Structure-function relations and evolution of ... · fructans mostly reach a DP of 10 to 200 and are very diverse in structure [3]. In plants fructans occur in many prominent orders

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Structure-function relations and evolution of fructosyltransferases

Denise Altenbach1 and Tita Ritsema2 1Zürich-Basel Plant Science Center, Universität Basel, Hebelstrasse 1 4055 Basel, Switzerland; 2Phytopathology, Universiteit Utrecht Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands

Abstract Plant fructosyltransferases (FTs) are divided in several groups according to the substrates they use and the type of glycosidic bond they form. FTs presumably evolved from vacuolar invertases by meansof small mutational changes. Invertases are dependent upon a catalytic triad for activity, a characteristic now also shown for FTs. Enzymatic specificity of FTs is encoded in the large subunit and partially resides in the sucrose-binding box. Single amino acid changes in FTs and vacuolar invertase revealed that enzymespecificity with respect to glycosidic bond formed and hydolysis versus transfer ratio are easily modulated.

Correspondence/Reprint request: Dr. Tita Ritsema, Phytopathology, Universiteit Utrecht, Sorbonnelaan 16 3584 CA Utrecht, The Netherlands. E-mail: [email protected]

Denise Altenbach & Tita Ritsema 2

In FTs a binding site for the acceptor substrate seems to have evolved, apparently at the rim of the (donor) substrate-binding pocket. 1. Fructans: Occurrence, structure and physiological function The disaccharide sucrose consists of glucose and fructose and is the main transport sugar in all plants. It can furthermore serve as reserve carbohydrate. Sucrose – dissolved in large quantities in the vacuole – and starch – stored in insoluble from in the amyloplasts or temporary in the chloroplasts – are by far the most common reserve carbohydrates in higher plants. Apart from these, about 15 % of flowering plants use fructans as reserve carbohydrate [1-4]. Fructans are “extensions of sucrose”: They consist of linear or branched fructose chains attached to sucrose. As highly water soluble molecules fructans are predominantly stored in the vacuole [5]. Depending on the plant species, fructans mostly reach a DP of 10 to 200 and are very diverse in structure [3]. In plants fructans occur in many prominent orders like the Asterales, the Liliales, and the Poales, among which are representatives of economic importance (e.g. wheat, barley, onion) [2,6,7]. Fructans are classified according to their differences in glycosidic linkage type (Fig. 1). Linear fructans called inulins are composed of ß(2-1) linked fructosyl units. They typically occur in the order of Asterales (e.g. chicory). Linear fructans containing primarily or exclusively ß(2-6) linkages occurring in many forage grasses (Poaceae), are called phleins [8]. Grasses often contain mixed fructan-types where ß(2-1) and ß(2-6) fructosyl linkages are combined within one molecule. These fructans, which occur for example in wheat and barley, are called graminans (branched fructans). Graminans sometimes are of even more complex structures where the fructose chains, linked ß(2-1) and ß(2-6), are elongated on two sites of the starter sucrose: at the C1 of the fructose, and/or at the C6 of the glucose residue (e.g. Lolium perenne; Fig. 1)[9]. Fructans elongated at two sites of the sucrose started unit are called neo-series fructan. They most often occur as inulin neo-series and are widespread in the Liliales and Asparagales (e.g. onion and garlic and asparagus) [10]. In plants, fructan mainly serves as a reserve carbohydrate. Storing fructan instead of sucrose as soluble reserve carbohydrate has several advantages: as soluble polysaccharide fructans are osmotically less active than sucrose, and can therefore be stored in much higher concentrations. Since fructans are highly water soluble and accumulate in the vacuole, the largest cell compartment, storage of very large quantities is possible. In sink organs like roots, tubers, bulbs or stems, as well as in source organs like mature leaves, high fructan concentrations (up to 70% of dry weight) can be stored. Generally fructans are stored if photosynthetic carbon production exceeds demands, and are mobilized if carbon and energy is needed. An example is the rapid

Enzymology of fructosyltransferases 3

Figure 1. Representation of sucrose and the derived first representatives of different fructan types. The enzymes for synthesis are indicated using their abbreviations. Arrows indicate the possible sites of fructose chain elongation for the different groups. Abbreviations: 1-SST (sucrose:sucrose 1-fructosyltransferase); 6-SST (sucrose:sucrose 6-fructosyltransferase); 6-SFT (sucrose:fructan 6-fructosyltransferase); 1-FFT (fructan:fructan 6-fructosyltransferase); 6-FFT (fructan:fructan 6-fructosyltransferase); 6G-FFT (fructan:fructan 6G-fructosyltransferase).


