Báo cáo khoa học: Crystal structures of the apo form of b-fructofuranosidase from Bifidobacterium longum and its complex with fructose pot

Crystal structures of the apo form of b-fructofuranosidase
from Bifidobacterium longum and its complex with
fructose
Anna Bujacz, Marzena Jedrzejczak-Krzepkowska, Stanislaw Bielecki, Izabela Redzynia and
Grzegorz Bujacz
Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Poland
Introduction
Bifidobacteria are found in human and animal gastro-
intestinal tracts, as well as in the oral cavity and the
vagina [1]. They are among the first bacteria that colo-
nize the sterile digestive system of newborns and they
become predominant micro-organisms ($ 95% of the
colonic flora) in breast-fed infants [2].
In infants, the most frequently detected bifidobacteria
species are Bifidobacterium breve, Bifidobacterium infan-
tis, Bifidobacterium bifidum and Bifidobacterium longum.
The latter one also inhabits the intestines of adults,
despite the fact that the composition of bifidobacterial
species changes and the amount of bifidobacteria
decreases with age [3–6]. They are Gram-positive, nons-
porulating and nonmotile rods, classified as lactic acid
bacteria, due to their ability to anaerobically ferment
carbohydrates [7,8]. These bacteria are known as micro-
Keywords
b-fructofuranosidase;
Bifidobacterium longum; crystal structure;
glycoside hydrolase family GH32; lactic acid
bacteria
Correspondence
A. Bujacz, Institute of Technical
Biochemistry, Faculty of Biotechnology and

Food Sciences, Technical University of Lodz,
Stefanowskiego 4 ⁄ 10, 90-924 Lodz, Poland
Fax: 48 42 6366618
Tel: 48 42 6313494
E-mail:
(Received 13 January 2011, revised 25
February 2011, accepted 15 March 2011)
doi:10.1111/j.1742-4658.2011.08098.x
We solved the 1.8 A
˚
crystal structure of b-fructofuranosidase from Bifido-
bacterium longum KN29.1 – a unique enzyme that allows these probiotic
bacteria to function in the human digestive system. The sequence of b-fruc-
tofuranosidase classifies it as belonging to the glycoside hydrolase family
32 (GH32). GH32 enzymes show a wide range of substrate specificity and
different functions in various organisms. All enzymes from this family
share a similar fold, containing two domains: an N-terminal five-bladed
b-propeller and a C-terminal b-sandwich module. The active site is located
in the centre of the b-propeller domain, in the bottom of a ‘funnel’. The
binding site, )1, responsible for tight fructose binding, is highly conserved
among the GH32 enzymes. Bifidobacterium longum KN29.1 b-fructofura-
nosidase has a 35-residue elongation of the N-terminus containing a five-
turn a-helix, which distinguishes it from the other known members of the
GH32 family. This new structural element could be one of the functional
modifications of the enzyme that allows the bacteria to act in a human
digestive system. We also solved the 1.8 A
˚
crystal structure of the b-fruc-
tofuranosidase complex with b-
D-fructose, a hydrolysis product obtained

by soaking apo crystal in raffinose.
Database
Coordinates and structure factors have been deposited in the Protein Data Bank under acces-
sion codes:
3PIG and 3PIJ
Structured digital abstract
l
b-fructofuranosidase binds to b-fructofuranosidase by x-ray crystallography (View interaction)
Abbreviation
GH32, glycoside hydrolase family 32.
1728 FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS
organisms that are beneficial to their host and probiotic
properties have been shown for some strains. They
prevent the growth of putrefactive and pathogenic
bacteria by producing organic acids, bacteriocins and
bacteriocin-like compounds, e.g. lipophilic molecules
[2,9–13]. Another health-related property ascribed to
these bifidobacteria is decreasing the serum cholesterol
level [14–16]. Bifidobacteria are significant for the well-
being of their hosts by providing protection against colon
cancer [10,17–19], boosting the immune system and by
synthesizing vitamins and amino acids [10,20–22].
As a result of saccharolytic fermentation by bifido-
bacteria, short-chain carboxylic acids, mostly lactic
and acetic with a small percentage of formic acid, are
formed in various quantities, depending on the strain
and substrate [23–25]. Short-chain fatty acids and the
lactic acid stimulate the absorption of sodium, calcium,
magnesium [18,26–28] and water, improving intestinal
peristalsis and providing protection from constipations

[10,28]. Furthermore, all carboxylic acids are absorbed
in the colon and become a source of energy for the
host [2,29–31]. It has been shown that the lactic and
acetic acids can be converted to butyric acid by other
bacteria occupying the colon related to Roseburia and
Eubacterium [32,33]. Butyric acid regulates the prolifer-
ation of cells and is the preferred source of energy for
colonic epithelial cells [17,29,34].
The growth- and ⁄ or health-related activity of bifido-
bacteria is stimulated by poly- and oligosaccharides,
especially by inulin-type fructans [a-d-Glc-(1,2)-(b-d-
Fru)n; 2 £ n < 60] and raffinose [a-d-Gal-(1,6)-a-d-
Glc-(1,2)-b-d-Fru]. Natural sources of inulin-type
fructans are chicory, Jerusalem artichokes, asparagus,
wheat, garlic, onions, leeks, bananas, barley, tomatoes
and honey, whereas raffinose appears naturally in soya
beans and other pulses [28,35,36]. Inulin-type fructans
and raffinose are a common part of the human diet
because of their widespread occurrence in natural
products. These carbohydrates are not lost, despite the
fact that they are not hydrolysed by humans and ani-
mal digestive enzymes. Mammalian genomes do not
encode glycoside hydrolases of family 32 (GH32).
Instead, they use sucrose glucosidase, a different and
unrelated enzyme, to hydrolyse sucrose, allowing these
carbohydrates to reach the intestinal tract, where they
act as bifidogenic factors. Bifidobacteria can metabo-
lize fructans of the inulin type as well as raffinose
because they synthesize b-fructofuranosidase.
The enzyme b-fructofuranosidase (EC 3.2.1.26), also

known as saccharase or invertase, occurs commonly in
bacteria, yeast and plants [37–40]. The typical inverta-
ses catalyse liberated b-d-fructofuranose from the non-
reducing terminus of the b-d-fructofuranosides such as
sucrose, raffinose, fructooligosaccharides or inulin.
Among these carbohydrates, sucrose is the most pre-
ferred, whereas the others are hydrolysed with much
lower efficiency [38], except for the majority of charac-
terized b-fructofuranosidases derived from bifidobacte-
ria. These enzymes display the ability to hydrolyse
fructooligosaccharides faster than sucrose [41–44].
The presented enzyme of probiotic B. longum KN29.1
has never been structurally investigated. The described
crystal structure revealed a new secondary structure ele-
ment – an N-terminal a-helix, which can explain the
adaptation to functioning in the digestive system.
Results and Discussion
Identification of the B. longum KN29.1
b-fructofuranosidase-encoding gene
The gene encoding b-fructofuranosidase from B. lon-
gum KN29.1 was cloned into vector pET303 ⁄ CT-His.
The use of this vector made it possible to obtain
recombinant b-fructofuranosidase, which contained
eight additional amino acids (L, E and 6 · H) at the
C-terminal end of the molecule in comparison with the
native protein.
The amino acid sequence (518 amino acids) of b-fruc-
tofuranosidase from B. longum KN29.1 determined on
the basis of 1557 nucleotides (the complete ORF of
gene b-fructofuranosidase) was compared with other

