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: 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. 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 [...]... 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 crystallization drop with the apo crystals of b-fructofuranosidase After... Materials and methods Conclusion Cloning, expression and purification of recombinant b-fructofuranosidase 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 Bifidobacterium longum KN29.1 was obtained from the collection of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences,... 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 family enzyme structures known to date, the cleft formed between the b-propeller and the b-sheet domain in the. .. replacement with Phaser [53] using a hybrid model created from two similar structures: (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-... 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 configuration on the. .. the anomeric carbon of fructose The distance ˚ of 5.7 A between the carboxyl oxygens of the catalytically 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 recognition The enzyme has a wide range of substrate specificity,... mass of the recombinant b-fructofuranosidase 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 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... firstly introduced in the arp ⁄ warp program [54] and later manually Progress of the refinement was monitored, and the models were validated using Rfree testing [80] The quality of the final structures was assessed with procheck [81] The final refinement statistics are shown in Table 1 The refined atomic coordinates and structure factors for the native b-fructofuranosidase and its complex with fructose have been... short, has the same shape in all investigated structures and contacts the b-sandwich domain The loop L2–3 ⁄ 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 L2–2¢s and L3¢–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... 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, FEBS Journal 278 (2011) 1728–1744 ª 2011 The Authors Journal compilation ª 2011 FEBS A Bujacz et al Crystal structure of B longum b-fructofuranosidase Table 5 Kinetic parameters in the hydrolysis of substrates . Crystal structures of the apo form of b-fructofuranosidase from Bifidobacterium longum and its complex with fructose Anna Bujacz,. 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