breakdown of fructan stored in the leaf base upon defoliation of grasses, providing energy and building stones for the re-growth of leaves [11]. If carbon fixation in a leaf exceeds export and demands, accumulation of sucrose can lead to a feedback inhibition of photosynthesis. In this situation the ability to synthesize fructan is a physiological advantage, since vacuolar fructan synthesis lowers the concentration of sucrose in the cell and thus, prevents sugar-induced feedback inhibition of photosynthesis [6,12]. For most plants the main reserve carbohydrate is starch. It can accumulate in “source”-leaves as transitory starch in the chloroplasts, or in reserve organs in amyloplasts. Like the accumulation of fructan, storage of transitory starch can lower the sucrose concentration in the leaf, but storage of equivalent amounts of starch as observed with fructans, would inevitably obstruct the chloroplasts and consequently interfere with photosynthesis [1]. Starch biosynthesis decreases dramatically when the temperature drops below 10 °C, whereas photosynthesis and fructan production are much less sensitive to low temperature [7]. Thus, plants having the possibility to accumulate fructans instead of or in addition to starch can optimally react to environmental conditions andtherefore have physiological advantages. Fructans are furthermore thought to be able to protect plants against drought and freezing stress. This assumption is supported by the observation that fructan-accumulating plants are especially abundant in temperate and arid climate zones with seasonal frost or drought periods, and are almost absent in tropical regions [2]. Because the cell membranes are primary targets for both freezing and desiccation injuries [13], fructans are supposed to be involved in the stabilization of membranes. Indeed, it was shown using in vitro systems, that fructo-oligosaccharides enhance membrane stability during freezing and cellular dehydration through their affinity to phospholipids [13-16]. In planta it was shown, that tobacco (Nicotiana tabaccum) transformed with a bacterial levansucrase, had enhanced drought and freezing resistance [17-19]. Other functions found for fructan metabolism is partitioning of assimilates induced by biotic or abiotic factors [6]. A rapid sequence of accumulation and breakdown of fructans in the growth zone of barley leaves [20] and during anthesis in Campanula rapunculoides [21] and daylily [22] flowers are examples that lead to the assumption that fructans play a role in cell expansion. Depolymerization of fructans probably contributes to the osmotic driving force involved in cell expansion. Interestingly also algae (green algae or Chlorophyta) and microorganisms are capable of synthesizing fructans [2]. Bacterial strains from genera such as Bacillus, Lactobacillus, and Streptococcus produce fructan extracellularly [2,23-25]. Bacterial fructans are called levans and are generally composed of ß(2-6) linked fructosyl residues linked to a terminal sucrose and can reach a DP of up to 100’000. Remarkably, also ß(2-1) linked fructans and branched


fructans are found. There are also reports on the synthesis of extracellular fructans by fungi, e.g. Aspergillus and Penicillium [26,27]. In microorganisms, fructans that are extracellularly produced might act as adhesive material around plant roots or leaves [28]. Also for oral fructan synthesizing streptococci such as Streptococcus mutans, fructans are believed to serve as a glue and readily mobilized carbohydrate, enhancing the formation of dental plaque [29]. Interest in fructans increased during the last decade due to health-promoting effects of fructans for humans. Inulin, mainly isolated from chicory roots, is added to a variety of products like yoghurt and bread as a food additive. Long chain fructans act as emulsifiers and give a better mouth feeling to products like fat-free yoghurt. Short chain fructans and oligofructose can serve as sweeteners. Fructans act as soluble food fibers, because the human digestive tract contains no enzymes to degrade the ß(2-1) and ß(2-6) glycosidic linkages. Therefore, fructans pass from the small intestine into the large intestine without being digested. In the bowel fructans are utilized preferably by beneficial bacteria such as Bifidobacterium and Lactococcus. This effect of advantageously altering the balance in the bacterial flora of the intestine is thought to increase gut health [30]. Further beneficial effects of fructan to human health are reported such as an increased calcium resorption, or a lowering of the concentrations of thriglycerides, cholesterol, and insulin [31-34]. 2. Enzymes involved in fructan metabolism The central transport sugar in plants sucrose is the starting point of fructan metabolism (Fig. 1). Sucrose is synthesized in the cytoplasm by the sequential action of sucrose-phosphate synthase and sucrose-phosphate phosphatase, and it can be reversibly cleaved by sucrose synthase, or irreversibly hydrolyzed by invertases [35]. Invertases in plants exist in several isoforms with different biochemical properties and subcellular locations [36]. In plants invertases, also called ß-fructosidases, consist of different groups; acid invertases, consisting of vacuolar invertases and cell-wall invertases, and neutral/alkaline invertases, also called cytosolic invertases. Besides cleaving sucrose, also hydrolysis of low DP fructans as well as of raffinose and stachiose has been reported using acid invertases. In contrast, alkaline invertases are sucrose specific [37]. For the understanding of fructan metabolizing enzymes the acid invertases are of special importance. Synthesis of plant fructans requires fructosyltransferases (FTs) that catalyze the transfer of fructosyl units from a donor substrate (sucrose or fructan) to an acceptor substrate (sucrose or fructan). Synthesis is always initiated by 1-SST (sucrose:sucrose 1-fructosyltransferase), producing the shortest fructan with a ß(2-1) linkage called 1-kestose (and glucose), from two molecules of sucrose. In this case sucrose serves as both a fructosyl donor and acceptor (Fig. 1; Fig. 2).


