These bacteria are known as micro-Keywords b-fructofuranosidase; Bifidobacterium longum; crystal structure; glycoside hydrolase family GH32; lactic acid bacteria Correspondence A.. The u
Trang 1from 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: anna.bujacz@p.lodz.pl
(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.
Trang 2organisms 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 Mamani-malian 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 StarDE3 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
Trang 33PIG MTDFTPETPVLTPIRDHAAELAKAEAGVAEMAAKRNN - RWY P YH IASNG GW I NDPNGL CF Y KG RW H FYQ LH P G TQ WG - PMH WGH V
1UYP - LFK P YH FFPIT GW M NDPNGL IFW KG KY H FYQ YN P RKPE WG - NIC WGH A
1Y4W - FNYDQ - PYRGQ YH FSPQKN W NDPNGL LYH N TY H F Q YN P G IE WG - NIS WGH A
3KF5 - SIDLSVDTSEYNR P LI H FTPEK GW M NDPNGL FYDKTAKLW H LYF Q YN P NATA WG QPLY WGH A
2AC1 - NQ - PYRTGF H FQPPKN W NDPNG PMI Y KG IY H FYQ WN P G AV WG - NIV W H
2ADE - QIEQ - PYRTG YH FQPPSN W NDPNG PML Y Q VY H FYQ YN P YAATF G DVII WGH A
3PIG S T ML NW KRE P IMFA P SL EQEKD G FSGSAVID D - G DLRFY YTG HRWANG HDNTGGDW QVQ MT A P N
1UYP V D LVH W RHL P VALY P DD ETH G FSGSAV EK - - G KMFLV YT YYRDPT - HNKGEKET Q CVVMSE N
1Y4W I E LTH W EEK P VALLARGFGSDVTEMY FSGSAV A V NTSGFGKDGK - TPLVAM YT SYYPVAQTLPSGQTVQEDQ Q Q SI A YSLD
3KF5 T N LVH W DEHEIAIG P EH DNE G FSGS I V H NTSGFFNSSIDPNQRIVAI YT NNIPD - N T DI A FSLD
2AC1 T T LI NW DPH P PAIF P SA PFDIN G CW SGSA T LP N - G KPVIL YTG IDPK - NQ QVQ NI A P N LS
2ADE V Y LV NW IHLDPAIY P TQ EADSKSCW SGSA T LPG - NIPAML YTG SDSK - SR QVQ DL A P N
3PIG - - E TSA TK Q - GMI I DC P T DKVD - HHY RDPK - VW KTG DT W M TF G VSSADKR G QMW L FS S D MVR W EYE - RV LF QHP
-1UYP - GLDFV K YDG - NPV I SK P - PE - EGT HAF RDPK - NRSN GE W M VL G SGKDEKI G RVL L YT S D LFH W KYE - GAI F EDE
-1Y4W - GLTW T TYDAANPV I PN P PSP - - EAEY - QNF RDP F V FWHDESQK W VVVTSIAE - LHKLAIYT S DNLKD W KLV - SE - GPYN
3KF5 - GYTF TK YEN - NPV I DVS S - NQF RDPK - FWHEDSNQ W M VVSKSQ - EYKIQIFG S ANLKN W VLN - SN - SSG
-2AC1 DP - Y REWK K SPL - NPLMAPD AVNGINASSF RDP TTA W LGQD - KK W RVII G SKI - HRR G LAITYT S D FLK W EKSPEP L HYD
2ADE - LSDPF L REWV K HPK - NPL I TP P - EGVKDDCF RDP STA W LGPD - GV W RIVV G GDR - DNN G MAF L YQ S D FVN W KRYDQP L SSA
3PIG DPDVFML ECPDF F IKD - - - GNE KWV IGF S AMGSKPSGFMNRNV S NAG Y IGT WEP - GGE F P ET - E
1UYP T TKEI ECPD LVRIG - E DILIY S ITS - TNSVLFSM G ELKE GKLNVEK
-1Y4W AQGG - VW ECP GLVKLPL - DSG - NST KWV ITSGLNPG - GPPGTVG S GTQ Y FV G EFDG - - F P DADTVYPGNST
3KF5 Y YGNQY ECP GLIEVPIEN - - DKS KWV MFLAINPG - SPLGG S INQ Y FV G DFDG - - F P DD - SQ
2AC1 - DGSGMW ECPDF F VTR - - GSNGVETSSFGEPNEIL K V LKI S LDD - TKHDY Y IGT YDRVKDK F P DN - GFK
2ADE - DATGTW ECPDF Y VPL - - STNGLDTSVYG - GSVRH V MKAGFE - GHDW Y IGT YSPDREN F P QNGLSLTGSTL
3PIG FRLW D G HNY YA P S - - VD G R QIVY GW MSPFV Q PI - MQDD GW C QLT LPR EIT L GD - D - G DVVTA P
1UYP RGLL D G TDF YA A T - - GT D R VVVI GW LQSWLRTG LY - TKRE GW N VMS LPR ELYVE - N - N ELKVK P
1Y4W ANWM D G PDF YA AAGYNG - LS LNDHVHI GW MNNWQ YGANI - T YP W RSAMAI PR HMA L KT - IGSKA - TLVQQ P
3KF5 TRFV D G KDF YA F T - SEVE H GVLGLA W ASNWQ YADQV - T NP W RSSTS L R NYT L RYVHTNAETKQ - L TLIQN P
2AC1 MDGTAPRY D G KY YA SKT F F DSAKN - RILW GW TNE - S SVEDDVEK GW S IQTI PR KIW L DR - S - G KQLIQW P
2ADE DLRY D G QF YA SKS F F DDAKN - RVLWA W VPE - D SQADDIEK GW A LQSF PR ALWIDR - N - G KQLIQW P
3PIG V E E LR ED - TLDHGS - VT L DMDGEEIIA - D A EAVEIEMTIDLAA - S TAERA GL KIH A TE
1UYP V E LLA LR KR - KVFETA - KS - GTFLL - VKENSYEIVCEFSG - EIE L RM - GNE