amino acid sequences deposited in the National Center
for Biotechnology Information [45] using the blast [46]
program. This alignment showed the highest sequence
identity in the range 71–100% to the other bifidobacte-
ria b-fructofuranosidases and below 46% sequence
identity with invertases from less closely related organ-
isms, e.g. Corynebacterium glucuronolyticum ATCC
51867 b-fructofuranosidase (ZP_03919487), Escherichi-
a coli B354 sucrose-6-phosphate hydrolase
(ZP_06654343), Lactobacillus antri DSM 16041
sucrose-6-phosphate hydrolase (ZP_05745308) revealed
46, 38 and 36% sequence identity, respectively. The
alignment of b-fructofuranosidase from B. longum
KN29.1 with the known crystal structures of GH32
showed only 22–28% amino acid sequence identity
(Fig. 1). On the basis of amino acid sequence similarity,
the protein from B. longum KN29.1 has been classified
to family 32 of the glycoside hydrolases as b-fructofura-
nosidase [47,48].
A high level of expression of b-fructofuranosidase
was obtained in E. coli BL21 StarÔDE3 using the
MagicMedia expression medium. The yield of the puri-
fied recombinant protein was $ 420 mg from 1 L of
culture medium. Purification of recombinant protein
A. Bujacz et al. Crystal structure of B. longum b-fructofuranosidase
FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS 1729
3PIG MTDFTPETPVLTPIRDHAAELAKAEAGVAEMAAKRNN-RWYPKYHIASNGGWINDPNGLCFY KGRWHVFYQLHPYGTQWG-PMHWGHV
1UYP LFKPNYHFFPITGWMNDPNGLIFW KGKYHMFYQYNPRKPEWG-NICWGHA
1Y4W FNYDQ-PYRGQYHFSPQKNWMNDPNGLLYH NGTYHLFFQYNPGGIEWG-NISWGHA
3KF5 SIDLSVDTSEYNRPLIHFTPEKGWMNDPNGLFYDKTAKLWHLYFQYNPNATAWGQPLYWGHA

2AC1 NQ-PYRTGFHFQPPKNWMNDPNGPMIY KGIYHLFYQWNPKGAVWG-NIVWAHS
2ADE QIEQ-PYRTGYHFQPPSNWMNDPNGPMLY QGVYHFFYQYNPYAATFGDVIIWGHA
3PIG SSTDMLNWKREPIMFAPSL EQEKDGVFSGSAVIDDN GDLRFYYTGHRWANG HDNTGGDWQVQMTALPDN
1UYP VSDDLVHWRHLPVALYPDD ETHGVFSGSAVEK-D GKMFLVYTYYRDPT HNKGEKETQCVVMSEN
1Y4W ISEDLTHWEEKPVALLARGFGSDVTEMYFSGSAVADVNNTSGFGKDGK TPLVAMYTSYYPVAQTLPSGQTVQEDQQSQSIAYSLD
3KF5 TSNDLVHWDEHEIAIGPEH DNEGIFSGSIVVDHNNTSGFFNSSIDPNQRIVAIYTNNIPD NQTQDIAFSLD
2AC1 TSTDLINWDPHPPAIFPSA PFDINGCWSGSATILPN GKPVILYTGIDPK NQQVQNIAEPKNLS
2ADE VSYDLVNWIHLDPAIYPTQ EADSKSCWSGSATILPG NIPAMLYTGSDSK SRQVQDLAWPKN
3PIG -D ELTSATKQ GMIIDCP T-DKVD-HHYRDPK-VWKTG DTWYMTFGVSSADKRGQMWLFSSKDMVRWEYE-RVLFQHP-
1UYP GLDFVKYDG-NPVISKP PE-EGT HAFRDPK-VNRSN GEWRMVLGSGKDEKIGRVLLYTSDDLFHWKYE-GAIFEDE-
1Y4W -D GLTWTTYDAANPVIPNPPSP-Y-EAEY-QNFRDPF-VFWHDESQKWVVVTSIAE LHKLAIYTSDNLKDWKLV-SE-FGPYN
3KF5 -G GYTFTKYEN-NPVIDVS S NQFRDPK-VFWHEDSNQWIMVVSKSQ EYKIQIFGSANLKNWVLN-SN-FSSG-
2AC1 DP YLREWKKSPL-NPLMAPD AVNGINASSFRDPTTAWLGQD-KKWRVIIGSKI-HRRGLAITYTSKDFLKWEKSPEPLHYD
2ADE -LSDPFLREWVKHPK-NPLITPP EGVKDDCFRDPSTAWLGPD-GVWRIVVGGDR-DNNGMAFLYQSTDFVNWKRYDQPLSSA
3PIG DPDVFMLECPDFFPIKD-K-D GNEKWVIGFSAMGSKPSGFMNRNVSNAGYMIGTWEP-GGEFKPET E
1UYP T TKEIECPDLVRIG EKDILIYSITS TNSVLFSMGELKE GKLNVEK
1Y4W AQGG-VWECPGLVKLPL-DSG NSTKWVITSGLNPG GPPGTVGSGTQYFVGEFDG-T-TFTPDADTVYPGNST
3KF5 Y-YGNQYECPGLIEVPIEN-S DKSKWVMFLAINPG SPLGGSINQYFVGDFDG-F-QFVPDD SQ
2AC1 -DGSGMWECPDFFPVTR-F-GSNGVETSSFGEPNEILKHVLKISLDD TKHDYYTIGTYDRVKDKFVPDN GFK
2ADE -DATGTWECPDFYPVPL-N-STNGLDTSVYG GSVRHVMKAGFE GHDWYTIGTYSPDRENFLPQNGLSLTGSTL
3PIG FRLWDCGHNYYAPQSF-N-VD G-RQIVYGWMSPFV Q PI-PMQDDGWCGQLTLPREITLGD D G-DVVTAP
1UYP RGLLDHGTDFYAAQTF-F-GT D-RVVVIGWLQSWLRTG LY-PTKREGWNGVMSLPRELYVE N N-ELKVKP
1Y4W ANWMDWGPDFYAAAGYNG-LS LNDHVHIGWMNNWQ YGANI-PT YPWRSAMAIPRHMALKT IGSKA-TLVQQP
3KF5 TRFVDIGKDFYAFQTF-SEVE H-GVLGLAWASNWQ YADQV-PT NPWRSSTSLARNYTLRYVHTNAETKQ L-TLIQNP
2AC1 MDGTAPRYDYG-KYYASKTF-F-DSAKN-RRILWGWTNE-S S SVEDDVEKGWSGIQTIPRKIWLDR S GKQLIQWP
2ADE DLRYDYG-QFYASKSF-F-DDAKN-RRVLWAWVPE-T D SQADDIEKGWAGLQSFPRALWIDR N GKQLIQWP
3PIG VAEMEGLRED-TLDHGS-VTLDMDGEEIIA-D D-AEAVEIEMTIDLAA STAERAGLKIHATE
1UYP VDELLALRKR-KVFETA-KS GTFLL-D VKENSYEIVCEFSG EIELRM-GNE
1Y4W QEAWSSISNKRPIYSRTFKTLS-EGSTNTT-T T-GETFKVDLSFSAK SKASTFAIALRASA
3KF5 V-LPDSINVV-DKLKKKNVKLTNKKPIKTN-FKGS-TGLFDFNITFKVLNLNVS PGKTHFDILI-NSQ

