulsed ures were generated with PYMOL (44). Data collection and
amperometric detection equipped with a Carbo-PacPA-100 refinement statistics are reported in Table 1. The homology
analytical column (250 ϫ 4 mm), a GP40 gradient pump, and an model of the kinase domain was derived with the Phyre server
AS3500 auto-sampler. The protocol used is the same as (45).
described by Cervera Tison et al. (33), except that the A buffer
Fluorescence Measurements—All experiments were per-
was composed of 5 mM NaOAc and 80 mM NaOH. To evaluate formed at 25 Ϯ 0.1 °C using a SAFAS flx Xenius 5117 spectro-
the activity of the AgaSK on ␣-galactosides (melibiose and raf- fluorometer. All spectra were corrected for buffer fluorescence.
finose), the initial slopes of progress curves were used to deter- Fluorescence measurements were routinely carried out for 2 ml
mine the catalytic efficiency (kcat/Km) of the reaction, following of AgaSK at 0.1 ␮M in 50 mM Hepes/KOH buffer, pH 6, con-
the equation of Matsui et al. (34). All assays were carried out in taining 1.0 mM MgCl2. Increasing concentrations of ATP (from
triplicate.
25 to 250 ␮M) were added, and the emission fluorescence was
Crystallization—Crystals of the glycosidase domain of scanned in the range of 310–380 nm, upon excitation at 282
AgaSK (AgaSK-tru) were grown at room temperature by the nm. ATP binding was monitored by the variation of tryptophan
vapor diffusion method in hanging drops consisting of equal intrinsic fluorescence of AgaSK produced by each addition of
volumes (1 ϩ 1 ␮l) of full-length AgaSK solution at 7 mg/ml and ATP. Corrections for the inner filter effect of the ligand were
crystallization solution composed of 14% (w/v) polyethylene performed under the same conditions by using N-acetyl-tryp-
glycol 8000 and 0.1 M Hepes, pH 7.5. Due to spontaneous in situ tophanamide (Sigma). Peak integration was carried out at each
proteolysis, only crystals of the ␣-galactosidase domain could ATP concentration, and the observed changes in fluorescence
be obtained. Crystals of the complex between AgaSK-tru and intensity were used for the calculation of affinity. Data collec-
galactose were obtained by soaking crystals of AgaSK-tru tion was performed in triplicate, and the curve fitting of the data
obtained as described above in a stabilizing solution containing was performed by using the GraFit 4.0 software, as described by
18% (w/v) polyethylene glycol 8000, 0.1 M Hepes, pH 7.5, and 20 Forouhar et al. (46).
mM galactose.
Kinase Activity Measurements—Sugar phosphorylation
Data Collection and Structure Determination—Crystals assays were carried out in a total volume of 10 ␮l. 0.44 ␮M
selected for data collection were rapidly transferred into the AgaSK was incubated for 45 min at 37 °C with 2 mM of the
40816 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 47•NOVEMBER 25, 2011

AgaSK, a Bifunctional Galactosidase/Sucrose Kinase
TABLE 1
Data collection and refinement statistics
AgaSK-tru
AgaSK-tru-galactose complex
Data processing
Beamline
ID14–1
ID29
Space group
I222
I222
Cell dimensions, a, b, c (Å)
Resolution (Å)a
105.06, 111.65, 154.89
36–1.45 (1.53–1.45)
454,881
105.02, 111.60, 155.29
47–1.35 (1.42–1.35)
1,029,088
198,265
No. of observations
No. of unique observations
158,241
b
Rsym
0.077 (0.459)
11.0 (2.2)
0.093 (0.470)
11.8 (3.0)
Mean I/␴(I)
Redundancy
2.9 (2.6)
5.2 (5.1)
Completeness (%)
99.0 (80.2)
10.50
98.6 (98.1)
7.99
B-factor from Wilson plot (Å2)
Refinement
Resolution (Å)
36–1.45 (1.488–1.45)
13.01 (24.0)
47–1.35 (1.385–1.35)
12.43 (19.40)
Rworkc (%)
Rfreed (%)
14.96 (25.8)
14.17 (20.09)
No. of reflections used in refinement
No. of free reflections
r.m.s. deviationse
150,207 (10,384)
8033 (525)
188,194 (13,810)
10,028 (735)
Bond length (Å)
0.013
1.45
0.014
1.01
Bond angles (°)
Chiral volume (Å 3)
Mean B-factors (Å2)
Main/side chain
0.091
0.093
11.03/13.31
22.85/21.25
9.64/12.08
8.29
Galactose
Solvent/other ligands
r.m.s. deviations on B-factors (Å2)
Main chain
25.02/20.60
0.534
1.491
0.589
1.480
Side chain
Ramachandran plot statisticsf
% of residues in favored regions
% of residues in allowed regions
% of outliers
97.35
99.86
0.14
97.63
99.86
0.14
a Throughout the table, the values in parentheses are for the outermost resolution shell.
