Báo cáo khoa học: Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes doc

REVIEW ARTICLE Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes Wim Van den Ende 1 , Willem Lammens 1,2 , Andre ´ Van Laere 1 , Lindsey Schroeven 1 and Katrien Le Roy 1 1 K.U.Leuven, Laboratory for Molecular Plant Physiology, Heverlee, Belgium 2 K.U.Leuven, Laboratory for Biocrystallography, Leuven, Belgium Introduction Fructans are polymers of fructose (Fru), and an exten- sion of sucrose (Suc), and they occur in many plant species mainly belonging to Asteraceae, Liliaceae and Poaceae [1,2]. Apart from their function as a vacuolar storage carbohydrate, fructans may protect plants from drought and cold stress by stabilizing cellular membranes [3–5]. Furthermore, they might participate in vacuolar antioxidant mechanisms [6], consistent with earlier [7] and more recent [8,9] observations on transgenic plants carrying fructan-synthesizing genes. Recently, substantial efforts have been made to under- stand fructan metabolism and its regulation in impor- tant cereals [10–12] and forage grasses [13]. Different fructan types (inulin, levan, graminan, neoseries) can be distinguished by the type of linkage present between the fructosyl residues [b(2-1) versus b(2-6)] and by the position of the glucose (Glc) residue [14]. Depending on the plant species, fructans are produced by different types of fructan biosynthetic enzymes (a minimum of two enzymes) or fructosy- ltransferases (FTs), which have probably evolved from vacuolar-type invertases (VIs) [15]. Sucrose:sucrose 1- fructosyltransferase (1-SST) seems to be a key enzyme in both monocots and dicots for initiating the fructan polymerization process. However, 1-SST might not ful- fil a prominent role in species exclusively producing levan-type fructans [16]. Critical-elongation FTs include fructan:fructan 1-fructosyltransferases (1-FFTs; inulin Keywords fructan; fructosyl transferase; invertase; structure–function; sucrose Correspondence W. Van den Ende, K.U.Leuven, Laboratory for Molecular Plant Physiology, Kasteelpark Arenberg 31, B-3001, Leuven-Heverlee, Belgium Fax: +32 16321967 Tel: +32 16321952 E-mail: wim.vandenende@bio.kuleuven.be (Received 18 June 2009, revised 7 August 2009, accepted 19 August 2009) doi:10.1111/j.1742-4658.2009.07316.x Plant family 32 glycoside hydrolase enzymes include hydrolases (cell wall invertases, fructan exohydrolases, vacuolar invertases) and fructosyltrans- ferases. These enzymes are very similar at the molecular and structural levels but are functionally different. Understanding the basis of the func- tional diversity in this family is a challenging task. By combining structural and site-directed mutagenesis data, Asp239 in AtcwINV1 was identified as an amino acid critical for binding and stabilizing sucrose. Plant fructan exohydrolases lack such an Asp239 equivalent. Substitution of Asp239 led to the loss of invertase activity, while its introduction in fructan exohydro- lases increased invertase activity. Some fructan exohydrolases are inhibited by sucrose. The difference between the inhibitor (fructan exohydrolase) and the substrate (invertase) binding configurations of sucrose can be explained by the different orientation of Trp82. Furthermore, the evolutionary hydro- lase ⁄ transferase transition could be mimicked and the difference between S-type fructosyltransferases (sucrose as donor) and F-type fructosyltransfe- rases (fructan as donor) could be unravelled. Abbreviations 1-KEH, 1-kestose exohydrolase; CWI, cell wall invertase; FEH, fructan exohydrolase; FFT, fructan:fructan fructosyltransferase; Fru, fructose; FT, fructosyltransferase; GH32, glycoside hydrolase family 32; Glc, glucose; SFT, sucrose:fructan fructosyltransferase; SST, sucrose:sucrose fructosyltransferase; Suc, sucrose; VI, vacuolar invertase; WT, wild-type. 5788 FEBS Journal 276 (2009) 5788–5798 ª 2009 The Authors Journal compilation ª 2009 FEBS synthesis), sucrose:fructan 6-fructosyltransferases (6- SFTs; graminan synthesis) and fructan:fructan 6 G -fructosyltransferases (6 G -FFTs; inulin neoseries) [15]. Plants degrade fructans using fructan exohydrolas- es (FEHs). Similarly to FTs, many different types of FEHs can be discriminated [17]. Intriguingly, these FEHs seem to be present in all plants, including those not accumulating fructans at all. Unravelling the function of these ‘defective invertases’ (containing FEH side activity) is a hot topic in this research area. While fructans are degraded by FEHs, the cleavage of Suc into Glc and Fru is catalyzed by two classes of acid- type plant invertases: the cell wall invertases (CWIs) and the VIs. Phylogenetically, two different groups can be differentiated in plants: the CWI ⁄ FEH group on the one hand and the VI ⁄ FT group on the other hand. All the different types of FTs, FEHs, CWIs and VIs are grouped together with microbial b-fructosidases (degrading both sucrose and fructans) in the glycoside hydrolase family 32 (GH32) (http://www.cazy.org) [18]. GH32 can be combined with GH68 in the clan GH-J. GH68 harbours bacterial invertases, levansuc- rases and inulosucrases. Recently, several 3D struc- tures have been unraveled within GH32 [15]. All these proteins consist of an N-terminal five-bladed b-propel- ler domain (GH32 and GH68) followed by a C-termi- nal domain formed by two b-sheets (only in GH32). The active site is present within the b-propeller domain and is characterized by the presence of three highly conserved acidic groups (present in the WMNDPNG, RDP and EC motifs). The Asp from the first motif acts as a nucleophile, the Asp from the second motif is believed to be a transition state stabilizer and the Glu residue from the EC motif acts as an acid ⁄ base catalyst, playing a crucial role in the catalytic mecha- nism [19–21]. According to the )n to +n subsite nomenclature proposed by Davies et al. [22], hydroly- sis takes place between the )1 and +1 subsites. For instance, when Suc binds as a donor substrate, the Fru moiety positions at the )1 subsite and the Glc unit is bound at the +1 subsite. The availability of the 3D structures helped greatly in the design of very specific site-directed mutagenesis experiments, which were carried out with the aim of understanding the molecular basis for the different substrate specificities within plant GH32 enzymes. Here we summarize the recent progress in this area. Understanding substrate specificity within the CWI ⁄ FEH group Differences in the active sites of AtcwINV1 and Ci1-FEHIIa The first plant enzyme 3D structures that became available within GH32 were Cichorium intybus 1-FEH IIa (Ci1-FEHIIa) [23] and AtcwINV1 (a CWI from Arabidopsis thaliana) [24]; both are hydrolases differing strongly in donor substrate specificity. Indeed, Ci1-FE- HIIa cannot degrade sucrose, while sucrose is the preferential substrate for AtcwINV1. Multiple aligment studies with other plant GH32 members revealed that both AtcwINV1 and Ci1-FEHIIa contain eight conserved motifs in the active-site region (Fig. 1A). Additionally, one hypervariable loop is located very close to the acid–base catalyst, and this loop is clearly different between AtcwINV1 (KISLDDTKH) and Ci1-FEHIIa (KADFEG H), the Ci1-FEHIIa showing a double deletion in this area (Fig. 2). AtcwINV1 shows an Asp239⁄ Lys242 couple (strong hydrogen A B Fig. 1. Schematic representation of AtcwINV1 showing eight conserved regions in the vicinity of the active site. The amino acids involved in catalysis and in binding the terminal fructose are in red. A hypervariable loop that comes close to the acid–base catalyst is shown in blue (A). Details of the active sites of Ci1-FEHIIa and AtcwINV1 in complex with sucrose [26]. The positions of Fru ()1 subsite) and Glc (+1 subsite) are indicated with arrows. Please note the difference in the hypervariable loop and the different orientation of both the Trp82 and the Glc moiety in Ci1-FEHIIa compared with AtcwINV1 (B). The figures were prepared using PYMOL [39]. W. Van den Ende et al. Donor and acceptor substrate selectivity in GH32 FEBS Journal 276 (2009) 5788–5798 ª 2009 The Authors Journal compilation ª 2009 FEBS 