Figure 2. Main activities of known fructosyltransferases in plants. The degree of polymerization (DP) is indicated for fructans with m ≥3 and n ≥2 (if n = 2 the molecule is sucrose, this is strictly speaking not a fructan). Chain elongation to higher DP fructan occurs via the action of 1-FFTs (fructan:fructan 1-fructosyltransferases), 6-SFTs (sucrose:fructan 6-fructosyl transferases) and/or 6G-FFTs (fructan:fructan 6G-fructosyltransferases), depending on glycosidic bonds present in the fructan types synthesized by different plant species. Inulin type fructans are synthesized by the elongation of 1-kestose via successive attachment of fructosyl units by the action of 1-FFT (Fig.2) [38-40]. 1-FFT uses one fructan as a fructosyl donor and attaches it to another fructan or sucrose, thereby shortening one fructan and elongating another one. Sucrose can be used as fructosyl acceptor but not as donor substrate. For different Asterales it was shown that their 1-FFTs determine the inulin chain length [41,42]. The two enzymes 1-SST and 1-FFT can only form inulin, ß(2-1) linked fructans, but cereals such as wheat and barley form other types of fructan, graminans, that have primarily ß(2-6) linkages between the fructosyl units [43]. The only enzyme so far known to form ß(2-6) linkages in cereals, 6-SFT, has been purified and cloned first from barley [44,45]. The preferred substrates of 6-SFT are sucrose and 1-kestose leading to the formation of the tetrasaccharide bifurcose which is the smallest branched fructan, and glucose


(Fig 1; Fig. 2). In the presence of sucrose as the only substrate, the activity of 6-SFT is mainly hydrolytic, leading to the production of glucose and fructose, and only 20% of total activity is directed into the production of 6-kestose. Evidence for the existence of a 1-FFT in barley was obtained by Lüscher and coworkers while purifying barley 1-SST [46]. In wheat a 1-FFT was cloned that preferentially makes ß(2-1) branches on fructan molecules containing ß(2-6) links [47]. Studies of oat fructan showed, that also fructans of the neo-series occur in grasses [48]. However, no 6G-FFT -the enzyme known to form these fructans in liliaceae- has been cloned to date in grasses. 6G-FFT from onion can use both sucrose and low DP inulin as fructosyl acceptors, whilst fructosyl donors can be 1-kestose and low DP inulin, but not sucrose (Fig. 2) [49]. The two FTs known in onion are 1-SST and 6G-FFT. It was shown, that transgenic tobacco BY2 cells expressing onion 6G-FFT and incubated with 1-kestose produced the same fructan-pattern as it is found in onion bulbs [50]. Thus, no 1-FFT seems to be needed in onion for the formation of higher DP fructans of the neo-series. In contrast, in the neo-series fructan producing asparagus, 1-SST, 6G-FFT and 1-FFT have been shown to be involved in fructan synthesis [51,52]. Breakdown of fructan is thought to proceed via fructan exo-hydrolases (FEH; (Fig. 2)), since increased FEH activity correlates with fructan breakdown [53-56]. FEHs degrade fructan polymers by splitting off terminal fructosyl residues. Up to now, no evidence for fructan endohydrolases has been found in plants. FEHs preferentially hydrolyzing ß(2-1)-bonds (1-FEH) and/or ß(2-6)-bonds (6-FEH) have been distinguished [53,54,57-59]. Generally sucrose has a strong inhibitory effect on FEHs and seems not to be hydrolyzed [60]. Surprisingly, FEH genes and activities have been recently detected in non-fructan plants, where they might play a role in defense, acting on microbial (exogenous) fructans [61,62]. Glycoside hydrolases have been classified into 87 families based on the similarity of their overall amino acid sequences, assuming that this reflects both structural and mechanistic relationships [63]. The database of “carbohydrate-active enzymes” (CAZY, http://afmb.cnrs-mrs.fr/-cazy/CAZY/index.html) groups bacterial invertases and levansucrases into glycoside hydrolase (GH) family 68, whilst fungal and plant invertases and FTs fall into GH family 32. Both these families are members of the glycoside hydrolase clan GH-J [63,64]. The overall sequence homology between the two families is less than 15% although they catalyze very similar reactions. 3. Evolution Utilization of sucrose as a source of carbon and energy depends on its cleavage into hexoses. Sucrose can be hydrolyzed to its components glucose and fructose by the enzyme invertase. In plants, invertases are ubiquitous.


