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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Unraveling the Difference between Invertases and Fructan Exohydrolases: A Single Amino Acid (Asp-239) Substitution Transforms Arabidopsis Cell Wall Invertase1 into a Fructan 1-Exohydrolase^1,[C]

Laboratory of Molecular Plant Physiology, Institute of Botany and Microbiology (K.L.R., W.L., B.D.C., A.V.L., W.V.d.E.) and Laboratory of Biocrystallography (W.L., M.V., A.R.), Katholieke Universiteit Leuven, B–3000 Leuven, Belgium

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Schematic structure of Suc (A) and 1-kestose (B).

Both vacuolar and cell wall invertases are grouped together with fructan biosynthetic and degrading enzymes and microbial -fructosidases in family 32 of glycoside hydrolases (GH32; www.cazy.org). Family GH32 can be combined in the clan GH-J together with the related family GH68 (Naumoff, 2001), harboring bacterial invertases, levansucrases, and inulosucrases. These enzymes use Suc as the preferential donor and transfer the fructosyl moiety to a variety of acceptors, including water (Suc hydrolysis), Suc, and fructan (levan or inulin). Within this GH-J clan, three-dimensional (3-D) structures of six enzymes have been determined, namely, the levansucrases from Bacillus subtilis (Meng and Fütterer, 2003; Protein Data Bank [PDB] code 1OYG) and Gluconacetobacter diazotrophicus (Martinez-Fleites et al., 2005; PDB code 1W18), an exoinulinase from Aspergillus awamori (Nagem et al., 2004; PDB code 1Y4W), a -fructosidase from Thermotoga maritima (Alberto et al., 2004; PDB code 1UYP; Alberto et al., 2006; PDB code 1W2T), a fructan 1-exohydrolase (1-FEH) from chicory (Cichorium intybus; Verhaest et al., 2005b; PDB code 1ST8), and, most recently, a cell wall invertase from Arabidopsis (Verhaest et al., 2006; PDB code 2AC1). All these proteins show a common fold; they consist of an N-terminal five-bladed -propeller domain (GH32 and GH68) followed by a C-terminal domain formed by two -sheets (only in GH32). Superposition of these structures indicates that the active sites are located in the -propeller domain. The active site is characterized by the presence of three highly conserved acidic groups that play a crucial role in the catalytic mechanism for the hydrolysis of the glycosidic bond (Reddy and Maley, 1990, 1996; Batista et al., 1999; Yanase et al., 2002).

A phylogenetic tree containing deduced amino acid sequences from vacuolar and cell wall invertases and fructan biosynthetic and breakdown enzymes revealed that FEHs are more related to cell wall invertases than to fructan biosynthetic enzymes, which, in turn, are more related to vacuolar invertases. FEHs and cell wall invertases are believed to have evolved from a common ancestor (Van den Ende et al., 2000). FEHs and invertases are both hydrolases but use different preferential donor substrates. Suc is the preferential donor substrate for cell wall invertases, but other donor substrates, such as 1-kestose (Fig. 1B), raffinose, and stachyose, were also reported to be degraded to a certain extent (Tymowska-Lananne and Kreis, 1998; De Coninck et al., 2005). It was reported that the side activity against raffinose is determined by a single amino acid (Goetz and Roitsch, 1999). Plant FEHs seem to be unifunctional enzymes, only degrading fructans but not Suc (Van Laere and Van den Ende, 2002), in contrast with microbial FEHs that can degrade Suc as well. Depending on the linkage type that is hydrolyzed, 1-FEH or inulinase (-2,1 linkages) and 6-FEH or levanase (-2,6 linkages) can be distinguished.

To gain insight into (1) which amino acids determine the preference of Glu-Fru linkage (Suc, raffinose, stachyose) hydrolysis versus Fru-Fru linkage (1-kestose and higher degree of polymerization [DP] fructans) hydrolysis and, more particularly, (2) which amino acids are important for the binding and degradation of Suc, site-directed mutagenesis studies were performed focusing on the identification of putative important residues as determined by sequence and 3-D structure comparisons. To our knowledge, this is the first report specifically considering the difference between GH32 hydrolases only differing in donor substrate specificity (Suc versus fructan). All studies were performed on Arabidopsis cell wall Invertase1 (AtcwINV1; Arabidopsis gene code At3g13790), a genuine cell wall invertase (De Coninck et al., 2005).