5789 bond), which is absent in Ci1-FEHIIa (Fig. 1B) and also in all characterized FEHs (Fig. 2A). However, such Asp ⁄ Lys or Asp ⁄ Arg couples are typically observed among CWIs (Fig. 2B), although with some exceptions (e.g. AtcwINV5). Another striking difference is the presence of a Ser residue in Ci1-FEHIIa (after the MLYTG motif), while AtcwINV1 contains an Ile at this position (Fig. 1). Intriguingly, among all plant FEHs character- ized to date, we found a nice correlation between the identity of the amino acid at this position and the degree of inhibition by Suc. Indeed, all FEHs that contain a small amino acid (such as Gly or Ser) at this position are strongly inhibited by Suc (Fig. 3; [25]). A last prominent difference in the active site between the two structures is the different orientation of the Trp82 residue (Fig. 1B); this, of course, remained undetected during multiple sequence alignments. Trp82 and Ser101 are very close in space in the active site of Ci1-FEHIIa (Fig. 1B). A B Fig. 2. Multiple alignment of a selection of FEHs (A) and CWIs (B) in the region surrounding the hypervariable loop. The position of the Asp ⁄ Lys or Asp ⁄ Arg couple is indicated in bold in CWIs (B). FEHs (A) and AtcwINV5 (B) contain alternative amino acids at these positions (bold) or deletions in this area. Functionally characterized enzymes are marked by an asterisk. Fig. 3. Comparison of the GWAS and MLYTG motifs in FEHs and their inhibition by sucrose. Donor and acceptor substrate selectivity in GH32 W. Van den Ende et al. 5790 FEBS Journal 276 (2009) 5788–5798 ª 2009 The Authors Journal compilation ª 2009 FEBS Asp239 fulfils a crucial role for binding Suc as a substrate in the CWI ⁄ FEH group Knowing these prominent differences in the active sites of FEHs and invertases, we used a mutagenesis approach to alter the donor substrate specificity within plant GH32 enzymes. The Asp239 in AtcwINV1 was changed into an Ala, a Phe and an Asn (Fig. 4; [21]). Additionally, Lys242 was mutated into a Leu. The invertase activity vanished in the D239A and D239F mutants but remained more or less intact in the D239N mutant. The kinetic parameters of the purified mutant enzymes were determined and compared with those obtained for the wild-type (WT) invertase (Table 1; [21]). In both the D239A and D239F mutants, the K m increased by 6–11-fold, respectively, inferring an important role for the Asp239 residue in substrate binding. The lower substrate affinity of D239F compared with D239A can result from the more extended steric hindrance of the bulky Phe. A similar tendency was observed for k cat values, which decreased 10–20-fold, suggesting that Asp239 is also important for efficient catalysis. By contrast, the K m and k cat values of the D239N mutant differed only slightly from those of the WT enzyme, convincingly demonstrating that an acidic group is not essential at this position and can be replaced by an Asn. Conclu- sively, these data showed that the presence of an addi- tional Asp or Asn residue, adjacent to the Glu203 proton donor, is important for optimal binding and efficient catalysis of Suc. To investigate whether the results obtained for the Asp239 mutants were caused by the substitution of this Asp residue itself, or were indirectly caused by the disturbance of the tight Asp239–Lys242 interaction, a K242L mutant was constructed and the kinetic proper- ties of the purified enzymes were determined (Table 1). The K m of the K242L mutant enzyme increased by a factor comparable to those of the Asp239 mutants. However, the k cat value only decreased by two-fold in comparison with the WT enzyme. Therefore, a crucial role for the Lys242 residue itself can be excluded. However, regarding the conserved interaction between Asp239 and Lys242, it is plausible that Lys242 is necessary to keep Asp239 in the correct orientation towards the active site. When using 1-kestose as a substrate, the results pre- sented in Table 1 show that the kinetics of the mutants (D239A, D239F) did not change substantially com- pared with the WT enzyme. So, the WT AtcwINV1 A B Fig. 4. Production of Glc and Fru from Suc by wild-type AtcwINV1 and several Asp239 mutants (D239A, D239F and D239N), as revealed by high-pressure anion-exchange chromatography with pulsed amperometric detection (HP-AEC-PAD; Dionex, Sunnyvale, CA, USA). The reaction conditions were as follows: incubation of 50 ng of purified enzyme with 10 m M Suc in 50 mM acetate buffer, pH 5.0, for 30 min at 30 °C (A). Comparison of specific enzymatic activities [s.a. in (mol fructose).