b
R
sym ϭ ͚hkl(͚ ͉Ihkl-͗Ihkl͉͘)/͚ ͉͗Ihkl͉͘.
hkl
c Rwork ϭ ͚hklʈFoi͉ Ϫ k͉Fcʈ/͚ ͉Fo͉.
hkl
d Rfree is calculated for randomly selected reflections excluded from refinement.
e Root mean square deviation from ideal geometry.
f Ramachandran plot statistics have been calculated with the MolProbity server.
tested sugar, 0.4 mM ATP, 1 mM MgCl2, and 0.05 ␮l of
Electrospray Ionization Quadrupole Time-of-Flight Mass
[␥-33P]ATP (0.5 ␮Ci) in 50 mM Hepes/KOH, pH 6. The phos- Spectrometry—ESI-Q-TOF-MS experiments were performed
phorylation reaction was stopped by incubating the samples for on a Micromass Q-TOF Ultima Global mass spectrometer
5 min at 80 °C and then placing them on ice. 1.5 ␮l of the reac- (Waters, Manchester, UK) in positive and negative ion mode.
tion mixtures were analyzed by TLC using silica gel plates Samples were diluted 1000-fold in 1:1 MeOH/H2O and intro-
(Merck). Plates were developed at room temperature in a satu- duced at a flow rate of 2.5 ␮l/min into the electrospray ion
rated chamber containing n-propyl alcohol and water (8:2, v/v) source (the ion spray voltage was maintained at 3.1 kV). For
as a solvent system. After development, the plates were dried collision-induced dissociation experiments, argon was used as
and exposed overnight to autoradiography. The plates were the collision gas. The collision energy was optimized for each
then sprayed with 0.1% orcinol dissolved in 5% H2SO4 and sample. The fragments were annotated according to the
heated at 150 °C for 15 min for visualization of the sugar spots. nomenclature of Domon and Costello (47). Mass data were
Matrix-assisted Laser Desorption Ionization Time-of-Flight recorded using MassLynx 4 (Waters). The m/z range was
Mass Spectrometry—MALDI-TOF-MS experiments were per- 150–1200.
formed on an Autoflex III TOF/TOF mass spectrometer
RESULTS AND DISCUSSION
(Bruker Daltonics, Bremen, Germany), equipped with a Smart-
beam laser (355 nm, 200 Hz) in positive and negative ionization
R. gnavus E1 Is Able to Metabolize Raffinose in the Intestinal
mode with a reflector detection. The matrix was prepared by Tract—R. gnavus E1 is a Gram-positive bacterium found in the
diluting 50 mg of 2,5-dihydroxybenzoic acid in 490 ␮l of a solu- human alimentary tract. To study its ability to hydrolyze non-
tion of H2O/acetonitrile 1:1. 10 ␮l of N,N-dimethylaniline (pur- digestible carbohydrates, and in particular ␣-linked galacto-
chased from Fisher Scientific) were added to the mixture. Sam- sides, we first tested its ability to grow on melibiose and raffi-
ples were diluted 10-fold and mixed with matrix solution of nose as a sole carbon and energy source. We observed that
N,N-dimethylaniline/2,5-dihydroxybenzoic acid in a 1:1 ratio R. gnavus E1 was able to grow on these two ␣-galactosides, sug-
(v/v). 1 ␮l of the mixture was deposited on a polished steel gesting that the bacterium possesses not only a suitable sugar
MALDI target plate. Mass data were recorded using FlexCon- uptake system but also an intracellular ␣-galactosidase and an
trol and processed using FlexAnalysis (Bruker Daltonics). The efficient metabolic pathway for stepwise degradation of the
m/z range was 180–1000.