2AC1 VREVERLRTKQVKNLRN-KVLKSGSRLEVYGV T-AAQADVEVLFKVRDLEKADVIEPSWTDPQLICSKMNVSVKSGLGPFGLMVLASK
2ADE VEEIEELRQN-QVNLQN-KNLKPGSVLEIHGI A-ASQADVTISFKLEGLKEAEVLDTTLVDPQALCNERGASSRGALGPFGLLAMASK
3PIG D GAYTYVAYDGQ IGRVVVDRQAMAN G DRGYRAAPLTDAELAS GKLDLRVFVDRGSVEVYVNG
1UYP S EEVVITKSR DELIVDTTRSGV S GGEVRKSTV EDEA TNRIRAFLDSCSVEFFFND
1Y4W NF TEQTLVGYDFA KQQIFLDRTHSGD VSFDET FASVYHGPLT PDST GVVKLSIFVDRSSVEVFGGQ
3KF5 ELNSSVDSIKIGFDSS QSSFYIDRH-IPN VEFPRKQFFTDKLAAYL EPLDYDQDLRVFSLYGIVDKNIIELYFND
2AC1 NL EEYTSVYFRIFKARQNSNKYVVLMCSDQSRSSLKEDN DKTTYGAFVD INPH QPLSLRALIDHSVVESFGGK
2ADE DL KEQSAIFFRVFQNQLGRY SVLMCSDLSRSTVRSNI DTTSYGAFVD IDPRS EEISLRNLIDHSIIESFGAG
476
414 475
413356
292 355
291228
156 227
15587
1 86
526
3PIG GHQVLSSYSYASE G PRAIKLVAE-SG SLKVDSLKLHHMKSIGLELEHHHHHH
1UYP -SIAFSFRIHPEN VYNILSVK SNQVKLEVFELENIW L
1Y4W GETTLTAQIFPSS D AVHARLAST-GG TTEDVRADIYKIASTW
3KF5 GTVAMTNTFFMGE GKYPHDIQIVTD-TEEPLFELESVIIRELNK
2AC1 GRACITSRVYPKLAIGK SSHLFAFNYGYQ SVDVLNLNAWSMNSAQI S
2ADE GKTCITSRIYPKFVNNE EAHLFVFNNGTQ NVKISEMSAWSMKNAKF-VVDQS
Fig. 1. Structural alignment [85] of Bifidobacterium longum KN29.1 b-fructofuranosidase (PDB ID: 3PIG) with all known crystal structures of
GH32: Thermotoga maritima b-fructosidase (PDB ID: 1UYP), Arabidopsis thaliana invertase (PDB ID: 2AC1), Cichorium intybus fructan-1-exo-
hydrolase IIa (PDB ID: 2ADE), Aspergillus awamori exo-inulinase (PDB ID: 1Y4W), b-fructofuranosidase from Schwanniomyces occidentalis
(PDB ID: 3KF5). Amino acids are coloured according to the similarity level: red, highest similarity; yellow, one residue difference; green, two;
blue, three. Secondary structure elements are shown as: cylinders, a-helix; arrows, b-strand; line, loop.
Crystal structure of B. longum b-fructofuranosidase A. Bujacz et al.
1730 FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS

was confirmed by SDS ⁄ PAGE analysis. The gel
revealed a single band around 60 kDa, corresponding
to the molecular mass calculated on the basis of the
amino acid sequence. On the basis of the deduced
amino acid sequence, the molecular masses of the
native and recombinant protein were 58 091 and
59 156 Da, respectively. The results of MS of recombi-
nant b-fructofuranosidase are close to the theoretical
value of the molecular mass. A peak of molecular ion
at m ⁄ z = 58 879 Da was observed on the MALDI-
TOF mass spectrum. The calculated isoelectric point
pI was 4.87 and 5.03 for the native and recombinant
protein sequences, respectively. The pI increase was
caused by additional His-tag residues.
Crystallization
The crystals used in this study were grown by the
hanging drop vapour diffusion method at 20 °C. The
initial crystallization conditions were established from
poly(ethylene glycol) ⁄ Ion Screen and Crystal Screen
(Hampton Research, Aliso Viejo, CA, USA). The
b-fructofuranosidase crystallized from medium relative
molecular mass poly(ethyleneglycol) in the presence of
a number of salts, with the best results obtained in
various concentrations of ammonium chloride. The
enzyme was very sensitive to acidity changes in crystal-
lization conditions and formed different crystal forms
depending on the pH value.
Several crystal forms of b-fructofuranosidase were
obtained. Needle-shaped crystals were probably
monoclinic and diffracted up to only 6 A

˚
resolution.
We also obtained orthorhombic crystals that diffract-
ed to 3.8 A
˚
, as well as well-diffracting hexagonal
crystals (better than 1.5 A
˚
). Although the latter grew
as beautiful, large hexagonal plates (0.3 · 0.3 ·
0.15 mm), they were severely twinned and disordered
in the c direction, making it impossible to index the
diffraction patterns. After optimization of the crystal-
lization conditions, we finally obtained trigonal crys-
tals diffracting to 1.8 A
˚
resolution. Crystals of
rhombohedral shape with dimensions of 0.1 · 0.1
· 0.15 mm were used for data collection of the apo
enzyme and its complex with fructose, the product of
hydrolysis. These data were utilized for successful
structure determination.
X-ray diffraction data were collected for the crystals
of the apo form of the enzyme and for the complex
with fructose using synchrotron radiation generated at
the BESSY (Berlin, Germany) beamlines BL_14.2 and
BL_14.1. Both crystals diffracted to around 1.8 A
˚
res-
olution and were isomorphous in the trigonal space

group P3
1
21. In both cases the diffraction data were
indexed, integrated and scaled with HKL2000 [49].
Table 1 shows the data collection and processing
statistics.
Structure determination and refinement
The crystal structure of b-fructofuranosidase from
B. longum KN29.1 was solved by molecular replace-
ment using a model created from two similar proteins:
invertase from Thermotoga maritima (PDB ID: 1W2T)
[50] and exo-inulinase from Aspergillus awamori (PDB
ID: 1Y4W) [51].
In order to create the search model for molecular
replacement, using the program clustalw [52] we
aligned the sequences representing known crystal struc-
tures of the GH32 family members to which our
enzyme should be similar. Unfortunately, the sequence
identity with B. longum KN29.1 ranged from only
22% to 28%. The search model was constructed based
on the largest sequence similarity and the smallest dif-
ference in length of the loop connecting the secondary
structure elements from two crystal structures. We
selected the catalytic part of the invertase (PDB ID:
1W2T) [50] b-propeller domain as a model of the cata-
lytic domain, because high similarity was observed
only in this region. This protein has a much smaller
b-sandwich domain with shorter loops, typical for
enzymes adapted for high temperature. The b-sand-
wich domain from exo-inulinase (PDB ID: 1Y4W) [51]

had a small difference in loop lengths and could be
used as a second part of the search model. Both struc-
tures were superimposed and the final model was built
by cutting off the side-chains and leaving the part
common to the protein sequence of the target protein.
This model was used to solve the crystal structure of
the native b-fructofuranosidase with the phaser [53]
program in a number of steps, changing various
parameters in molecular replacement. The refinement
of the distant model was very difficult and only the
phases from molecular replacement were used to build
the target structure in the arp ⁄ warp program [54]. The
final model was built in the program coot [55] using
advance options for adding missing fragments and fit-
ting them to the electron density maps.
The crystal structure of the complex of b-fructofura-
nosidase with fructose was solved by molecular
replacement using the crystal structure of the native
enzyme as the search model. The solution gave the
model in a different orientation, which indicated that
both data sets have inconsistent indexing. Diffraction
data of the complex were reindexed and rescaled to the
same cell system as the apo form. Next, the model of
the apo crystal structure was refined against complex
A. Bujacz et al. Crystal structure of B. longum b-fructofuranosidase
FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS 1731
data by a rigid body procedure. Despite the fact that
the crystal of b-fructofuranosidase was soaked in raffi-
nose, a fructose molecule was found bound inside the
b-propeller domain. That observation indicated that