(mol enzyme) )1 .(s) )1 ] of the same wild-type and mutant AtcwINV1 enzymes as a function of increas- ing Suc concentrations (B). The figure is reproduced from that presented in a previous publication [21]. Table 1. Kinetic parameters for the hydrolysis of sucrose and 1-kestose by purified WT AtcwINV1, D239A, D239F, D239N and K242L mutant enzymes. The table was reproduced from a previous publication [21]. Sucrose K m (mM) k cat (s )1 ) k cat ⁄ K m (mM )1 Æs )1 ) AtcwINV1 0.35 ± 0.05 59 ± 4 168.6 D239A 2.1 ± 0.2 6 ± 0.3 2.9 D239F 4.5 ± 0.5 3 ± 0.2 0.7 D239N 0.6 ± 0.07 61 ± 5 101.7 K242L 3.7 ± 0.4 29 ± 1 7.8 1-Kestose AtcwINV1 1 ± 0.1 21 ± 2 21.0 D239A 0.6 ± 0.03 21 ± 3 35.0 D239F 1.2 ± 0.1 20 ± 2 16.7 W. Van den Ende et al. Donor and acceptor substrate selectivity in GH32 FEBS Journal 276 (2009) 5788–5798 ª 2009 The Authors Journal compilation ª 2009 FEBS 5791 contains an intrinsic 1-kestose exohydrolase (1-KEH) activity, which remains intact in the D239A mutant. It can be concluded that the substitution of Asp239 very selectively destroys the invertase activity, but not the 1-KEH activity. The importance of a structurally equivalent Asp239 residue, and the presence of a conserved Asp ⁄ Lys or Asp ⁄ Arg couple to bind and stabilize Suc as the prefer- ential donor substrate in invertases, was confirmed by generating several mutated AtcwINV1–Suc complexes. These complexes nicely demonstrated the strong interaction between Asp239 and the Glc moiety of Suc (Fig 1B, [15,26]). By using the presence of an intact Asp239 homo- logue as a selective marker to discriminate between real and defective invertases, we predicted that three out of the six (i.e. 50%; AtcwINV3, 5 and 6; Fig. 5) of the so-called CWIs from this species are not real inver- tases but defective invertases (FEHs). This functional- ity has already been proven for AtcwINV3 (termed a 6-FEH) and AtcwINV6 (termed a 6&1 FEH) [27]. For the next challenge, it was questioned whether introduction of an Asp239 homologue in an FEH would result in the introduction of sucrose hydrolyzing activity. As the introduction of a structural and functional Asp239 homologue in Ci1-FEH IIa is com- plex because of the presence of a double deletion (see also Fig. 2A), site-directed mutagenesis experiments were performed on Beta vulgaris 6-FEH [28], which lacks such a deletion and which is characterized by the presence of a nonacidic Asp239 structural equivalent (a bold Phe residue: see Fig. 2). Compared with the WT sugar beet 6-FEH, a F233D mutant indeed showed substantial invertase activity, especially at higher Suc concentrations (Fig. 6A). Trp82 fulfils a crucial role for binding Suc as inhibitor in some FEHs Some FEHs are strongly inhibited by Suc, and others or not. Ci1-FEHIIa is a typical example of an FEH that is strongly inhibited. The presence of a small amino acid (Ser101) next to Trp82 in Ci1-FEHIIa is believed to be important for determining the orienta- tion of the Trp82 residue. The Glc moiety of Suc in Ci1-FEHIIa occupied a position clearly different [25] Fig. 5. Models of the active sites of the six putative CWIs from the model plant Arabidopsis thaliana. AtcwINV1, 2 and 4 contain a correctly orientated Asp239 homologue (indicated with an arrow) close to the acid–base catalyst Glu203 (AtcwINV1 terminology). AtcwINV3, 5 and 6 lack the Asp239 equivalent. The active site residues are marked in red. Modelling was performed based on the known 3D structures of Ci1-FEHIIa and AtcwINV1 using SWISS MODEL (http://swissmodel.expasy.org). The figures were prepared using PYMOL [39]. Donor and