1Y4W QEAWSSISNKRPIYSRTFKT L - EGSTNTT - T GETFKVDLSFSAK - S KASTFAIALR A SA
3KF5 V LPDSINVV - DKLKKKNVK L TNKKPIKTN - FKGS - TGLFDFNITFKVLNLNVS - PGKTHFDILI - NSQ
2AC1 V E E LR TKQVKNLRN - KV L KSGSRLEVYGV T A AQADVEVLFKVRDLEKADVIEPSWTDPQLICSKMNVSVK S GLGPF GL MVL A SK
2ADE V E E LR QN - QVNLQN - KN L KPGSVLEIHGI A A SQADVTISFKLEGLKEAEVLDTTLVDPQALCNERGASSRGALGPF GL LAM A SK
3PIG D GAY T V AY D GQ - IGRVVV DR QAMAN - G - D RGYRA A L TDA E LAS - GKLD LR V FVD RG SVE VYV N
1UYP S - EEVVITKSR - DELIV D TTRSGV - S - GGEVRKSTV - E DEA - TNRI R F D SC SVE FFF N
1Y4W NF - TEQ T V GY D FA - KQQIFL DR THSGD - VSFDET FASVYHGP L PDST - GVVK L SI FVD RS SVE VFGGQ
3KF5 ELNSSVDSIKIGF D SS - QSSFYI DR H IPN - VEFPRKQFFTDKLA A L - E PLDYDQDLRVFS L YGI VD KNII E LYF N
2AC1 NL - EEY T V YFRIFKARQNSNKYVVLMCS D QSRSSLKEDN - D KTTYG A FVD INPH - QPLS LR ALI D HSV VE SFGGK
2ADE DL - KEQSAIFFRVFQNQLGRY SVLMCS D LSRSTVRSNI - D TTSYG A FVD IDPRS EEIS LR NLI D HSII E SFGAG
476
413 356
291 228
155 87
526 3PIG G HQVLS S YS Y ASE - G PRA I KL V AE - SG SLKVDS L KLHH M S IGLELEHHHHHH
1UYP - SIAFSFRIHPEN - VYN I LS V - SNQVK L EVFELENIW - L
-1Y4W G ETTLTAQIFPSS - D AVHARLAST - GG TTEDVRADIYKIA S TW
-3KF5 G TVAMTNTFFMGE - GKYPHD I QI V TD - TEEPLFELESVIIRELNK
-2AC1 G RACIT S RV Y PKLAIGK SSHLFAFNYGYQ SVDVLN L NAWS M S AQI - S
-2ADE G KTCIT S RI Y PKFVNNE EAHLFVFNNGTQ NVKISEMSAWS M KNAKF - 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.
Trang 4was 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 20C 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 P3121 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
Trang 5data 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 ibLII–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.
Data collection
a = b = 90.0, c = 120
a = b = 86.8, c = 223.9
a = b = 90.0,c = 120
Refinement
Number of atoms (protein ⁄ solvent ⁄ Cl ⁄ fructose) 8365 ⁄ 1167 ⁄ 6 ⁄ 0 8388 ⁄ 989 ⁄ 5 ⁄ 24
rms deviations from ideal
Residues in Ramachandran plot (%)
a Values in parentheses correspond to the last resolution shell b Rint= P
h
P
j |Ihj) <I h >| ⁄ P
h
P
j Ihj, where Ihjis the intensity of observation j
of reflection h c R = P
h ||Fo| ) |F c || ⁄ P
h |Fo| for all reflections, where Foand Fcare observed and calculated structure factors, respectively.
R free is calculated analogously for the test reflections, randomly selected and excluded from the refinement.
Trang 6GenBank, 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 configurareten-tion 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 (frprod-uctose), 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.
Trang 7maps 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).
Hydrogen bonds
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).
Trang 8donated 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
L2¢–3¢ (Thr412-Tyr417) and L4¢–5¢ (Gln434-Arg441)
These two loops interact with the tips of four other
loops: L1–1¢ (Gly378-Asp375), L2¢–2 (Ala402-Arg404),
L3¢–4¢ (Gly424-Gly427) and L6–6¢(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.
Trang 9where 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 longBifidobacteri-um 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 longumKN29.1 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-gumKN29.1 b-fructofuranosidase, six-fold (KM= 31.4 mm) and 49-fold (KM= 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 (ibLI–V) and the loops L2–3 between b-strands 2 and 3 in each blade This structural
Trang 10ele-(1) (2)
A
B
(3)
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).