the crystallized enzyme performed hydrolysis of the
2,1-glycosidic bond of raffinose, trapping the active site
pocket fructose, the product of the reaction.
General description of the b-fructofuranosidase
crystal structure
The enzyme consists of two domains: the N-terminal
five-blade b-propeller domain that includes the cata-
lytic site, as well as the b-sandwich C-terminal domain
(Fig. 2A). The b-sheets creating the b-propeller are
located radially and pseudosymmetrically around the
central axis. Each of them consists of four antiparallel
b-strands, in a typical ‘W’ assembly, connected by
loops. Some of the loops are short and create hairpin
turns, the others are extended, and two (loop 2–3 in
blade II and loop 2–3 in blade V) have helical turns
on the top. The latter loop is greatly extended and
also forms an additional b-strand interacting with
strand 1 of blade I (Fig. 2B). The interblade loop
ibL
II–III
is also relatively long and has a single helical
turn on the top. The overall shape of this domain is
cylindrical with the ‘funnel’ shape channel inside of it,
which resembles the shape of a fishtrap. The active
site is located at the bottom of the axial funnel cre-
ated by the interblade loops and the loops between
b-strand 2–3 of each blade. The second funnel on the
opposite side of the cylinder is shallower and is
formed by loops 2–3 and 3–4 connecting antiparallel
b-strands. The first strand in each blade is closest to

the central axial channel, running from the deepest
part of it to the opposite side of the molecule. Each
fourth strand, located on the external surface of this
module, is connected with the first strand of the next
blade by an interblade loop. This domain contains
one additional structural element, an a-helix, sticking
to the surface of the cylinder in the area of blades I
and II. Such an extension is reported for the first time
for an enzyme belonging to the GH32 family. All
b-fructofuranosidases from bifidobacteria, deposited in
Table 1. Data collection and structure refinement statistics.
Native 3PIG Complex BFF ⁄ fructose 3PIJ
Data collection
Radiation source BL.14.2. BESSY ⁄ Berlin BL.14.1. BESSY ⁄ Berlin
Wavelength (A
˚
) 0.9184 1.0000
Temperature of measurements (K) 100 100
Space group P3
1
21 P3
1
21
Cell parameters (A
˚
) a = b = 87.1, c = 223.9
a = b = 90.0, c = 120
a = b = 86.8, c = 223.9
a = b = 90.0,c = 120
Resolution range (A

˚
) 40.0–1.87 (1.94–1.87)
a
40–1.80 (1.86–1.80)
Reflections collected 499 046 438 938
Unique reflections 81 348 84 167
Completeness (%) 98.6 (87.3) 92.2 (56.0)
Redundancy 6.1 (2.8) 5.2 (1.4)
<I> ⁄ <rI> 19.2 (2.0) 13.8 (1.95)
R
int
b
0.090 (0.445) 0.097 (0.291)
Refinement
No. of reflections in working ⁄ test set 77200 ⁄ 4078 79944 ⁄ 4216
R ⁄ R
free
(%)
c
14.90 ⁄ 19.99 15.30 ⁄ 20.80
Number of atoms (protein ⁄ solvent ⁄ Cl ⁄ fructose) 8365 ⁄ 1167 ⁄ 6 ⁄ 0 8388 ⁄ 989 ⁄ 5 ⁄ 24
rms deviations from ideal
Bond lengths (A
˚
) 0.020 0.020
Bond angles (°) 1.69 1.73
<B> (A
˚
2
) 24.47 24.93

Residues in Ramachandran plot (%)
Most favoured regions 87.1 88.3
Allowed regions 11.9 10.8
Generously allowed 0.7 0.7
Disallowed 0.3 0.2
a
Values in parentheses correspond to the last resolution shell.
b
R
int
=
P
h
P
j
|I
hj
) <I
h
>| ⁄
P
h
P
j
I
hj
, where I
hj
is the intensity of observation j
of reflection h.

c
R =
P
h
||F
o
| ) |F
c
|| ⁄
P
h
|F
o
| for all reflections, where F
o
and F
c
are observed and calculated structure factors, respectively.
R
free
is calculated analogously for the test reflections, randomly selected and excluded from the refinement.
Crystal structure of B. longum b-fructofuranosidase A. Bujacz et al.
1732 FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS
GenBank, possess this additional N-terminal element
[42]. Because it is a feature typical of probiotic bacte-
ria adapted to live in a human digestive system, this
helix may allow the enzyme to act in such an environ-
ment, but the precise function of that element is so
far unknown.
The C-terminal domain has a b-sandwich architec-

ture and is composed of two six-stranded antiparallel
b-sheets. In both b-sheets the sixth strand is located
between strands 1 and 2. Although the antiparallel pat-
tern of both b-sheets is preserved, the network of loops
connecting the strands is complicated; six loops con-
nect b-strands from both b-sheets (1–1¢ 1¢–2 2–2¢ 5¢–3
5–6¢ 6¢–6) and five loops connect b-strands within the
same b-sheet (2¢–3¢ 3¢–4¢ 4¢–5¢ 3–4 4–5) (Fig. 2B). The
loop 5¢–3 forms a one-and-a-half-turn helix on the top.
The interface between both b-sheets is formed by the
predominately hydrophobic residues. The two domains
are connected by a hinge region with a two-turn helix.
Interactions between the b-propeller and b-sandwich
domains involve contacts of IV and V blades and
b-strands (1–6) from the internal b-sheet of the b-sand-
wich domain. The last four amino acids of the native
sequence at the C-terminus and the His-tag protrude
from the b-sandwich domain and interact with the N-
terminal b-propeller domain on the side surface of
blade I, which can be described as a second hinge
region.
Active site of b-fructofuranosidase
The active site forms a clearly delineated pocket, which
is fully consistent with the exo mode of hydrolysing
inulin by this enzyme (Fig. 3A). It is located in the
funnel created by the five blades of the b-propeller on
its axis (Fig. 2A). The first strand of each blade creates
an internal surface of the ‘whirl’ and all five strands go
from the substrate entrance direction, ending on the
opposite site of the cylindrical N-terminal domain. The

catalytically active residues, Asp54 and Glu235, are
located on the first strand on blades I and IV (Fig. 3B,
C). The distance between the carbonyl oxygens of
these two residues, 5.7 A
˚
, proves that the enzyme
belongs to the group of hydrolases acting with reten-
tion of substrate configuration. The other residues
involved in interactions with the fructofuranoside ring
are Asn53, Gln70, Trp78, Ser114, Arg180 and Asp181
(Fig. 3C). The interactions of the blades in the axis
area are of polar character. The water channel in the
apo form of the enzyme runs along the axis of the
b-propeller through the whole domain and is only
blocked by Cys236 in the area of the subsite )1 bind-
ing the fructose molecule.
A number of structures of the complexes with prod-
uct (fructose), substrates and inhibitors have been
reported for the GH32 family enzymes. All complexes
exhibited a very similar position for the terminal
fructosyl moiety at the )1 subsite [50,51,56–58]. The
residues surrounding the )1 binding site are highly
conserved and the hydrogen bonds with fructose are
also maintained (Table 2). The other binding places
located in the entrance of the ‘funnel’, +1, +2 and
+3, show lower sequence homology and interact with
the substrate less tightly.
Comparison of apo structure and complex
with fructose
The rms deviation aligned pairs of Ca atoms