acceptor substrate selectivity in GH32 W. Van den Ende et al. 5792 FEBS Journal 276 (2009) 5788–5798 ª 2009 The Authors Journal compilation ª 2009 FEBS from the ones observed in the bacterial levansucrase [29] and in AtcwINV1 [26]. The Glc moieties are moved away from each other by ± 3 A ˚ , but neverthe- less they still stay in the same plane more or less alongside each other (Fig. 1B). The configuration of the Glc moiety of Suc present in Ci1-FEHIIa can be considered as the ‘inhibitor con- figuration or inhibitor-binding modus’, whereas the Suc orientation in levansucrase or AtcwINV1 is termed ‘substrate configuration or substrate-binding modus’, because Suc acts as an inhibitor in Ci1-FEHIIa and as a substrate for the other enzymes. Interestingly, chang- ing the Ser101 from Ci1-FEHIIa into a Leu also resulted in an increased invertase activity (Fig. 6B), strongly suggesting that the nature of this amino acid is essential to discriminate whether Suc will bind as a substrate rather than as an inhibitor. A W82L mutant had invertase activity similar to that of the WT enzyme (Fig. 6B). It is a challenging task to figure out whether defective invertases occurring in nonfructan- accumulating plants would also be able to bind Suc in the inhibitor configuration [30]. Transforming a VI into a high-affinity FT Destroying the hydrogen bond network in the WMNDPNG motif is essential to create an FT from TaVI Both FTs (sucrose ⁄ fructan as acceptor substrate: trans- ferase activity) and VIs (water as acceptor: hydrolase activity) occur within plant GH32 members. A rather limited increase in transfructosylation capability was already realized by mutagenesis adjacent to the nucleo- phile in the b-fructosidase or WMNDPNG motif of onion VI [31], but the mutant enzyme still mainly behaved as an invertase with fully saturable kinetics for hexose production (hydrolytic reaction) and no full saturation was observed for 1-kestose production (transfer reaction). Triticum aestivum (wheat) is an eco- nomically important species and an ideal model plant for using to perform detailed structure–function work on VIs and FTs. Moreover, it is a unique fact that Ta1-SST, Ta6-SFT, Ta1-FFT and TaVI recombinant enzymes (each of which were derived after hetero- logous expression in Pichia pastoris) are all available [32,33], which results in an excellent system for site- directed mutagenesis, especially because the percentage of identity is very high among TaVIs and TaFTs [34]. In an attempt to understand the evolution of an FT from an ancestral VI, multiple sequence alignments of VIs and Suc-splitting FTs (S-type FTs such as 1-SSTs and 6-SFTs) revealed prominent differences in two conserved VI regions, namely the WMNDPNG and GWAS motifs [34]. Because 1-SSTs of Poaceae always contain a Tyr instead of a Trp in the WMNDPNG motif, the W23Y TaVI mutant was constructed. In addition, because 1-SSTs of dicotyledonous plants and 6-SFTs from Poaceae contain a Ser instead of an Asn adjacent to the nucleophile, the TaVI N25S and W23Y+N25S mutants were designed. Intriguingly, in all plant invertases (VIs as well as CWIs), a well-defined hydrogen bond network is con- served between the Trp23 and Asn25 equivalents and the nucleophile. Strikingly, modelling studies indicated that this hydrogen bond network is destroyed in all plant FTs [34]. Therefore, we wanted to test the hypothesis of whether the destruction of the hydrogen bond network in TaVI would result in a mutant enzyme with increased FT capability. Comparing transfructosylation capabilities of WT and mutant TaVIs Reaction mixtures containing the purified heterolo- gously expressed TaVI, Ta1-SST and Ta6-SFT WT and A B Fig. 6. Specific sucrose-hydrolyzing activities [s.a. in (mol fruc- tose).(mol enzyme) )1 .(s) )1 ]ofBeta vulgaris wild-type 6-FEH and F233D mutant enzymes as function of increasing sucrose concen- trations (A). Specific sucrose-hydrolyzing activities [s.a. in (mol fruc- tose).(mol enzyme) )1 .