	RESULTS AND DISCUSSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

The Active Site of AtcwINV1 Consists of Three Highly Conserved Amino Acids

By multiple-sequence alignments of members of GH32, GH68, and GH43 (harboring -xylanases, -xylosidases, -L-arabinases, and -L-arabinofuranosidases), and GH62 (harboring some -L-arabinofuranosidases), three highly conserved amino acids, at an equivalent position in all the enzymes, can be identified (Pons et al., 2004). The general GH reaction mechanism requires at least two critical acidic residues, a proton donor, and a nucleophile. The reaction involves two major mechanisms resulting in either an overall retention (GH32 and GH68) or inversion (GH43) of the anomeric configuration (Davies and Henrissat, 1995). Several mutation experiments on enzyme members of these families and on a plant invertase in particular (Goetz and Roitsch, 2000), revealed a crucial role for Asp and Glu residues in substrate hydrolysis (Reddy and Maley, 1990, 1996; Batista et al., 1999; Nurizzo et al., 2002; Yanase et al., 2002; Meng and Fütterer, 2003; Ozimek et al., 2004; Altenbach et al., 2005; Ritsema et al., 2006). Recently, the 3-D structures of chicory 1-FEH IIa/AtcwINV1 were resolved, and the acidic residues Asp-22/Asp-23, Asp-147/Asp-149, and Glu-201/Glu-203 were considered as nucleophile, transition-state stabilizer, and acid/base catalyst, respectively (Verhaest et al., 2005b, 2006). These residues belong to the conserved NDPNG, RDP, and EC regions within the GH32 family (Fig. 2 ; Naumoff, 2001). Because the Asp of the RDP motif is considered to be too distant to take part in the catalytic process (Meng and Fütterer, 2003), we focused on Asp-23 and Glu-203 from AtcwINV1 and mutated them into Ala residues. The total activity, measured as the release of Fru from Suc, of wild-type and mutant enzymes, were compared and their kinetic parameters determined (Table I ). Compared to the wild-type enzyme, the k_cat for both mutant enzymes drastically decreased (600- to 900-fold), whereas the K_m was modified to a lesser extent (8-fold). The large reduction in k_cat values for both D23A and E203A mutants confirms that these residues are essential for catalysis in AtcwINV1. In accordance with previous studies on yeast (Saccharomyces cerevisiae) invertase (Reddy and Maley, 1996) in which the nucleophile and proton donor were identified, we conclude that the Asp-23 and Glu-203 residues in AtcwINV1 act as the nucleophile and proton donor in the hydrolysis reaction of Suc.

View larger version (7K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Multiple sequence alignment (using EMBL-EBI ClustalW) of three conserved regions of some plant GH32 enzymes. The following sequences were used for the alignment: AtcwINV1, chicory vacuolar invertase, 1-FEH IIa, 1-SST, 1-FFT, and an invertase from yeast. The three crucial catalytic residues are shown in bold. Arrows indicate the residues subjected to mutagenesis. Numbering refers to AtcwINV1.

A comparison of the AtcwINV1 and chicory 1-FEH IIa structures reveals some marked differences in their active site regions (Fig. 3 ). In AtcwINV1, an extra acidic residue (Asp-239) is observed in the vicinity of the conserved acid/base catalyst Glu-203 (Fig. 3A). An acidic amino acid is also found in 1-FEH IIa (Glu-234), but the acidic chain is twisted over 180° and oriented away from the active site (Fig. 3B). This different orientation is most probably due to the presence of a double deletion in the vicinity of this residue (Fig. 4 ). The presence of an Asp-239 homolog is highly conserved in cell wall invertases, a noticeable exception being Arabidopsis cell wall invertase 5 (Fig. 4). Besides cell wall invertases, vacuolar invertases in general also share this feature (data not shown). It can be hypothesized that the presence of an Asp-239 homolog contributes to the specificity of Suc recognition of plant acid invertases. By contrast, analogous to chicory 1-FEH IIa, all other known FEHs are generally characterized by the absence of such a structural Asp-239 homolog. In some cases, an acidic residue is present, but, analogous to chicory 1-FEH IIa, always surrounded by deletions (Fig. 4). Taking into account the orientation of Glu-234 in chicory 1-FEH IIa away from the active site, most probably due to a double deletion, it can be assumed that the presence of deletions in this region might be a reason for the absence of a structural Asp-239 homolog in FEHs.