If the protein sequence of any plant acid invertase is compared to sequences of proteins from the kingdom of bacteria using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) the highest similarities are detected with bacterial invertases, levanases, inulinases, sucrose 6-phosphate hydrolases and levansucrases (Fig. 3). Similarly, comparison of a plant acid invertase sequence against proteins from the fungal kingdom, retrieves the sequences of invertases, inulinases, and of fructosyltransferases. If the protein sequence of a plant acid invertase is compared to sequences of plant proteins, the first sequences retrieved correspond to invertases, FTs, and FEHs. There are five significantly similar proteins found in the kingdom of Protista (e.g. Leishmania), and only one in Archaea (Haloarcula marismortui). Interestingly, there are no significant similarities when plant acid invertases are compared to all available sequences of animal proteins. Animals hydrolyze sucrose using the enzyme sucrase, which is a structurally totally unrelated enzyme. Plants contain also neutral/alkaline invertase. This enzyme has no significant similarity in its amino acid sequence with acid invertases. Interestingly only neutral invertases and no acid invertases are reported for blue algae (cyanobacteria) such as for example Anabaena variabilis [65]. There are no homologues of neutral invertases in any other kingdom. In summary, the enzyme class of acid invertases and FTs is predominantly found in the kingdoms of bacteria, fungi and plants. Their abundance in these only distantly related organisms implies an extremely large protein family.

Figure 3. Kingdoms harboring “acid invertase-like” enzymes.


In the plant kingdom invertases with different biochemical properties and subcellular locations are omnipresent [37]. In contrast, FTs are of rather limited distribution and restricted to a few although partially very large plant families [2]. Comparison of the amino acid sequences of plant FTs and acid invertases revealed a very high degree of identity [3,45]. The occurrence in unrelated and ‘young’ plant families as well as the close homology of the enzymes indicate that the capacity for fructan synthesis is a relatively novel trait that most probably developed independently in several genera. If vacuolar invertases are presented in a phylogenetic tree together with FTs, FEHs and cell-wall invertases, they cluster with FTs whilst FEHs cluster with cell-wall invertases (Fig. 4). The following evolutionary process from invertases to FTs was proposed [66]: an ancestral invertase gene duplicated before the divergence of monocots and dicots. One duplicate evolved into cell-wall invertase isoforms and FEHs, and the other evolved into the vacuolar invertases and various FTs. Because of the high degree of similarity of the amino acid sequences, it was speculated that vacuolar invertases were recruited for generating FTs by means of small mutational changes [2,3,45,67].

Figure 4. (picture taken from [4]): Phylogenetic tree of fructosyltransferases, fructan exohydrolases and invertases in monocots (m) and dicots (d). acGFT, Alium cepa 6G-FFT (Y07838); acINV, Allium cepa invertase (AJ006067); acSFT, Agropyron cristatum 6-SFT (AF211253); acSST, Allium cepa 1-SST (AJ006066); aoINV, Asparagus officinalis invertase (AF002656); asSST, Allium sativum 1-SST (AY098442); atINV, Arabidopsis thaliana invertase (AY142666); ciCIN, Cichorium intybus cell wall invertase (Y11124); ciFEH1, Cichorium intybus 1-FEH I (AJ242538);


Figure 4. Legend continued ciFEH2, Cichorium intybus 1-FEH II (AJ295033); ciFFT, Cichorium intybus 1-FFT (U84398); ciINV, Cichorium intybus invertase (AJ419971); ciSST, Cichorium intybus 1-SST (U81520); csFFT, Cynara scolymus 1-FFT (AJ000481); csSST, Cynara scolymus 1-SST (Y09662); faSST, Festuca arundinaceae 1-SST (AJ297369); htFFT, Helianthus tuberosus 1-FFT (AJ009756); htSST, Helianthus tuberosus 1-SST (AJ009757); hvSFT, Hordeum vulgare 6-SFT (X83233); leCIN, Lycopersicon esculentum cell wall invertase (AF506006); leINV, Lycopersicon esculentum invertase (D11350); lpSFT 6-SFT Lolium perenne (AF494041); lpSST, Lolium perenne 1-SST (AF492836); osCIN, Oryza sativa cell wall invertase (AB073749); osINV, Oryza sativa invertase (AF019113); psSFT, Poa secunda 6-SFT (AF192394); taCIN, Triticum aestivum cell wall invertase (AF030420); taFEH, Triticum aestivum FEH (AJ508387); taSFT, Triticum aestivum 6-SFT (accession number AB029887); taSST, Triticum aestivum 1-SST (AB029888); toSST, Taraxacum officinale 1-SST (AJ250634); zmCIN, Zea mays cell wall invertase (U17695); zmINV, Zea mays invertase (U16123). 4. Molecular and biochemical properties