(s) )1 ] of wild-type Ci1-FEHIIa and S101L mutant enzymes as a function of increasing sucrose concentrations (B). Results are the mean (± SE) for three replicates. The figure is reproduced from that presented in a previous publication [30]. W. Van den Ende et al. Donor and acceptor substrate selectivity in GH32 FEBS Journal 276 (2009) 5788–5798 ª 2009 The Authors Journal compilation ª 2009 FEBS 5793 mutant TaVI enzymes were compared after incubation with 500 mm Suc as a single substrate (Fig. 7). The WT TaVI showed no significant production of 1-kestose. The W23Y mutant synthesized a small amount of 1-kestose, comparable to the amount produced by the WT Ta6-SFT. Additionally, the WT Ta6-SFT also produced a small amount of 6-kestose. Compared with the W23Y mutant, the N25S mutant produced a greater amount of 1-kestose. Interestingly, a synergistic effect is observed when the two mutations are com- bined (W23Y+N25S), resulting in a strongly increased 1-kestose synthesis and a decreased Fru to Glc ratio. The WT Ta1-SST, showing nearly zero hydrolytic activity at 500 mm Suc, is shown for comparison (Fig. 7; [34]). The percentage of 1-kestose (transfer to Suc) over the total of Fru (transfer to water: hydrolysis) and 1-kestose is a measure of the transfructosylation capac- ity. Typically, the transfructosylation capacity increases with increasing substrate concentration; this is the case for all enzymes tested (Fig. 8; for details see [34]). A clear shift from hydrolysis to transfructosylation was observed for the mutant TaVIs, especially at higher Suc concentrations. This change in acceptor substrate specificity was most prominent for the double mutant W23Y+N25S, showing a 17-fold increase in transfruc- tosylation capacity and reaching a maximal transfruc- tosylation capacity of more than 50% (Fig. 8). It can be hypothesized that this capacity can be further increased by changing the WGW motif (Fig. 1) into a WGY motif, because the W to Y transition was also shown to increase the transfructosylation capability [35]. We refer to Schroeven et al. [34] for a detailed kinet- ical analysis on the WT and mutant enzymes. Conclu- sively, it is demonstrated, for the first time, that it is possible to create a FT (a 1-SST in this particular case) with fully saturable kinetics (1-kestose production) and a very high affinity for Suc (the K m is more than 10 times lower compared with that of the WT Ta1-SST). Such mutated enzymes are extremely useful for bio- technological applications, such as producing tailor- made fructans in transgenic plants or in bioreactors. Transforming a 6 G -FFT into a 1-SST 1-SST and 6 G -FFT enzymes in Lolium perenne FTs might be crucial for plant survival under stress conditions in species where fructans represent the major form of reserve carbohydrate, such as in peren- nial ryegrass (Lolium perenne). Perennial ryegrass is an economically important species (the number 1 plant in fructan research at this moment) and forms an ideal system for conducting detailed structure–function work on FTs and VIs. Indeed, many cDNAs have become available from this species in the last 5 years [36,37]. Moreover, the overall identity between these cDNAs is very high, and recombinant enzymes are available through heterologous expression in P. pastoris. The Fig. 7. High-pressure anion-exchange chromatography with pulsed amperometric detection (HP-AEC-PAD; Dionex) chromatograms of reaction mixtures of WT Triticum aestivum TaVI, Ta6-SFT, Ta1-SST and mutant VI W23Y, N25S and W23Y+N25S enzymes. All enzymes were incubated with 500 m M Suc at 30 °C for 10 min (VI and mutants derived thereof), 30 min (6-SFT) and 2 h (1-SST). 