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Overview of the 3-D structure of the active site of AtcwINV1 (PDB code 2AC1; A) and chicory 1-FEH IIa (PDB code 1ST8; B). Figures were prepared with PyMol (Delano, 2002). The sequence motifs containing the indicated residues are shown in Figures 2, 4, and 7: Asp-23/Asp-22 (DPN region), Asp-149/Asp-147 (RDP region), Glu-203/Glu-201 (EC region), Asp-239/Glu-234, Lys-242 (LDDTKH/FEG--H region), Trp-19/Trp-20 (WMN region), Trp-47/Phe-46 (WGN/FGD region), and Trp-82/Trp-82 (WSGSAT region). [See online article for color version of this figure.]

View larger version (39K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Multiple sequence alignment of the Asp-239/Lys-242 region (shown in bold) in cell wall invertases and all known plant FEHs. The following sequences were used for the alignment: Arabidopsis cell wall invertase 1, Solanum tuberosum cell wall invertase 1, Nicotiana tabacum cell wall invertase, S. tuberosum cell wall invertase 2, S. tuberosum cell wall invertase 3, tomato cell wall invertase Lin5, Vicia faba cell wall invertase, Pisum sativum cell wall invertase, V. faba cell wall invertase 2, Chenopodium rubrum cell wall invertase, Arabidopsis cell wall invertase 2, Arabidopsis cell wall invertase 4, Daucus carota cell wall invertase 1, Oryza sativa cell wall invertase, wheat cell wall invertase, Zea mays cell wall invertase 1, Z. mays cell wall invertase 2, Z. mays cell wall invertase 3, Arabidopsis cell wall invertase 5, Arabidopsis 6-FEH (ancient cell wall invertase 3), Beta vulgaris 6-FEH, chicory 1-FEH I, chicory 1-FEH IIa, chicory 1-FEH IIb, Campanula rapunculoides 1-FEH, Arabidopsis 6&1-FEH (ancient cell wall invertase 6), wheat 1-FEH w1, wheat 1-FEH w2, wheat 6&1-FEH, Hordeum vulgare 1-FEH, wheat 6-KEH w1, wheat 6-KEH w2, Lolium perenne 1-FEH, and wheat 6-FEH. Functionally characterized enzymes are marked with an asterisk.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Multiple sequence alignment of the three conserved hydrophobic residues (shown in bold) of some cell wall invertases: Arabidopsis cell wall invertase 1, S. tuberosum cell wall invertase 1, N. tabacum cell wall invertase, S. tuberosum cell wall invertase 2, S. tuberosum cell wall invertase 3, V. faba cell wall invertase, P. sativum cell wall invertase, V. faba cell wall invertase 2, O. sativa cell wall invertase, wheat cell wall invertase, Z. mays cell wall invertase 3, Z. mays cell wall invertase 1, and Z. mays cell wall invertase 2.

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. A, Chromatographic pattern of AtcwINV1 wild-type and Asp-239 mutant enzymatic activities. Reaction conditions were as follows: incubation of 50 ng purified enzyme with 10 mM Suc in 50 mM acetate buffer, pH 5.0, during 30 min at 30°C. B, Comparison of specific enzymatic activities [specific (enzymatic) activity in (mol Fru) x (mol enzyme)–1 (s)–1 and (nkat) x (mg protein)–1] of AtcwINV1 wild-type and Asp-239 mutant purified proteins in function of increasing substrate concentrations.