released monosaccharides. By activity assays using extracts of
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
JOURNAL OF BIOLOGICAL CHEMISTRY 40817

AgaSK, a Bifunctional Galactosidase/Sucrose Kinase
TABLE 2
R. gnavus E1 grown on several sugars, as well as Tryptone, we
confirmed the presence of a constitutively expressed enzyme,
capable of hydrolyzing PNPG.
Specific activity for hydrolysis of different natural or synthetic mole-
cules by AgaSK
Parameters are calculated from the release of nitrophenolate from nitrophenylated
To find the enzyme(s) and the corresponding gene(s) respon-
sible for this carbon source utilization, an analysis of the R. gna-
vus E1 genome was undertaken. This analysis revealed an
8.5-kb cluster, composed of six genes, potentially involved in
raffinose and sucrose metabolism (supplemental Fig. 1). The
first ORF, agaR, could encode a transcriptional regulator of the
AraC family that is widespread among bacteria and that regu-
lates genes having diverse functions, ranging from carbon
metabolism to stress response (48). The following five genes are
transcribed in the opposite direction of the first ORF. The prod-
ucts of agaE, agaF, and agaG could constitute part of an ABC
importer specific for raffinose. Indeed, they present significant
homologies with the MsmFII, MsmGII, and MsmEII proteins,
the high affinity solute-binding protein, and the transmem-
brane domains of an ABC transporter involved in raffinose
uptake in L. acidophilus NCFM (10). However, the gene encod-
ing for the corresponding nucleotide-binding domain, nec-
essary for an active ABC transporter (49), was not found in
this cluster, but a gene with significant homology to the gene
encoding this nucleotide-binding domain in L. acidophilus
NCFM has been found elsewhere on the genome. The fourth
gene encodes for a putative ␣-galactosidase, hereafter called
AgaSK, and the last gene encodes for a putative sucrose
phosphorylase, an enzyme that converts sucrose to fructose
and glucose-1-phosphate.
substrates, and of galactose from natural substrates.
Substrate
Specific activity
Ϫ1
unitsꢁmg
PNPG
162 Ϯ 43
420 Ϯ 8
Melibiose
Raffinose
231 Ϯ 7
Stachyose
213 Ϯ 19
21 Ϯ 2
Locus bean gum
Guar gum
11 Ϯ 0.6
0.96 Ϯ 0.6
1.80 Ϯ 4.7
0.70 Ϯ 0.3
Xylose ␣-D-PNP
Glucose ␣-D-PNP
ONPGa
a ONPG, o-nitrophenyl-alpha-D-galactopyranoside.
Then, its ability to hydrolyze carbohydrates, and in particular
␣-1,6 glycosidic linkages, was tested toward different molecules
(Table 2). AgaSK efficiently hydrolyzed the ␣-galactosidic link-
age in PNPG, melibiose, raffinose, and stachyose, but the activ-
ity was low toward o-nitrophenyl-␤-D-galactopyranoside,
p-nitrophenyl-␣-D-glucopyranoside, p-nitrophenyl-␣-D-xylo-
pyranoside, locus bean, and guar gum. The release of products
from raffinose and melibiose was analyzed by high performance
anion exchange chromatography coupled with pulsed ampero-
metric detection (supplemental Fig. 4). Galactose, sucrose, and
glucose, but not fructose, were present in the hydrolysates of
raffinose and melibiose. We then determined the enzymatic
parameters of AgaSK, and we observed that the enzyme was
active between pH 4.0 and 6.5 and exhibited highest activity on
PNPG at pH 6.0 and 50 °C, with a specific activity of 150 Ϯ 17
units/mg. Using PNPG as substrate, the pH profile and the
kinetic parameters, Km ϭ 1.37 Ϯ 0.20 mM and kcat/Km ϭ 1850 Ϯ
24 sϪ1.mMϪ1, were similar to those of other GH36 ␣-galactosi-
dases described previously (23–25, 27, 52). All these results
indicate that AgaSK is an ␣-galactosidase specific for ␣-1,6
linkages.