between the apo crystal structure and the complex
with fructose are 0.16 and 0.12 A
˚
for molecules A
and B, respectively. A single fructofuranose molecule
is clearly visible in the difference electron density
A
B
Fig. 2. Structure of Bifidobacterium longum KN29.1 b-fructofura-
nosidase: (A) ribbon diagram, (B) topology scheme.
A. Bujacz et al. Crystal structure of B. longum b-fructofuranosidase
FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS 1733
maps in both monomers (Fig. 3B). The conformation
of the active site residues in both structures is practi-
cally unchanged after fructofuranose binding that
involves Asn53, Asp54, Gln70, Asp181 and Glu235
(Fig. 3C). The movement of the side-chains of amino
acids surrounding the fructose molecule is < 0.4 A
˚
,
with the sole exception of the oxygen Oe2 from
Glu235, which moves by 0.85 A
˚
due to formation of
a hydrogen bond with the fructofuranose molecule.
Six water molecules occupy the )1 binding site in the
native enzyme.
The catalytic mechanism of hydrolysis
The fructose present in the crystal structure of the
complex is the result of hydrolysis of raffinose. The

conformation of the sugar observed in the electron
density map indicated a b-d-fructofuranoside ring
(Fig. 3B). This observation proves that the enzyme
operates with retention of configuration, described for
the other GH32 family members [38]. The catalytic
mechanism involves two steps, in which the covalent
fructosyl–enzyme intermediate is formed and is hydro-
lysed via oxocarbenium ion-like transition states
(Fig. 4). In the first step of the enzymatic reaction, a
nucleophilic attack is performed on the anomeric car-
bon of the sugar substrate by the carboxylate of the
Asp54 acting as the primary nucleophile, forming a
covalent fructose–enzyme intermediate. A proton is
A
B
C
Table 2. Hydrogen bonds between fructose and b-fructofuranosi-
dase in the active site (PDB ID: 3PIJ).
Fructose Enzyme
Hydrogen bonds
Chain A Chain B
O6 Asn53 Nd2 2.96 2.93
Trp78 Ne1 3.06 3.01
Gln70 Oe1 2.69 2.65
O1 Asp54 Od1 2.73 2.82
O3 Glu235 Oe2 2.86 3.14
Arg180 Ne 2.85 2.73
Asp181 Od2 2.54 2.58
O2 Glu235 Oe2 2.66 2.72
O4 Asp181 Od1 2.64 2.67

Ser114 N 3.09 2.97
O5 Asp54 Od2 3.08 3.15
Asn53 Nd2 3.22 3.22
Fig. 3. Active site of b-fructofuranosidase from Bifidobacterium lon-
gum: (A) potential surface with fructose in the active site ‘funnel’,
(B) electron density map for fructose and surrounding side-chains,
(C) hydrogen bonds with numbering of residues interacting with
fructose (the hydrogen bond lengths are in Table 2).
Crystal structure of B. longum b-fructofuranosidase A. Bujacz et al.
1734 FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS
donated by Glu235 to the glycosyl leaving group. In
the second step (deglycosylation), a water molecule
guided by Glu235 performs a nucleophilic attack on
the anomeric carbon of fructose. The leaving group is
carboxylate of Asp54.
Dimerization
The asymmetric units of the crystals of both the apo
and complexed form of the enzyme consist of dimers
with noncrystallographic two-fold symmetry (Fig. 5).
The two-fold axis of each dimer is perpendicular to the
crystallographic three-fold axis and is approximately
collinear with the crystallographic two-fold axis. How-
ever, its height does not correspond to the one-third of
the unit cell dimension. The dimerization interface is
located at the b-sandwich domain. The interactions in
this interface are mostly polar and involve the loops
L
2¢–3¢
(Thr412-Tyr417) and L
4¢–5¢

(Gln434-Arg441).
These two loops interact with the tips of four other
loops: L
1–1¢
(Gly378-Asp375), L
2¢–2
(Ala402-Arg404),
L
3¢–4¢
(Gly424-Gly427) and L
6–6¢
(Glu498-Gly500). The
buried surface area of the dimer interface is 5525 A
˚
2
per monomer, suggesting that dimerization is a result
of crystal packing, especially as gel filtration experi-
ments show that monomers are present in solution.
The monomeric form of the investigated protein was
confirmed by dynamic light scattering, which revealed
that b-fructofuranosidase in the solution is unambigu-
ously in the monomeric stage (99.9%).
A recently released structure of b-fructofuranosidase
from Schwanniomyces occidentalis (PDB ID: 3KF3)
[59] and (PDB ID: 3KF5) [59] shows a different type
of dimer in which the C-terminal domain interacts
with the b-propeller domain from the other monomer
[59,60]. Although a different way of assembly, this
dimer has comparable surface interface area 5283 A
˚

2
per monomer. This compact dimer is probably typical
for yeast b-fructofuranosidases. Biological data show a
similar type of dimerization of the same enzyme from
Saccharomyces cerevisiae [61].
Structural comparison of GH32 from bacteria,
fungi and plants
Five crystal structures of GH32 have been published
to date. They include an extracellular invertase from
T. maritima [62], exo-inulinase from A. awamori [51],
fructan-1-exohydrolase from Cichorium intybus [72],
invertase from S. occidentalis [59] and cell wall invert-
ase from Arabidopsis thaliana [63,71]. The b-fructofura-
nosidase from B. longum represents only the second
crystal structure of GH32 from a bacterial source.
GH32 from different groups of organisms (bacteria,
fungi and plants) have different biological function.
For the majority of bacteria the GH32 enzymes hydro-
lyse polyfructans to provide monosaccharides as
a source of energy. This is especially important for
probiotic bacteria colonizing the digestive system,
Fig. 4. Hydrolysis of raffinose by b-fructofuranosidase with the double displacement mechanism of reaction leading to retention of configura-
tion on the anomeric carbon of the b-(2,1)-
D-fructofuranose.
Fig. 5. Dimer of b-fructofuranosidase from Bifidobacterium longum,
the noncrystallographic two-fold axis is approximately perpendicular
to the plane of the picture.
A. Bujacz et al. Crystal structure of B. longum b-fructofuranosidase
FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS 1735
where they can utilize unhydrolysed polysaccharides.

For fungi, polysaccharide hydrolysis is important for
colony development as well as an energy source,
whereas plants use these enzymes predominantly for
processing storage material. This enzyme may play a
role in the plant pathogen response because its highest
expression level can be induced after infection [64,65].
In plants, fructan exohydrolases might evolve from
catalytically more restricted invertases [66].
The protein sequence comparison [52] (Fig. 1) shows
that GH32 from bacteria, fungi and plants exhibit rela-
tively low sequence identity. Superposition of the
structures of the members of the GH32 family reveals
similarity in the secondary structure area, but shows
differences in the length and conformation of the loops
(Fig. 6). Table 3 shows rms deviations and percentage
identity between aligned pairs of corresponding Ca
atoms. The plant enzymes are the longest, whereas
b-fructofuranosidases from yeast and B. longum are 20
residues shorter. An exception is invertase from
T. maritima, which is 100 residues shorter, probably
due to its adaptation to high temperature. The
sequence similarity is higher between GH32 from bac-
teria, fungi and plants (Table 3). However, the slightly
higher sequence similarity is not reflected in structural
similarity. A structure alignment shows that rms devia-
tion of aligned pairs of Ca atoms is usually above 2 A
˚
.
The exception is high sequence and structural similar-
ity between plant enzymes, which shows 53% of