A superposition of the B. subtilis levansucrase and AtcwINV1 structures strongly suggests that Glu-340 in levansucrase can be considered as the functional (but not structural) homolog of Asp-239 in AtcwINV1 (Fig. 6A ). The presence of a Glu or Gln besides the proton donor Glu-342 is strictly conserved in levansucrases, implying a possible important role for this residue in binding and/or degradation of Suc in these enzymes. Indeed, it was shown that Glu-340 forms strong H-bridges with the Glc moiety of Suc (O3 and O4 groups) in the active site of B. subtilis levansucrase (Fig. 6B; Meng and Fütterer, 2003). As a consequence of these interactions, we can presume that Glu-342 is not hindered by these groups in its proton donation to the glycosidic oxygen. Regarding this superposition, it can be assumed that the Glu-340 residue is important for both Suc binding and degradation in B. subtilis levansucrase, although this was not checked by site-directed mutagenesis in this system. By analogy with these findings, our results on the Asp-239 AtcwINV1 mutants could be explained (Fig. 6C). Although the exact position of Suc in the active site of AtcwINV1 is unknown and the structure of the AtcwINV1-Suc complex remains to be determined, the orientation of Suc in AtcwINV1 can be predicted (Fig. 6C) based on the position of Suc in the active site of B. subtilis levansucrase because the position of the terminal fructosyl at the –1 subsite is equal for all GH32 and GH68 enzymes (Verhaest et al., 2007). An Asp-239/Glu-340 functional homolog can also be found in G. diazotrophicus levansucrase (Gln-399) and T. maritima invertase (Glu-188). On the contrary, chicory 1-FEH IIa lacks this additional residue. Like all other known plant FEHs, 1-FEH IIa is unable to degrade Suc. Moreover, Suc is a strong inhibitor of most FEHs, including 1-FEH IIa (De Roover et al., 1999). Unlike plant FEHs, microbial FEHs are able to degrade Suc to a certain extent (Nagem et al., 2004). Although A. awamori exoinulinase lacks a structural Asp-239 homolog, the function of this residue could probably be partly taken over by Trp-335.

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. A, Superposition of the active sites of AtcwINV1 (turquoise) and B. subtilis levansucrase (green; PDB code 10YG) showing similarities in the catalytic region. B, Details of Suc (orange) in the active site of B. subtilis levansucrase Glu-342A mutant (PDB code 1PT2) in which Ala-342 was replaced in silico by Glu-342 (wild-type orientation; PDB code 1OYG). C, Illustration of the putative position of Suc in the active site of AtcwINV1 based on the known structural complex of B. subtilis levansucrase with Suc. Putative Suc-protein interactions are indicated with dashed lines. Figures were prepared with PyMol (Delano, 2002).

Conserved Trp Residues Are Important for Stable Substrate Binding

Besides polar forces that are involved in the carbohydrate recognition of various enzymes, it has been reported that other interactions can play a distinctive role. Depending on the stereochemistry of the carbohydrate monomers, the presence of a number of apolar C-H groups (aromatic residues) can play an important role by stabilizing the sugar-enzyme complexes by means of interactions between the aromatic and sugar rings through van der Waals contacts and CH- stackings (Van der Veen et al., 2001; Muraki, 2002; Chavez et al., 2005).

At the rim of the AtcwINV1 active site, there is a prominent presence of three Trp residues, namely, Trp-20, Trp-47, and Trp-82, together forming an aromatic zone (Fig. 3A). These residues belong to the conserved (among invertases) WMNDPN, WGN, and WSGSAT regions (Fig. 7 ). Mutagenesis experiments were performed in which these Trp residues were changed into a Leu. Determination of the kinetic parameters of the purified mutant proteins and comparison with the wild-type enzyme revealed an important role for these hydrophobic residues in substrate binding (Table III ). K_m values increased 200, 600, and 120 times for the W20L, W47L, and W82L mutants, respectively, in comparison with the wild-type enzyme. The especially high value of the W47L mutant is not surprising because this residue is very close to the active site and the hydrophobic ring has a good orientation for interacting with the substrate. As a consequence of the very unstable substrate binding in all three mutants, the release of the Glc and Fru residues out of the active site probably takes place at a higher velocity, giving rise to an increase of the k_cat values in two of the three cases.