Both vacuolar invertases and FTs are formed as vacuolar targeted preproenzymes with an N-terminal signal sequence and a propeptide that are cleaved off after protein folding and final targeting [45,68]. The common feature of plant acid invertases and FTs is to consist of an approximately 80 kD protein, which is cleaved during maturation yielding a N-terminal large subunit and a C-terminal small subunit (Fig. 5) [45,69-71]. Whether or not this cleavage has a physiological function is not clear. As to the physiological functions of the subunits, it was shown that the small subunit is needed to get active FTs but that the specificity of the enzyme is encoded in the large subunit [72]. In contrast to this, FEHs are not cleaved in two subunits. Also bacterial fructosyltransferases consist of one subunit only. At the biochemical level, further similarities between plant FTs and vacuolar invertases are evident. FTs use sucrose as a substrate, just as vacuolar invertases. Vacuolar invertases cleave sucrose, just as FTs do with their donor substrate. The different FT enzymes may differ in their preferential fructosyl donor and acceptor substrates. Besides their main activity, each FT generally catalyses also fructosyl- transfers from and to alternative substrates, albeit at a lower efficiency. This depends on the type and substrate concentrations provided, the temperature and the ionic strength [3,43,73]. For example barley (Hordeum vulgare) 6-SFT and tall fescue (Festuca arundinacea; re-classified as Schedonorus arundinaceus) 1-SST exhibit invertase activity in addition to their main activity. When sufficient amounts of sucrose and 1-kestose are present, 6-SFT guides 80% of its total activity into fructan synthesis, forming bifurcose, and only 20% into the hydrolysis of sucrose. If only sucrose is available as substrate, 6-SFT acts almost purely as a hydrolase [45]. Thus the


Figure 5. Scheme of barley 6-SFT cDNA. Highly conserved motifs are indicated.

enzyme is not only highly homologous to vacuolar invertases at the level of amino acid sequence but also retains considerable invertase activity. On the other hand invertases are well known to exhibit some FT activity under certain conditions, forming 1-kestose at high concentrations of sucrose [74,75]. Thus FTs harbor the intrinsic capacity to act as hydrolases and vice versa. 5. The catalytic triad

The general enzymatic mechanism proposed for glycoside hydrolase family 32 is a ping-pong mechanism via an enzyme-fructosyl intermediate (Fig. 6), as was first proposed for GH68 [76]. It involves the protonation of the glycosidic oxygen followed by a nucleophile attack on the anomeric carbon of the sugar substrate by a carboxylate group. The reaction requires three acidic amino acids: (I) The catalytic nucleophile for the covalent binding of the fructose residue, (II) an acid/base catalyst that functions as a proton donor and, (III) an amino acid that is not directly involved in catalysis but acts as a transition state stabilizer. Alignments and experimental studies with yeast extracellular invertase and bacterial levansucrase, such as affinity labelling, site-directed mutagenesis and random mutagenesis propose three conserved regions involved in the reaction containing the following motifs: The ß-fructosidase motif (Fig. 6), the EC-motif and the RDP motif (Fig. 5). The ß-fructosidase motif [77] consists of the amino acids NDPNG for all known acid invertases, whereas it is quite variable in FTs (NDPNG, SDPNG, ADPNA etc.) (Fig. 7) [78]. The ß-fructosidase motif was shown to be essential for activity in yeast invertase (GH32) where Asp23 was identified as the catalytic nucleophile [77, 79]. With respect to the catalytic nucleophile in GH68, mutational studies have been reported for FTs of Lactobacillus reuteri 121 and Bacillus subtilis levansucrase [80, 81].


Figure 6. Cartoon of the ping-pong mechanism proposed for FTs. Depicted is the activity of 1-SST on sucrose. A) the donor sucrose binds in the substrate pocket, B) sucrose is cleaved and glucose is released whereas fructose resides in the pocket, C) the sucrose acceptor substrate binds, D) the acceptor substrate is coupled to fructose resulting in the product 1-kestose.

Figure 7. Comparisons of N-terminal 33 amino acids of the mature protein of several vacuolar invertases and FTs. The sucrose-binding boxes are boxed, the ß-fructosidase motive is boldface. The numbers represent the position of the conserved Tyr (Y) residue at the end of the sequences. L, Liliaceae; P, Poaceae; A, Asteraceae; off., officinale; tuber., tuberosus. Accesion numbers; Allium cepa INV, AJ006067; A. cepa 6G-FFT, ACY07838; Asparagus officinale INV, AF002656; Hordeum vulgare INV, AJ623275; Lolium perenne INV, AY082350; Cichorium intybus INV, AJ419971; A. cepa 1-SST, AJ006066; H. vulgare 1-SST, AJ567377; L. perenne 1-SST, AY245431; Triticum aestivum 1-SST, AB029888; C. intybus 1-SST, U81520; Helianthus tuberosus 1-SST, AJ009757; H. vulgare 6-SFT, X83233; L. perenne 6-SFT, AF494041; T. aestivum 6-SFT, AB029887; C. intybus 1-FFT, U84398; H. tuberosus 1-FFT, AJ009756; A. officinale 6G-FFT, AB084283.