To determine whether AgaSK could be involved in the ␣-ga-
lactosidase activity detected previously, we analyzed whether it
is expressed in vivo. In particular, RT-PCR with mRNA from
the cecal content obtained from germ-free mice associated with
R. gnavus E1 was thus performed. A high level of agaSK tran-
script was detected, suggesting the presence of the enzyme in
the intestinal tract (supplemental Fig. 2).
AgaSK Possesses ␣-Galactosidase Activity—Sequence analy-
sis of AgaSK suggests that it does not contain a signal peptide
and that it is composed of two domains (supplemental Fig. 3).
The N-terminal domain encompasses residues 1–720, and a
BLAST search (50) reveals homology with ␣-galactosidases
belonging to family GH36 of the CAZy classification (14). Sur-
prisingly, the C-terminal domain (residues 721–935) shares
sequence homologies with several kinases acting on small mol-
ecules, like phosphoribulokinases, uridine kinases, or adenylyl
sulfate kinases, and contains a Walker A motif ((G/A)X4GK(T/
S)) (51), typical for nucleotide-binding proteins. The presence
of these two domains indicates that AgaSK would not be a clas-
sical ␣-galactosidase but could be a bifunctional enzyme com-
posed of two domains, each having their own enzymatic
property.
AgaSK Is a Tetrameric Protein—Full-length AgaSK was used
for crystallization trials, but due to spontaneous in situ prote-
olysis, only crystals of a truncated form, AgaSK-tru, corre-
sponding to the ␣-galactosidase domain, could be obtained.
The structure of AgaSK-tru was solved by x-ray crystallography
to 1.45 Å resolution, and the structure of a complex with galac-
tose was obtained at 1.35 Å resolution (Table 1). The three-
dimensional structure of AgaSK-tru discloses three domains:
an N-terminal ␤-supersandwich domain (residues 2–327) fol-
lowed by a canonical (␤/␣)8 barrel (residues 328–626) and a
C-terminal ␤-sheet domain (residues 627–720) (Fig. 1A).
AgaSK-tru forms a tetramer in the crystal structure by applica-
tion of two crystallographic 2-fold axes, which is consistent
with results from size-exclusion chromatography (Fig. 1B). The
association of AgaSK-tru into a tetramer results in a buried
surface of 21,770 Å2 as calculated by the PISA server (43), rep-
resenting 26% of the total tetramer surface area. A virtually
To undertake a thorough biochemical characterization of
AgaSK, recombinant AgaSK was heterologously expressed in
E. coli BL21 (DE3). After purification, the molecular mass of the
protein as determined by SDS-PAGE was about 105 kDa, con- identical tetrameric assembly can be observed in the crystal
sistent with its theoretical mass of 105,510 Da, deduced from structures of L. brevis ␣-galactosidase LbR11 and L. acidophilus
the peptide sequence. The molecular mass of native AgaSK, as NCFM LaMel36A, reflecting 35 and 46% sequence identity,
estimated by size-exclusion chromatography, was about 490 respectively, with AgaSK, whereas T. maritima TmGalA (28%
kDa, indicating that AgaSK is a tetramer in solution.
sequence identity with AgaSK) presents itself as a monomer.