1-Kestose as a Substrate for AtcwINV1: The D239A Mutant Acts as a FEH

Cell wall invertases can, besides Suc, degrade other substrates like 1-kestose, raffinose, and stachyose, although to a much lesser extent. Because 1-kestose is the preferential substrate of chicory 1-FEH IIa (De Roover et al., 1999) and an Asp-239 homolog is missing in 1-FEH IIa (Fig. 3B), we investigated whether the breakdown of 1-kestose by AtcwINV1 was also affected by mutagenesis on the Asp-239 residue. The kinetic parameters for the degradation of 1-kestose were determined for the wild-type, D239A, and D239F mutant proteins (Table IV ). There was no marked difference in K_m and k_cat values between the wild-type and D239F mutant proteins. D239A was seriously affected in its ability to degrade Suc (Fig. 5; Table II), but retained its ability to degrade 1-kestose, supporting the idea that plant FEHs lacking invertase activity can evolve from cell wall invertases by a minimal number of mutations or deletions. Similarly, degradation of raffinose and stachyose was affected to the same extent as observed for Suc (data not shown). Higher DP inulin-type fructans were found to be very poor substrates for AtcwINV1 (De Coninck et al., 2005) and for the mutant D239A enzyme (data not shown), so the mutant D239A enzyme can in fact be considered as a special 1-FEH preferentially degrading 1-kestose, the smallest inulin-type fructan (a 1-kestose exohydrolase [1-KEH]). Enzymes that preferentially degrade fructan trisaccharides were recently discovered in fructan plants. Specific 6-kestosidase (6-KEH) enzymes have been purified and cloned from wheat (Triticum aestivum; Van den Ende et al., 2005) and 1-KEH activities were reported in onion (Allium cepa; Benkeblia et al., 2005).

Asp-239 as a Reliable Determinant for Prediction of Functionality within the Group of Plant Cell Wall Invertases/FEHs

Without exception, all functionally characterized FEHs lack an Asp-239 homolog and are unable to break down Suc (Van den Ende et al., 2000, 2001, 2003a, 2003b, 2005; De Coninck et al., 2005; Van Riet et al., 2006). On the contrary, the presence of such an Asp-239 homolog seems to be a general feature of cell wall invertases. Unfortunately, only four so-called cell wall invertases are functionally characterized as real invertases (Sturm and Crispeels, 1990; Roitsch et al., 1995; Fridman et al., 2004; De Coninck et al., 2005). Functional characterization and reliable modeling studies (data not shown) now revealed that three of six of the previously termed cell wall invertase genes of Arabidopsis do not encode real invertases, but defective invertases/FEHs with different substrate specificities (De Coninck et al., 2005). Based on mutagenesis data, extensive functional characterization, and modeling studies on many FEHs, Asp-239 can be proposed as a reliable marker to predict whether a new, uncharacterized member within the cell wall invertase/FEH group encodes a real invertase or a defective invertase/FEH.

	CONCLUSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
The GH32 family harbors -fructosidases, invertases, FEHs, and various types of fructosyltransferases, differing only in their donor and acceptor substrate specificities. High levels of amino acid sequence similarities between these enzymes reveal evolutionary relationships. Fructan biosynthesizing enzymes are closely related to vacuolar invertases (Vijn and Smeekens, 1999; Francki et al., 2006; Ritsema et al., 2006), whereas FEHs are closely related to cell wall invertases, suggesting that they both evolved from a common -fructosidase ancestor (Van den Ende et al., 2000). Site-directed mutagenesis-based data on AtcwINV1 demonstrate an important role for the Asp-239 residue in both binding and hydrolysis of Suc. A hydrophobic zone at the rim of the active site further stabilizes the optimal binding of Suc. Moreover, an D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose. In general, GH32 family enzymes containing an Asp-239 functional homolog have Suc as a preferential donor, whereas enzymes lacking this homolog use fructans as their donor substrate. Asp-239 is proposed as a reliable determinant for the identification of noncharacterized members within the group of cell wall invertases/FEHs.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Cloning and Site-Directed Mutagenesis