The Glu204 of the EC-motif in yeast invertase (GH32) is proposed as proton donor during sucrose hydrolysis [77,79]. This conclusion is strengthened further by site-directed mutagenesis and structural analysis of Bacillus subtilis levansucrase (GH68), where the mutation of Glu342 to Ala completely abolished enzyme activity [81]. The mechanism proposed by Reddy and Maley for the cleavage of sucrose, basically involves the above mentioned nucleophile, attacking the fructose of sucrose, and a proton donor which might be the glutamate of the EC-motif. In the first step glucose is released and fructose is bound to the nucleophile in an ester linkage. Then this ester linkage can be hydrolyzed using water and thereby releasing the fructose. This mechanism for sucrose hydrolysis is also proposed for the polymerizing activity of members of GH family 32 and GH family 68, how subsequent transfer to the acceptor substrate is achieved is up-to-date unclear. All studies available concerning the highly conserved RDP motif were done with enzymes from family GH68. Studies in bacterial levansucrase propose that the Asp in this motif forms a transient covalent fructosyl intermediate or acts as a transition state stabilizer [82-84]. Some amino acids in the vicinity of the RDP motif, namely Arg331 in Bacillus subtilis levansucrase and the His296 in Zymomonas mobilis levansucrase, were shown to be important for maintaining the enzyme’s polymerizing activity. In contrast, the hydrolyzing activity was not affected by changing these amino acids [84,85]. The above described studies were performed using extracellular yeast invertase or bacterial FTs, enzymes showing relatively low overall homologies to plant acid invertases and FTs. But since the ß-fructosidase motif, the EC-motif and the RDP motif are also very conserved in plant FTs, a similar mechanism for sucrose hydrolysis and/or the fructosyl-transfer to sucrose and/or fructan can be expected. Similarly as proposed in the model of yeast invertase, the corresponding glutamate (EC-motif) of plant FTs could serve as a proton donor after a nucleophile attack on sucrose and/or fructan by the aspartate of the ß-fructosidase motif. Recently two papers shed more light on the role of the presumed nucleophile, proton donor and transition state stabilizer. Modification of the presumed nucleophile of a plant FT into Glu resulted in a total loss of activity [78]. Furthermore, the proposed transition-state stabilizer is also essential for enzyme activity in plant FTs [86]. A very interesting phenomenon was observed when mutations in the proton donor were analyzed. This study was done in a 1-SST where replacing the Glu with Ala resulted in an enzyme that did not react with sucrose (the preferred substrate of 1-SSTs), but showed still residual activity when 1-kestose -normally the product of the reaction- was fed [86].


6. Structure-function relationships FTs can differ in their donor and acceptor substrate specificities as well as in the type of glycosidic bond they create (Fig. 1 and Fig. 2). What structural components cause these specificities is not easily detected from the primary sequences. Several papers dealing with enzyme specificities of FTs have recently been published. One set uses onion 6G-FFT and concentrate on the sucrose-binding box region [78,87], two others use barley 6-SFT and festuca 1-SST [72,86]. The 1-SST versus 6-SFT activity is studied using chimerical enzymes consisting of part of the 6-SFT combined with part of the 1-SST. Specificity resides in the large subunit, but expression of the large subunit on its own does not give an active enzyme [72]. Therefore, it can be concluded that the C-terminal small subunit is needed for enzymatic activity, probably by enhancing protein stability, but does not contribute to the specificity of the reaction catalyzed. Furthermore, also the N-terminal 108 amino acids of 6-SFT -containing the sucrose-binding box- could be replaced by the homologous region of 6-SFT without altering enzyme specificity [86]. Chimeras containing the N-terminal 36 amino acids of either onion 1-SST or invertase fused to the C-terminus of onion 6G-FFT showed a more diverse picture. The chimera containing invertase did retain full 6G-FFT specificity, but the chimera harboring 1-SST did not since the capacity to synthesize neo-series fructan was lost [87]. This shows that the sucrose-binding box region is able to influence product specificity of FTs. Single amino acid mutations of amino acids in the sucrose-binding box of 6G-FFT furthermore show that one single substitution of the amino acid N-terminal of the nucleophile can already influence enzymatic specificity of FTs quite dramatically [78,87]. Another very intriguing question –also with respect to the presumed evolution of FTs from vacuolar invertases- is what structural components of the enzymes in GH32 determine if sucrose is used for glycosyltransfer or just hydrolyzed. The region in which the sucrose-binding box resides was investigated to study its influence on transferase activity. Replacing the amino terminal 33 amino acids (including the sucrose-binding box) of vacuolar invertase with the corresponding region of the FT 6G-FFT (both from onion) led to a shift in activity from sucrose hydrolysis to sucrose polymerization [88]. Subsequent research into the amino acids responsible for the enhanced polymerization activity revealed that amino acids at positions 3 N-terminal and 2 C-terminal of the nucleophile are essential for the shift towards transferase activity. Changing a single amino acid at either of these positions in the sucrose-binding box increases the transglycosilation capacity of invertase. Combining the two mutations had an additive effect on transglycosilation capacity. The mutations generated functionally correspond to natural variation present in the sucrose-binding boxes of known invertases and FTs. Therefore,