40818 JOURNAL OF BIOLOGICAL CHEMISTRY
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AgaSK, a Bifunctional Galactosidase/Sucrose Kinase
FIGURE 1. Structure of the ␣-galactosidase domain of AgaSK, tetrameric assembly, substrate-binding site, and model for full-length AgaSK. A,
graphic presentation of the ␣-galactosidase domain of AgaSK, with the N-terminal domain in deep blue, the catalytic (␤/␣)8 barrel in light blue, and the
C-terminal in marine blue. Galactose is shown as red sticks. B, tetrameric assembly of AgaSK-tru, with one monomer oriented and colored as in A and the
other monomers shown in pink, green, and yellow hues, respectively. C, the ␣-galactosidase active site. Residues interacting with the product are shown
as sticks, with carbons color-coded as in B. Galactose is shown as sticks with green carbons, and the catalytic residues Asp-478 and Asp-540 are shown as
sticks with yellow carbons. Oxygen and nitrogen atoms are colored in red and blue, respectively. Fo Ϫ Fc electron density calculated prior to incorporation
of galactose into the model and contoured at 4 ␴ is shown in green. D, the association of three monomers within the tetrameric assembly creates a deep
tunnel for substrate binding. The substrate-binding groove is shown in surface representation, with different monomers color-coded as in B. Galactose
is shown as sticks with yellow carbons, and a model of raffinose, derived from a superposition of AgaSK-tru with S. cerevisiae ␣-galactosidase 1, Mel1,
complexed with raffinose (PDB 3lrm), is shown as sticks with green carbons. E, model for full-length AgaSK. The tetrameric assembly is color-coded as in
B. The modeled C-terminal domains are colored in the same hue as the N-terminal domains of each monomer. The polypeptide chain at the junction of
both domains has to bend backwards toward the (␤/␣)8 barrel of an adjacent subunit to avoid steric clashes. This places each nucleotide-binding
domain in proximity to an ␣-galactosidase active site. Galactose and modeled ATP molecules are shown as red sticks. Two junctions between the
galactose and the kinase domain are indicated with red arrows.
The Active Site Exemplifies Substrate Specificity—The struc-
ture of AgaSK-tru in complex with the reaction product galac-
tose allows us to identify unambiguously the catalytic center,
which is located in a shallow pocket within the (␤/␣)8 barrel
domain (Fig. 1C). The identity of the catalytic residues can be
inferred from a superposition with the crystal structure of
T. maritima TmGalA, for which the catalytic nucleophile and
general acid/base had been identified previously to be Asp-327
and Asp-387, respectively (19). In AgaSK-tru, Asp-478 is found
at 3.0 Å from the anomeric center of the sugar and thus well
positioned for nucleophilic attack. Conversely, the general
acid/base Asp-540 is ideally poised to donate a hydrogen to the
sugar O1 hydroxyl, found at a distance of 2.95 Å.
FIGURE 2. Characterization of sugar phosphorylation by AgaSK. A and B,
Asp-478 is held in place by hydrogen bonds with the side
phosphorylation of several sugars was tested in reaction mixtures containing
AgaSK, ATP, MgCl2, [␥-33P]ATP (0.5 ␮Ci), and different sugars. The radioactive
chains of Trp-411 and Asn-480 and with the main chain nitro-
spots were detected on the autoradiogram (A). The sugar spots were
detected with 0.1% orcinol (B). Lanes 1, without AgaSK; lanes 2, with AgaSK;
lanes 3, with raffinose; lanes 4, with melibiose; lanes 5, with sucrose; lanes 6,
gen of Gly-520. The protonation state of Asp-540 is modulated
by interactions with the side chains of His-199 and Trp-537.
with glucose; lanes 7, with fructose; lanes 8, with galactose. C, phosphoryla-
The galactose, found in the standard 4C1 chair conformation, is
tion of raffinose and sucrose was tested in reaction mixtures containing
AgaSK, ATP, and MgCl2. Lane 1, withoutAgaSK; lane 2, with AgaSK; lane3, with
raffinose; lane 4, with raffinose and 10 ␮M Gal-DNJ; lane 5, with raffinose and
100 ␮M Gal-DNJ; lane 6, with sucrose; lane 7, with sucrose and 10 ␮M Gal-DNJ;
tightly enveloped by the enzyme and establishes an extensive
network of hydrogen bonds with surrounding residues, most of
them conserved in family GH36. The OH-2 hydroxyl interacts
lane 8, with sucrose and 100 ␮M Gal-DNJ. The sugar spots were detected with
with the enzyme via the side chain of the acid/base Glu-540 and 0.1% orcinol.