sequence identity and 1.2 A
˚
deviation between Ca
pairs. Additionally, these enzymes show very similar
distribution of the secondary structure elements, 527
residues from 537 Ca atoms can be superposed on
their analogues in related enzymes.
Enzymatic characterization of B. longum KN29.1
b-fructofuranosidase
All enzymatic properties were checked for the enzyme
naturally isolated from B. longum (without point muta-
tion) and later for the enzyme overexpressed in E. coli.
Enzymatic properties of the recombinant protein were
identical to those of the native B. longum KN29.1
b-fructofuranosidase, which suggests that the point
mutation does not affect the enzymatic activity. More
detailed information about the enzymatic properties of
both the native and the recombinant protein can be
found elsewhere (Jedrzejczak-Krzepkowska et al., sub-
mitted). The substrates most preferred by the enzyme
in hydrolysis are short-chain inulin-type fructans.
Bifidobacterium longum KN29.1 b-fructofuranosidase
showed a higher affinity for b-(2,1) linkages between
fructosyl units in short-chain inulin-type fructans than
between glucosyl and fructosyl units. This enzyme was
also able to hydrolyse inulin, but substrate specificity
decreased with the increasing degrees of polymeriza-
tion of the inulin-type fructans (Table 4). Bifidobacteri-
um longum KN29.1 b-fructofuranosidase, like most of
the characterized b-fructofuranosidases from bifidobac-

teria, belongs to the group of unique invertases
[41–44]. It is known that invertase and inulinase are
distinguished by the S ⁄ I value (ratio of sucrose to inu-
linase activity). The S ⁄ I value for a typical invertase is
high (>1600), whereas for a typical inulinase it is low
(commonly £10). The S ⁄ I ratio for B. longum KN29.1
b-fructofuranosidase is 1.7, indicating that this enzyme
can be considered to be an inulinase [67].
Bifidobacterium longum KN29.1 b-fructofuranosidase
shows different substrate specificity and function in
comparison with other enzymes from the family GH32
for which crystal structures are known. On the one
hand, B. longum KN29.1 b
-fructofuranosidase prefers
kestose, nystose-like S. occidentalis invertase and C. in-
tybus fructan-1-exohydrolase. On the other hand, this
enzyme can hydrolyse sucrose, in contrast to C. intybus
fructan-1-exohydrolase (Table 4). Schwanniomyces occi-
dentalis invertase produces several fructooligosaccha-
rides by transfructosylation [68] and shows higher
substrate affinity to sucrose and raffinose than B. lon-
gum KN29.1 b-fructofuranosidase, six-fold (K
M
= 31.4
mm) and 49-fold (K
M
= 64.6 mm), respectively. More-
over, T. maritima shows a higher affinity for raffinose
than for sucrose. The low affinity of B. longum KN29.1
b-fructofuranosidase to raffinose is probably affected by

the presence of a-(1,6) glycosidic bonds between glucose
and galactose next to the b-(2,1)-linkages between fruc-
tose and glucose in this trisaccharide. It was found that
unlike B. longum KN29.1 b-fructofuranosidase, the
invertase from A. thaliana [69] has a higher affinity for
sucrose than for 1-kestose (Table 5).
b-Fructofuranosidase B. longum KN29.1 is not able
to hydrolyse substrates such as levan polysaccharide
consisting of fructose units linked by b-(2,6)-glycosidic
bonds, whereas A. thaliana invertase, C. intybus
fructan-1-exohydrolase, as well as A. awamori exo-
inulinase [70] are capable of degrading levan via an
exo-type cleavage, releasing terminal fructosyl residues.
Substrate specificity of the GH32 family enzymes
Substrate specificity of GH32 enzymes is regulated on
three levels. The first one is based on the shape and
charge of the active site pocket (Fig. 3A), determined
by the conformation, length and sequence of the inter-
blade loops (ibL
I–V
) and the loops L
2–3
between
b-strands 2 and 3 in each blade. This structural ele-
Crystal structure of B. longum b-fructofuranosidase A. Bujacz et al.
1736 FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS
(1) (2)
A
B
(3)

(4) (5) (6)
Fig. 6. Structural comparison of GH32 enzymes. (A) Stereo view of aligned crystal structures of GH32: bacterial: (PDB ID: 3PIG) (Bifidobac-
terium longum) (1) dark violet, (PDB ID: 1UYP) (Thermotoga maritima) (2) light violet; plant: (PDB ID: 2AC1) (Arabidopsis thaliana) (3) dark
green, (PDB ID: 2ADE) (Cichorium intybus) (4) light green; fungal: (PDB ID: 1Y4W) (Aspergillus awamori) (5) orange; yeast: (PDB ID: 3KF5)
(Schwanniomyces occidentalis) (6) yellow. (B) Electrostatic potential of the enzymes oriented in such a way that the active site is in front of
the viewer. The numbering (1–6) corresponds to the enzymes listed in (A).
A. Bujacz et al. Crystal structure of B. longum b-fructofuranosidase
FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS 1737
ment differs in its length and sequence in all known
crystal structures of particular organisms, leading to
changes in the shape and in charge distribution. The
loop L
2–3 ⁄ I
in blade I has a very similar shape in all
compared enzymes. Interblade loop ibL
I–II
in plant
and bifidobacteria enzymes is bent to the edge of the
funnel and does not block the entrance to the active
site (Fig. 6A). In both the fungal enzymes and one
bacterial (T. maritima), this loop is straight and nar-
rows the active site entrance. The loop L
2–3 ⁄ II
in blade
II in both plant enzymes and one yeast ( S. occidentalis)
is short and does not block the active site. In both
bacterial enzymes and one fungal (A. awamori) this
loop is long, creating a kind of flap and modulating
access to the active site. A corresponding loop in the
exo-inulinase from A. awamori (PDB ID: 1Y4W) is

long and in open conformation, and consists of amino
acids having short side-chains. In the thermophilic bac-
teria, enzyme (PDB ID: 1W2T) has shorter loops with
‘straight’ conformation of the polar amino acids on
the top (Thr96, His97, Asn98, Lys99). This loop is
slightly shorter in the b-fructofuranosidase (PDB ID:
3PIG) than the one present in the fungal enzyme
(PDB ID: 1Y4W). By comparison to (PDB ID: 1UYP)
(T. maritima), the loop is in a ‘semi-closed’ conforma-
tion and makes a wide turn, partially covering the
active site. It has polar residues on the top, but also
bulky Trp134 located near the active site. In the plant
enzymes, this loop is very short and has the conserved
Lys104 on its top. Interblade loop ibL
II–III
has a simi-
lar length in all compared enzymes. Although the
loop’s shape is different in all GH32 enzymes, it does
not significantly influence the access to the active site.
The loop L
2–3 ⁄ III
is shorter in yeast enzymes and longer
in plant and bacterial ones, unlike the interblade loop
ibL
III–IV
, which is shorter in plant and bacteria and
elongated in yeast enzymes. The loop L
2–3 ⁄ IV
is short in
plant and bacterial T. maritima (PDB ID: 1UYP),

slightly longer in the fungi, and quite extended in bifi-
dobacteria enzymes. Interblade loop ibL
IV–V
is short,
has the same shape in all investigated structures and
contacts the b-sandwich domain. The loop L
2–3 ⁄ V
is
very long in all structures and forms a helical turn on
the top, interacting with b-strand 1 of blade I. In the
sandwich domain, loops L
2–2¢s
and L
3¢–4¢s
in plant
enzymes are 26 and 10 amino acids longer than in
bacterial and yeast enzymes, respectively. The longest
one forms a two-and-a-half-turn helix interacting with
the surface of the sandwich domain.
The active site pocket is narrowest and deepest in
the yeast and fungal enzymes. The plant enzyme’s
active site pocket is the widest and not so deep,
Table 3. Structural comparison of GH32 known crystal structures. The rmsd ⁄ number of aligned pairs of Ca ⁄ % identity. The alignment was
performed using the Dali Database Server [85].
Enzyme 3PIG (518aa) 1UYP (432aa) 1Y4W (517aa) 3KF5 (512aa) 2AC1 (537aa) 2ADE (537aa)
b-Fructofuranosidase Bifidobacterium longum
PDB ID: 3PIG
– 2.1 ⁄ 423 ⁄ 29 2.5 ⁄ 459 ⁄ 24 2.3 ⁄ 442 ⁄ 24 2.0 ⁄ 456 ⁄ 28 1.9 ⁄ 453 ⁄ 26
b-Fructosidase (invertase) Thermotoga
maritima PDB ID:1UYP