conversion of these amino acids seems central in the evolution of invertases to fructosyltransferases. Interestingly the amino acid N-terminal of the nucleophile is a mutation from Trp (invertase) to Tyr (6G-FFT). Many sugar-binding enzymes rely on aromatic amino acids for close contact with sugar residues, therefore, conserved aromatic amino acid residues can be considered as candidates influencing the enzymatic activities of hydrolases and/or transferases. Pons and coworkers [64] pointed out before that invertases have a Trp residue 3 amino acids N-terminal of the nucleophile, whereas fructosyltransferases have a Phe or Tyr, but never a Trp (Fig. 7). Taking the experimental results into this one can conclude that the link of Trp with hydrolase activity and Phe or Tyr with polymerase activity is a functional one. In contrast to this, the reciprocal mutations of Tyr to Trp in festuca 1-SST and onion 6G-FFT did not influence transferase activities [87,89]. Therefore it seems that for the development of transferase activities in invertase the mutation has great value, but once an enzyme has evolved to an optimized FT the amino acid has become less important. To shed some light on the influence of amino acids outside of the sucrose-binding box region, a mutational analysis was performed based on primary sequence comparisons between highly homologous hydrolases and FTs [89]. For this analysis acid invertase and 1-SST were chosen since both use sucrose as their preferred substrate. In an alignment including all known SSTs and selected vacuolar invertases from both fructan-producing as well as non-producing plants, nine amino acids dispersed along the sequence correlated with either hydrolase or transferase activity. Subsequent mutational analysis showed that also the mutation of single amino acids outside the sucrose-binding box region could already cause a considerable shift in the catalytic specificity and activity (Fig. 8). For invertase the one extra mutant that increased transferase activity is again a Trp (amino acid 440) to Tyr mutation. This finding strongly supports the theory that fructosyltransferases evolved from hydrolyzing acid invertases via small alterations in the amino acid sequence. The relative ease with which polymerisation capacity of invertases can be increased might furthermore explain the polyphyletic origin of the fructan accumulation trait [2].

Figure 8. Schematic overview of the distribution of amino acids that alter enzymatic specificity of FTs or vacuolar invertases. The positions are indicated by an asterix.


According to our view, the catalytic specificity of these enzymes basically involves a binding site for the fructosyl donor and a binding site for the fructosyl acceptor. The simplest reaction is catalyzed by invertases. They initially bind the fructosyl moiety of sucrose, upon cleavage glucose is released and in a second step fructose is released via a reaction with water. 1-SSTs also initially cleave the bond between the glucose and fructose moiety of sucrose and retain the fructosyl residue, but then they transfer the fructose to another sucrose -the acceptor substrate- (Fig. 6). In contrast to invertases, 1-SSTs must therefore harbor a binding-site for the fructosyl acceptor sucrose. This can be considered as an essential step in the evolution from vacuolar invertases towards FTs. A third enzyme which is also capable of using sucrose as fructosyl donor is 6-SFT. Like invertases, 6-SFTs cleave the bond between glucose and fructose of sucrose, maintain the fructosyl moiety, but transfer it to either sucrose or fructan (forming kestose or bifurcose). Like 1-SSTs, 6-SFTs must therefore have a binding-site for a fructosyl acceptor, but with a different specificity than 1-SSTs. 6G-FFTs and 1-FFTs lost the capacity to use sucrose as fructosyl donor, but instead use a fructan of DP≥3 as donor. The fructosyl acceptor can be either sucrose or a fructan. The basic difference compared to the previously described enzymes is that the donor-binding site can only accept fructan. Another mechanistic difference between 6-SFT, 6G-FFT and other FTs is the position of the acceptor substrate in the binding pocket. Since 6-SFT makes ß(2-6) glycosidic linkages and 6G-FFT couples fructose moieties to the glucose residue of the (starter) sucrose -instead of the fructose- the acceptor substrates have to be positioned differently according to the transferred fructose than in 1-SST and 1-FFT.