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
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AgaSK, a Bifunctional Galactosidase/Sucrose Kinase
FIGURE 3. Positive ESI-Q-TOF-MS mass spectra and chemical structure of phosphorylated sugar. All reaction products are deduced from the MS/MS data
at m/z 365.107 (sucrose monosodiated form), 445.072 (phosphorylated sucrose monosodiated form), 467.060 (phosphorylated sucrose disodiated form), and
203.018 (galactose monosodiated form). The structural features of phosphorylated sucrose are deduced from an ion m/z 445.059 MS/MS spectrum. A, full scan
MS of the sample from enzymatic reaction (solvent ions are indicated by black triangles). B, MS/MS of the sodiated ion at 445,1 with corresponding annotated
fragments. In two areas of the MS/MS spectrum, the peak intensity was scaled up 24 and 36 times, respectively. C, chemical structure of the phosphorylated
oligosaccharide annotated with corresponding fragments.
the main chain nitrogen of Gly-521. The OH-3 atom hydrogen mutant of Saccharomyces cerevisiae ␣-galactosidase 1, Mel1, in
bonds to Lys-476, which in turn stabilizes the axial OH-4, complex with raffinose (PDB 3lrm), allows us to define struc-
together with the side chains of Trp-411 and Asp-366. The tural elements possibly involved in aglycone binding. Phe-55,
OH-6 hydroxyl contacts the side chains of Asp-367 and Arg- from the N-terminal region of a neighboring subunit, inserts
443. Finally, the indole ring of Trp-336 provides a stacking plat- tightly into a pocket situated below the active site pocket,
form for the sugar. Some family GH36 members exhibit ␣-N- thereby anchoring adjacent loop regions in close proximity to
acetylgalactosaminidase activity, but in AgaSK-tru, the tight the putative ϩ1 and ϩ2 binding sites (53). Residues likely to
interactions of the substrate with the enzyme do not allow for interact with the aglycone part of raffinose are His-199, Asp-
exocyclic substitutions on the galactose moiety.
376, and Arg-443, as well as Asp-52, Arg-66, and the backbone
The Tetrameric Assembly Provides a Platform for Efficient Gly-53-Gly-54 of a neighboring subunit. Most importantly, the
Substrate Binding—Due to the tetrameric assembly of AgaSK- shallowness of the substrate-binding tunnel does not allow
tru, the shallow active site pocket extends toward a deep sub- accommodation of substrates with glycosidic linkages other
strate-binding tunnel formed by loop regions of the central than ␣-1,6, confirming the strict specificity of AgaSK for ␣-1,6-
(␤/␣)8 barrel and loop regions of the N- and C-terminal region linked galactose. Our observations let us conclude that the self-
of different subunits (Fig. 1D). A superposition, based on the association of AgaSK into a tetramer is likely to be physiologi-
position of galactose, of the structure of the AgaSK-tru galac- cally relevant because different subunits join near the active site
tose complex, with the structure of a family GH27 active site to provide a platform for efficient substrate binding.
40820 JOURNAL OF BIOLOGICAL CHEMISTRY
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AgaSK, a Bifunctional Galactosidase/Sucrose Kinase
FIGURE4. SucroseandraffinosepathwaysinR. gnavusE1(adaptedfromReidandAbratt(13)). Twopathwaysforsucrosetransportarewellcharacterized,
the PTS-dependent sucrose system (left) and the non-PTS transport system (middle). The raffinose pathway (right) could be another possibility to provide
sucrose to the bacterium.