2.1 ⁄ 423 ⁄ 29 – 2.0 ⁄ 422 ⁄ 29 2.1 ⁄ 408 ⁄ 25 2.1 ⁄ 417 ⁄ 26 2.0 ⁄ 415 ⁄ 28
Exo-inulinase Aspergillus awamori
PDB ID: 1Y4W
2.5 ⁄ 459 ⁄ 24 2.0 ⁄ 422 ⁄ 29 – 2.0 ⁄ 468 ⁄ 35 2.5 ⁄ 459 ⁄ 28 2.4 ⁄ 457 ⁄ 26
b-Fructofuranosidase Schwanniomyces
occidentalis PDB ID: 3KF5
2.3 ⁄ 442 ⁄ 24 2.1 ⁄ 408 ⁄ 25 2.0 ⁄ 468 ⁄
35 – 2.6 ⁄ 448 ⁄ 23 2.7 ⁄ 452 ⁄ 24
Invertase Arabidopsis thaliana PDB ID: 2AC1 2.0 ⁄ 456 ⁄ 28 2.1 ⁄ 416 ⁄ 26 2.5 ⁄ 461 ⁄ 28 2.6 ⁄ 449 ⁄ 23 – 1.2 ⁄ 527 ⁄ 53
Fructan 1-exohydrolase IIa Cichorium intybus
PDB ID: 2ADE
1.9 ⁄ 453 ⁄ 26 2.0 ⁄ 414 ⁄ 28 2.4 ⁄ 457 ⁄ 27 2.6 ⁄ 451 ⁄ 24 1.2 ⁄ 527 ⁄ 53 –
Table 4. Substrate specificity of Bifidobacterium longum KN29.1
b-fructofuranosidase, Arabidopsis thaliana invertase [71] and Cicho-
rium intybus fructan exohydrolase [86] (nd, not determined).
Substrate
Relative activity (%)
B. longum A. thaliana C. intybus
Sucrose 100 100 0
1-Kestose 263 35 100
Nystose 128 nd 75
Actilight
Ò
950P
a
200 nd nd
Raftiline
Ò
GR
b

114.6 nd nd
Inulin 59
c
383
Raffinose 6.7 nd 0
Levan 0 9 20
a
Actilight
â
950P – a mixture of fructooligosaccharides: 1-kestose
(GF
2
) 37 ± 6%, nystose (GF
3
) 53 ± 6%, 1-fructofuranosyl-D-nystose
GF
4
10 ± 6% and glucose + fructose + sucrose 5% was supplied by
Beghin Meiji (France).
b
Raftiline
â
GR ) inulin DP = 10: inulin $ 92%
and glucose + fructose + sucrose $ 8% from Orafti (Belgium).
c
Frutafit
â
TEX ) inulin DP2 Ä DP60, av. DP ‡ 22: inulin ‡ 99.5%
and glucose + fructose + sucrose £ 0.5% was obtained from
Sensus (the Netherlands).

Crystal structure of B. longum b-fructofuranosidase A. Bujacz et al.
1738 FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS
whereas in the bacterial enzymes it is deep and moder-
ately wide. The differences are visible in Fig. 6B, pre-
senting the shape and charge distribution of the
binding pocket in the compared enzymes from various
organisms. For the plant enzymes, the outlet of the
axial water channel on the opposite site of the active
centre is blocked by the specific loop L
1–2 ⁄ IV
, extend-
ing from Arg212 to Leu230 in A. thaliana (PDB ID:
2AC1) [71] and from Leu210 to Val225 in C. intybus
(PDB ID: 1ST8) [72].
The second level of controlling substrate specificity,
especially for long polysaccharides, is glycosylation,
which occurs in fungal, yeast and plant enzymes.
Except for the structure reported in this work and
the bacterial b-fructofuranosidase from T. maritima,
the other GH32 crystal structures all showed the
presence of glycosylation. The cleft formed between
the b-propeller and the b-sheet domain is postulated
as an inulin-binding site. Unlike in other GH32 fam-
ily enzyme structures known to date, the cleft formed
between the b-propeller and the b-sheet domain in
the A. thaliana cell wall invertase is blocked by carbo-
hydrates, suggesting that their presence in the cleft
prevents the binding of longer donor substrates
[71,73,74].
The third level of control, found only in the yeast

and fungal enzymes, is dimerization. For the yeast type
of dimer, the C-terminal b-sandwich domain is located
in the vicinity of the active pocket entrance. A muta-
tion study of some crucial residues in this domain
shows their role in controlling the binding of long inu-
lin-type substrate [59].
Conclusion
The high-resolution structure of b-fructofuranosidase
from B. longum is the first one for an enzyme of the
GH32 family from nonsporulating probiotic bacteria.
The protein is composed of two separated domains,
the larger N-terminal catalytic domain of the rare five-
bladed b-propeller fold and the smaller C-terminal
domain, folded as a b-sandwich. This enzyme possesses
one additional structural element, an N-terminal
a-helix, interacting with the b-propeller domain in the
area of the first and second blades. Structural compari-
son of the native enzyme with the enzyme ⁄ fructose
complex enables identification of the active site, and
residues Glu235 as the proton donor and Asp54 as the
nucleophile. These catalytic residues are located at the
first b-strand of blades 1 and 4 in the N-terminal
b-propeller domain, respectively. The presence of
fructose remaining in the active site of the complex
structure, obtained by raffinose soaking, proves that
the enzymes act with the overall retention of configura-
tion on the anomeric carbon of fructose. The distance
of 5.7 A
˚
between the carboxyl oxygens of the catalyti-

cally active acidic residues also corresponds to a group
of enzymes with the overall retention of the anomeric
configuration of the substrate.
The conserved RDP motif in the vicinity of the
active site of the enzyme is important for substrate rec-
ognition. The enzyme has a wide range of substrate
specificity, although it hydrolyses only b-(2,1) glyco-
sidic bond. The ability to utilize undigested oligosac-
charides is important for proper bifidobacteria
development and function, as well as for symbiosis
with the human organism.
Materials and methods
Cloning, expression and purification of
recombinant b-fructofuranosidase
Bifidobacterium longum KN29.1 was obtained from the col-
lection of the Institute of Animal Reproduction and Food
Research of the Polish Academy of Sciences, Division of
Table 5. Kinetic parameters in the hydrolysis of substrates by enzymes from family GH32 with determined crystal structure. b-fructofura-
nosidase from Bifidobacterium longum KN29.1; b-fructofuranosidase (invertase) from Thermotoga maritima [37]; invertase from Arabidop-
sis thaliana [71]; fructan-1-exohydrolase IIa from Cichorium intybus [86]; exo-inulinase from Aspergillus awamori [70]; b-fructofuranosidase
from Schwanniomyces occidentalis [59] (nd, not determined).
Substrate
K
M
(mM)
B. longum T. maritima A. thaliana S. occidentalis C. intybus A. awamori
Nystose 1.29 nd nd 1.6 ± 0.8 64 nd
Actilight 950P 2.65
a
nd nd nd nd nd