1-K, 1-kestose; 6-K, 6-kestose. The figure is reproduced from that pre- sented in a previous publication [34]. Fig. 8. The percentage of transfructosyla- tion at increasing concentrations of Suc (25– 1000 m M). From left to right: WT Ta1-SST, WT TaVI, TaVI mutants W23Y+N25S, N25S and W23Y. The figure is reproduced from that presented in a previous publication [34]. Donor and acceptor substrate selectivity in GH32 W. Van den Ende et al. 5794 FEBS Journal 276 (2009) 5788–5798 ª 2009 The Authors Journal compilation ª 2009 FEBS very high identity at the sequence level between Lp6 G - FFT ⁄ 1-FFT (an enzyme from L. perenne showing both 6 G -FFT and 1-FFT activities) and Lp1-SST inspired us to study, in depth, the molecular differences between these functionally different enzymes. Despite their high identity, these enzymes differ greatly in sub- strate specificity and product formation, the two most crucial differences being (a) Lp1-SST uses Suc as a donor substrate while Suc is a very poor donor sub- strate for the recombinant Lp6 G -FFT ⁄ 1-FFT, and (b) Lp1-SST can only create a b(2-1) linkage between two fructosyl residues while Lp6 G -FFT ⁄ 1-FFT can create both a b(2-1) linkage between two fructosyl residues and a b(2-6) linkage between a fructosyl residue and a glucosyl residue. Designing specific mutants based on multiple sequence alignments and modelling In the mature proteins of Lp6 G -FFT ⁄ 1-FFT and Lp1- SST, 83% of the amino acids are identical. As a first step to understand the difference at the molecular level between these enzymes, a multiple sequence alignment was made for the large subunits of Lp1-SST, Lp6 G - FFT ⁄ 1-FFT and all 6 G -FFT types of enzymes charac- terized to date [38]. Ten amino acids were unique in 6 G -FFTs and could not be found in Lp1-SST. Among those, only three amino acids (arrows in Fig. 9) were located in the vicinity of the active-site region, as revealed by modelling studies (Fig. 10). These amino acids are Asn340, Trp343 and Ser415 in Lp6 G -FFT ⁄ 1- FFT (Fig. 10A), which are replaced by Asp349, Arg352 and Asn424 in Lp1-SST (Fig. 10B). Both Asn340 and Trp343 are present in a hypervariable loop very close to the acid–base catalyst. Therefore, the single mutants N340D, W343R and S415N, the double mutants N340D+W343R, N340D+S415N and W343R+S415N, and finally the triple mutant, N340D+W343R+S415N, were constructed and trans- formed into P. pastoris for functional characterization and comparison with the WT enzymes Lp6 G -FFT ⁄ 1- FFT and Lp1-SST [38]. 1-SST activity in mutant and WT enzymes: transforming F-type into S-type The WT Lp6 G -FFT ⁄ 1-FFT showed a low, but intrin- sic, 1-SST activity (Table 2; [37]). The N340D, Fig. 9. Amino acid composition in the active-site motifs of WT Lp1- SST and WT Lp6 G -FFT ⁄ 1-FFT and mutants derived of the latter. Amino acids near the active site that are unique in Lp6 G -FFT ⁄ 1-FFT compared with Lp1-SST are shown in bold. Two of these amino acids (Asn340 and Trp343) are present in the hypervariable loop. A B Fig. 10. Localization of the considered amino acids in the active sites of the WT Lp6 G -FFT ⁄ 1-FFT (A) and WT Lp1-SST (B). The active site residues are marked in red, and the amino acids sub- jected to mutagenesis are indicated with an arrow. Modelling was performed based on the 3D structure of AtcwINV1 using SWISS MODEL (http://swissmodel.expasy.org). The figures were prepared using PYMOL [39]. W. Van den Ende et al. Donor and acceptor substrate selectivity in GH32 FEBS Journal 276 (2009) 5788–5798 ª 2009 The Authors Journal compilation ª 2009 FEBS 5795 W343R, S415N, N340D+S415N and W343R+S415N mutants also showed very low 1-SST activity. Most importantly, while the N340D+W343R mutant showed 6 G -FFT and 1-FFT activities that lay between those of the N340D and W343R single mutants [38], its 1-SST activity drastically increased (Table 2). The 1-SST activity increased further in the triple N340D+W343R+S415N mutant, while its intrinsic 1-FFT activity was further decreased. At high Suc concentrations (1.0 M), the triple Lp6 G -FFT ⁄ 1-FFT mutant produced the same amount of 1-kestose as the WT Lp1-SST [38], demonstrating, for the first time, the transformation of an F-type of enzyme into an S-type enzyme by introducing a functional Asp ⁄ Arg couple in the hypervariable loop. The interested reader is referred to a previous publication to read further details on the kinetics and substrate specificities of the different mutant and WT enzymes [38]. Donor substrate selectivity: similarities between the CWI ⁄ FEH and VI ⁄ FT subgroups The