AgaSK, an ␣-Galactosidase with a Kinase Activity—The The structural features of phosphorylated sucrose were deduced
presence of the Walker A motif in the C-terminal domain of from an MS/MS spectrum, and a set of specific related fragments
AgaSK (supplemental Fig. 3) suggested that it was also able to was obtained, which allowed to locate the phosphate on position
bind nucleotides. We took advantage of the presence of trypto- C6 of glucose (Fig. 3). Altogether these results show that AgaSK is
phans in the sequence of AgaSK to study nucleotide binding by also a sucrose kinase able to phosphorylate specifically sucrose on
intrinsic fluorescence measurements, and we showed that the C6 position of glucose.
AgaSK binds ATP (Kdapp ϳ61 Ϯ 13 ␮M) (supplemental Fig. 5).
Model of Full-length AgaSK—As all attempts to obtain crys-
We then tested its potential ability to phosphorylate several tals of full-length AgaSK failed, we used the Phyre server (45) to
sugars, especially sugars that are substrates of the ␣-galactosid- build a homology model for the kinase domain based on the
ase domain, in the presence of ATP and [␥-33P]ATP (Fig. 2A). crystal structure of putative fructose transport system kinase
Raffinose and melibiose were tested (lanes 3 and 4), and a new (YP_612366.1) from Silicibacter sp. TM1040 (PDB 3c8u). The
radioactive spot was observed only when raffinose was used as model of the kinase domain could not be joined to the crystal
substrate. Because raffinose is degraded by AgaSK into sucrose structure of AgaSK-tru in an arbitrary fashion as the tetrameric
and galactose, we also tested sucrose, galactose, and the two assembly positions the C-terminus of the galactosidase domain
monosaccharides composing the sucrose unit, glucose and from two adjacent subunits face to face (Fig. 1E). Due to these
fructose (lanes 5–8). We observed the same radioactive spot in steric constraints, the kinase model had to be placed in a way
lane 5 only, showing that sucrose was phosphorylated by such that the polypeptide chain at the galactosidase-kinase
AgaSK. Furthermore, to test whether AgaSK was able to phos- junction bends backwards toward the (␤/␣)8 barrel, thereby
phorylate both sugars, raffinose and sucrose, we added the locating the active sites of the kinase domains near the galacto-
␣-galactosidase inhibitor D-galacto-1-deoxynojirimycin to the sidase active sites of adjacent subunits within the tetramer (Fig.
phosphorylation reaction mix (Fig. 2B, lanes 4, 5, 7, and 8). In 1E). The proximity of the two active sites, as proposed in this
the presence of the inhibitor, phosphorylated sucrose was not pro- model, might assure efficient cross-talk between the two activ-
duced anymore when raffinose was used as substrate (lanes 4 and ities of AgaSK, but the full-length structure of this bifunctional
5 compared with lane 3), but phosphorylated sucrose was still enzyme will be necessary to corroborate our hypothesis.
observed when the substrate was sucrose (lanes 6–8). We there-
Raffinose and Sucrose Uptake and Metabolism—To date, two
fore conclude that in the presence of ATP, AgaSK specifically different pathways for sucrose utilization have been character-
phosphorylates the sucrose unit liberated by the hydrolysis of raf- ized in bacteria (13) (Fig. 4). The first involves a phosphoenol-
finose. This specific sucrose phosphorylation by AgaSK was con- pyruvate-dependent phosphotransferase system (PTS), where
firmedbyMALDI-MS(Fig. 3). Thecontrolsamplespectrumhigh- sucrose is phosphorylated into sucrose-6-phosphate and simul-
lighted raffinose and adducts of ATP (supplemental Fig. 6A), taneously transported into the cell. Sucrose-6-phosphate is
whereas the spectrum of the reaction products formed by AgaSK then cleaved by a hydrolase into glucose-6-phosphate and fruc-
showed two products and adducts of ADP (supplemental Fig. 6B). tose. In the second pathway, a PTS-independent transport sys-
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
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AgaSK, a Bifunctional Galactosidase/Sucrose Kinase
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