7. Three-dimensional structures The first crystal structure of a FT elucidated was that of a bacterial levansucrase from family GH68 [81]. From family GH32 the first crystal structure published was that of the extracellular invertase from the bacterium Thermotoga maritima [90] and no plant FT is crystallized up-to-now. Within GH family 32 the structures of the exo-inulinase from Aspergillus awamori and of the fructan-exohydrolase (1-FEH) from chicory are also elucidated [91,92]. Bacillus subtilis levansucrase was crystallized in both the ligand-free and -bound (sucrose) state. The exo-inulinase from Aspergillus awamori was crystallized in the fructose-bound state, and the invertase from Thermotoga maritima and the FEH from chicory in the ligand-free states only. All structures described above show a five-bladed ß-propeller with a deep negatively charged central pocket that binds the substrate (Fig. 9). Each blade consists of five ß-sheets that adopt the classical “W” topology of four antiparallel ß-strands that are named A to D. The N-terminal A strand lines the central cavity, and the C-terminal D-strand the periphery. The ß-sheets are


Figure 9. 3D presentation of Cichorium intybus 1-FEH (PDB accession 1ST8 ). The central pocket of the ß-barrel is the substrate-binding pocket that contains the catalytic triad. packed face-to-face and show a characteristic propeller blade-like twist. In contrast to the crystallized levansucrase, the structures from enzymes of GH32 have a second module, namely a C-terminal ß-sandwich (Fig. 9). This ß-sandwich consists of two sheets of six ß-strands. The two modules are linked via a linker. The structures provide a template for other members of GH family 32 such as acid invertases and plant FTs. The C-terminal ß-sandwich module corresponds roughly to the small subunit found in plant vacuolar invertases and FTs. The active site in the available structures is positioned at one end of the cavity at the center of the ß-propeller, with a funnel like opening towards the surface of the molecule. This central pocket is heavily negatively charged. The pocket in the crystal structure of B. subtilis accommodates a single sucrose molecule [81]. The fructosyl unit of sucrose is located at the bottom of the pocket with the glucose moiety on top. The pocket is composed almost exclusively of amino acids that belong to highly conserved sequence motifs which includes the catalytic triad. Thermotoga maritima invertase and chicory 1-FEH were crystallized only in the ligand-free state, their crystal structures revealed a glycerol molecule that was present in the active site. It mimics the O4’ and O6’ hydroxyl-groups of the fructose-moiety of sucrose or inulin respectively. This feature helped defining the precise position of the modeled substrate molecule in the active site. All 3D-structures reveal that


the Asp of the ß-fructosidase motif and the Glu of the EC-motif are in the ideal position to be in close contact with their substrate -sucrose or fructan. No crystal structure of a plant FT or a plant acid invertase is yet available. These plant enzymes are only up to 15% identical to enzymes of GH68 such as B. subtilis levansucrase and should rather be compared to members of their own family, namely GH32. From the available structures, chicory FEH is the closest relative to plant FTs and acid invertases with an amino acid sequence identity of 30 to 40%. What can be learned from the crystal structures published up to now? Based on the extensive overlap of the active site of the available 3D-structures, the fructosyl donor binding site can be relatively well specified. Especially because the exo-inulinase from Aspergillus awamori was crystallized in the fructose-bound state. The donor binding site includes the three motifs that were also emerging from biochemical studies, namely the NDPNG (ß-fructosidase), the RDP and the EC-motif. The reaction scheme in the case of 1-FEH has been interpreted as follows [92]: a fructan of the inulin type initially binds to the active site, where the glycosidic oxygen of the inulin is protonated by the Glu of the EC-motif. This step is followed by a nucleophile attack of the Asp in the NDPNG motif, forming a covalent fructosyl-intermediate. The third step of the reaction is the hydrolysis of the fructosyl-intermediate releasing fructose and the enzyme in its ligand-free state. The same scheme can be applied to invertases and plant FTs where instead of inulin, sucrose, 1-kestose or another fructan acts as fructosyl donor and binds to the Asp of the NDPNG motif. Thus the fructosyl donor binding step is basically solved. However, it is currently unknown what structures account for donor preferences. Furthermore, it is unknown where the fructosyl acceptor binds and what determines acceptor preferences. Interestingly, three of the mutations altering transglycosylating ability in 1-SST or vacuolar invertase are predicted to reside very close together at the entrance of the active-site pocket (Fig. 10), indicating a hotspot for enzymatic specificity in the 3D structure [88, 89]. The two mutations that directly line the sucrose-binding pocket are both Trp to Tyr mutations in invertase. In this region binding of the acceptor substrate in FTs might be determined. In order to discuss such key questions, one of the main goals in the near future should be the structural elucidation of a plant FT. A possible hindrance in the crystallization of a plant FT might be protein N-glycosylation. Chicory FEH, with only two glycosylation sites, is the only plant member of GH32 to be successfully crystallized so far. Crystallizing for example Festuca 1-SST, an enzyme carrying six potential glycosylation sites, might be a tedious but path-breaking task.


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Structure-function relations and evolution of ... · fructans mostly reach a DP of 10 to 200 and are very diverse in structure [3]. In plants fructans occur in many prominent orders