results show that Asn340 and Trp343 are impor- tant determinants for explaining the particular donor substrate characteristics of the WT Lp 6 G -FFT ⁄ 1-FFT and WT Lp1-SST. What are the structural equivalent amino acids in other FTs and in the structurally well characterized Ci1-FEHIIa and AtcwINV1? Figure 11 shows a multiple alignment of a selection of FTs together with Ci1-FEHIIa and AtcwINV1. Modelling studies proposed that the N340 ⁄ W343 couple is equiva- lent to the D239 ⁄ K242 couple in AtcwINV1. The presence of an Asp ⁄ Lys or an Asp ⁄ Arg couple in the hypervariable loop is believed to be essential for binding Suc in the CWI ⁄ FEH group (see above). Simi- larly, the same Asp ⁄ Arg couple seems to determine the preference for Suc as the donor substrate within the VI ⁄ FT subgroup. It can be concluded that the presence of a functional Asp ⁄ Lys or Asp ⁄ Arg couple (not disturbed by other amino acids in the area) determines the Suc donor substrate selectivity in all plant GH32 enzymes. In particular, all enzymes that are able to use Suc as a donor substrate (1-SSTs, 6-SFTs, VIs and CWIs) contain this couple, while it is absent in all typical F-type enzymes (FEHs, 1-FFTs and 6 G -FFTs) (Fig. 11). It is a matter of discussion whether this point of view can be extended to all microbial GH32 enzymes and to GH68 enzymes [38]. Conclusions The availability of the 3D structures of Ci1-FEHIIa and AtcwINV1 boosted structure–function research on plant GH32 members. Enzymes preferentially using Suc as the donor substrate (S-type enzymes) show a functional Asp ⁄ Arg or Asp ⁄ Lys couple in a hypervariable loop very close to the acid–base catalyst. Enzymes lacking Table 2. 1-SST activity (1-kestose production from 200 mM sucrose)* of Lp1SST, Lp6 G -FFT ⁄ 1-FFT and the mutants derived from Lp6 G -FFT ⁄ 1-FFT. The table was reproduced from that shown in a previous publication [38]. Enzyme Activity (nkat mg )1 protein) Lp6 G -FFT ⁄ 1-FFT 5.38 ± 0.66 Lp1-SST 32.22 ± 0.69 N340D 1.71 ± 0.13 W343R 2.04 ± 0.67 S415N 3.32 ± 0.31 N340D+W343R 15.17 ± 0.23 N340D+S415N 2.69 ± 0.16 W343R+S415N 2.28 ± 0.04 N340D+W343R+S415N 21.17 ± 0.17 Fig. 11. Multiple alignment of a selection of plant GH32 VI ⁄ FT members in the regions surrounding the amino acids under study (bold). Both S-type and F-type enzymes can be discriminated. For comparison, structurally characterized representatives of S-type (AtcwINV1) and F-type (Ci1-FEH IIa) enzymes within the plant GH32 CWI ⁄ FEH subgroup are also presented (underlined). We refer the reader to a previous reference [30] for more extended multiple alignments within the CWI ⁄ FEH subgroup. *The artificial Cys in barley 1-SST probably represents a PCR mistake because > 20 expressed sequence tags carrying a Tyr were detected. Donor and acceptor substrate selectivity in GH32 W. Van den Ende et al. 5796 FEBS Journal 276 (2009) 5788–5798 ª 2009 The Authors Journal compilation ª 2009 FEBS this couple have Fru as the preferential donor substrate. By contrast, all FTs contain altered WMNDPNG and WGW motifs (Fig. 11), while all hydrolases (invertases, FEHs) show an intact hypervariable loop within the WMNDPNG motif and an intact WGW motif (Fig. 11). In all cases, only a few amino acids in the vicinity of the active site seem to control the substrate specificity. Using these simple rules, we are now close to predicting the functionality of new GH32 plant enzymes. 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Morvan-Bertrand A, Van den Ende W & Prud’homme MP (2006) Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT) ⁄ fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.) J Exp Bot 57, 3961–3961 38 Lasseur B, Schroeven L, Lammens W, Le Roy K, Spangenberg G, Manduzio H, Vergauwen R, Lothier J, Prud’homme MP & Van den Ende . REVIEW ARTICLE Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes Wim Van den Ende 1 , Willem Lammens 1,2 , Andre ´ Van. Suc donor substrate selectivity in all plant GH32 enzymes. In particular, all enzymes that are able to use Suc as a donor substrate (1-SSTs, 6-SFTs, VIs and CWIs)