1-Kestose 4.74 nd 1 ± 0.1 1.3 ± 0.4 58 nd
Sucrose 31.45 64 0.35 ± 0.05 4.9 ± 1 nd nd
Raffinose 64.56 15 nd 1.3 ± 0.6 nd 8 ± 0.02
Inulin 8.87
b
19 nd nd nd 0.003 ± 0.0001
a
Assumed average molecular mass, 622.78 gÆmol
)1
.
b
mgÆmL
)1
.
A. Bujacz et al. Crystal structure of B. longum b-fructofuranosidase
FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS 1739
Food Science, Department of Food Microbiology, Olsztyn,
Poland. The b-fructofuranosidase gene was amplified using
TaKaRa Ex TaqÔ polymerase (Takara Bio Inc., Shiga,
Japan) and specific primers: forward primer 5 ¢-GCACG
C
TCTAGATGACTGACTTCACTCCTGAAACC-3¢ and
reverse primer 5¢-GCATT
CTCGAGCTCCAGTCCGATG
GACTTC-3¢ (the underlined nucleotides indicate the restric-
tion sites XbaI and XhoI, respectively, and the bold ones
indicate the initiation start codon). The b-fructofuranosi-
dase gene from B. longum KN29.1 was sequenced using the
AB13037 Genetic Analyzer (Applied Biosystems, Foster
City, CA, USA) and was deposited in GenBank [75]

(GenBank accession no. EF216677).
Expression of recombinant b-fructofuranosidase was car-
ried out in a bacterial expression system cloning using
expressive vector pET303 ⁄ CT-His and E. coli BL21 StarÔ
(DE3) from Invitrogen (Carlsbad, CA, USA). The recombi-
nant bacteria were grown in commercially available Magic-
MediaÔ E. coli Expression Medium (Invitrogen) for 20 h
at 37 °C. The cells were subsequently collected by centrifu-
gation (12 100 g, 20 min at 4 °C) and the biomass resus-
pended in 0.02 m sodium phosphate buffer pH 7.4. The
cell-free extract was sonicated, then centrifuged (13 400 g,
10 min, 4 °C). The recombinant b-fructofuranosidase was
then purified by a HisTrap HP column (16 mm ⁄ 25 mm;
Amersham Bioscience, Piscataway, NJ, USA) and concen-
trated by centrifugation on Viva-spin filters with a 10 kDa
membrane to 21 mgÆmL
)1
. The purity of the recombinant
b-fructofuranosidase preparation was controlled by
SDS ⁄ PAGE (12% polyacrylamide gel) according to the
Laemmli method [76].
The clearly visible electron density maps for one amino
acid showed a conflict with the sequence deposited in Gen-
Bank [75] (Phe240 is Ser). This was probably a consequence
of a mistake incorporated by the TaKaRa Ex TaqÔ poly-
merase and did not affect the enzymatic activity.
MS
The molecular mass of the recombinant b-fructofuranosi-
dase was determined using the MALDI-TOF method. The
analysis was carried out by the Centre of Molecular and

Macromolecular Studies, Polish Academy of Sciences,
Lodz, Poland.
Dynamic light scattering
The oligomerization state of b-fructofuranosidase from
B. longum KN29.1 was investigated by dynamic light scat-
tering. The dynamic light scattering measurements were car-
ried out at 18 °C on a Zetasizer lV instrument (Malvern
Instruments, Malvern, UK) at a wavelength of 830 nm
under measurement angle 90° . The experiment was per-
formed using protein at a concentration of 21 mgÆmL
)1
.
The obtained results showed that the investigated protein in
solution was in the monomeric form at a level of 99.9%.
Other parameters were also obtained: hydrodynamic radius
of 16.78 A
˚
, average radius of 34.02 A
˚
and ratio of the
shortest to the longest axis of 0.628.
Kinetic parameters of b -fructofuranosidase
The activity of b-fructofuranosidase was detected by mea-
suring the amount of liberated reducing sugars from differ-
ent substrates (1% w ⁄ v) in 50 mm sodium phosphate buffer
(pH 6.2) at 37 °C for 10 min. The hydrolysis products, glu-
cose and fructose, were determined by the Miller [77]
method. K
M
was determined by Lineweaver–Burk plots.

Reaction rates were measured for substrate concentrations
ranging from 0.125 to 100 mm, under the standard reaction
conditions (37 °C, 10 min, pH 6.2).
Crystallization, data collection and processing
The rhombohedrally shaped crystals used in this study were
grown by the hanging drop vapour diffusion method at
20 °C and reached the dimensions of 0.1 · 0.1 · 0.15 mm
after 3–4 weeks. The reservoir solution, containing 0.4 m
ammonium chloride, 0.1 m Mes pH 7.0, 18% poly(ethylene
glycol) 3350 was mixed with the protein solution at the
11 mgÆmL
)1
concentration in 10 mm Hepes buffer pH 7.5
with the ratio 1.5 lL : 1.5 lL. The crystals from the same
drop were used for data collection of the native enzyme
and its complex with the product of hydrolysis – fructose.
The crystals of raffinose ($ 0.1 mg) were added to the crys-
tallization drop with the apo crystals of b-fructofuranosi-
dase. After dissolving raffinose, the native enzyme crystals
were soaked for 2 h. A mixture of the well solution with
50% poly(ethylene glycol) 400 (v ⁄ v) in a 1 : 1 ratio was
used as a cryoprotectant during the diffraction experiment.
X-ray diffraction data were collected for crystals of apo
form enzyme and its complex with fructose using synchro-
tron radiation generated at the BESSY (Berlin, Germany)
beamlines BL_14.2 and BL_14.1. Both crystals diffracted
X-rays to around 1.80 A
˚
resolution and were solved in tri-
gonal space group P3

1
21. In both cases the diffraction data
were indexed, integrated, and scaled with HKL2000 [49].
Table 1 shows the data collection and processing statistics.
Structure determination and refinement
The crystal structure of apo b-fructofuranosidase from
B. longum was solved by molecular replacement with Phaser
[53] using a hybrid model created from two similar struc-
tures: (PDB ID: 1W2T) (from T. maritima) [50] b-propeller
domain and (PDB ID: 1Y4W) (from A. awamori) [51]
b-sandwich domain. The refined crystal structure of the apo
form was used as a model in the rigid body refinement of
the complex with fructose. The second diffraction data set
was rescaled to obtain the same setting of the crystallo-
Crystal structure of B. longum b-fructofuranosidase A. Bujacz et al.
1740 FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS
graphic axis as for the apo structure. In both cases, structure
refinement was carried out in refmac5 [78] using the maxi-
mum likelihood targets, including Translation, Libration,
Screw motion (TLS) parameters [79], defined separately for
each polypeptide chain. Model rebuilding was carried out in
coot [55] with water molecules firstly introduced in the
arp ⁄ warp program [54] and later manually. Progress of the
refinement was monitored, and the models were validated
using R
free
testing [80]. The quality of the final structures
was assessed with procheck [81]. The final refinement statis-
tics are shown in Table 1. The refined atomic coordinates
and structure factors for the native b-fructofuranosidase and

its complex with fructose have been deposited in the Protein
Data Bank (PDB ID: 3PIG) and (PDB ID: 3PIJ), respec-
tively. All crystallographic calculations were performed
using the ccp4 suite of programs [82] and phenix [83].
Molecular illustrations were prepared using pymol [84].
Acknowledgements
We would like to thank Dr Alexander Wlodawer for
valuable comments and editorial corrections. We are
grateful to European Synchrotrons Facilities for provid-
ing their beamlines: MaxLab, Lund, Sweden and SLS,
Villigen, Switzerland for initial crystals measurements
and BESSY, Berlin, Germany for the final diffraction
data collection. This work has been partially supported
by the European Social Fund and the Polish State
within the frame of ‘Mechanizm WIDDOK’ programme
(contract number Z ⁄ 2.10 ⁄ II ⁄ 2.6 ⁄ 04 ⁄ 05 ⁄ U⁄ 2⁄ 06).
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