Arabidopsis (Arabidopsis thaliana) AtcwINV1 (gene accession no. At3g13790) was cloned into the pPICZA vector as described by De Coninck et al. (2005). Single amino acid substitutions were generated following the Quick Change protocol (Stratagene), using the pPICZA-AtcwINV1 construct as a template. For site-directed mutagenesis, the following forward oligonucleotide primers (and complementary reverse primers) were used: 5'-GGATGAACGCTCCTAATGGG-3' (D23A), 5'-GGAAGTGGAATGTGGGCATGTCCTGATTTTTTC-3' (E203A), 5'-GAAAATAAGTTTGGCCGACACGAAACATG-3' (D239A), 5'-GTTGAAAATAAGTCTGTTCGACACGAAACATG-3' (D239F), 5'-GTTGAAAATAAGTCTGAACGACACGAAACATG-3' (D239N), 5'-GTTTGGACGACACGCTACATGATTATTAC-3' (K242L), 5'-CCCCCAAAAATTTGATGAACGATCC-3' (W20L), 5'-GGAGCCGTGTTGGGTAACATC-3' (W47L), and 5'-CAACGGATGCCTGTCCGGTTCAG-3' (W82L; mutations are in bold). After DpnI digestion, an additional purification step was performed (QiaQuick PCR purification kit; Qiagen) and 3 µL of the purified plasmids were used for transformation of Escherichia coli TOP10 cells. Mutations were confirmed by sequence analysis.

Expression and Purification

The methylotrophic yeast Pichia pastoris was used for extracellular gene expression as described in De Coninck et al. (2005). Purification of the protein from the culture supernatant was achieved by 80% ammonium sulfate precipitation. After centrifugation (8,500g, 30 min at 4°C), the pellet was redissolved in 50 mM sodium acetate buffer, pH 5.0. The solution was further centrifuged (10,000g, 10 min at 4°C) to spin down undissolved material. The supernatant was subsequently dialyzed in 50 mM sodium acetate buffer, pH 5.0, for 4 h at 4°C. As a final purification step, the dialyzed solution was loaded onto a Mono S column as described in Verhaest et al. (2005a). The purity of the protein was checked by SDS-PAGE and a single band could be observed after gel staining with Coomassie Blue. Concentration measurements of purified enzymes were performed using the Bradford method with bovine serum albumin as a standard (Sedmak and Grossberg, 1977). The amount of pure protein yielded from one P. pastoris expression culture varied depending on the P. pastoris clone and ranged between 50 and 200 µg.

Enzyme Assays

For kinetic determination, appropriate aliquots of enzyme were mixed with Suc or 1-kestose (final concentration ranging from 250 µM to 1 M) in 50 mM sodium acetate buffer, pH 5.0. Reaction mixtures were incubated at 30°C for different time periods. Sodium azide (0.02% [v/v]) was added to prevent microbial growth. Total enzyme activity was determined by measuring the amount of released Fru by anion-exchange chromatography with pulsed amperometric detection (Van den Ende and Van Laere, 1996). For all reactions, different time points were analyzed and only data from the linear range were used. The experiments were repeated three times with consistent results. Kinetic parameters (K_m and k_cat) were determined using the linear Hanes plot.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: AY079422 (Arabidopsis cell wall invertase 1), AJ419971 (chicory [Cichorium intybus] vacuolar invertase), AJ295033 (chicory 1-FEH IIa), U81520 (chicory 1-SST), U84398 (chicory 1-FFT), P10594 (yeast [Saccharomyces cerevisiae] invertase), Q43171 (Solanum tuberosum cell wall invertase 1), X81834 (Nicotiana tabacum cell wall invertase), Q9M4K7 (S. tuberosum cell wall invertase 2), Q9M4K8 (S. tuberosum cell wall invertase 3), AJ272304 (tomato [Lycopersicum esculentum] cell wall invertase Lin5), Z35162 (Vicia faba cell wall invertase), AK118343 (Arabidopsis cell wall invertase 2), AB049617 (Arabidopsis cell wall invertase 4), X69321 (Daucus carota cell wall invertase 1), AF063246 (Pisum sativum cell wall invertase), Z35163 (V. faba cell wall invertase 2), X81792 (Chenopodium rubrum cell wall invertase), AB073749 (Oryza sativa cell wall invertase), AF030420 (wheat [Triticum aestivum] cell wall invertase), AF050129 (Zea mays cell wall invertase 1), AF050128 (Z. mays cell wall invertase 2), AF050631 (Z. mays cell wall invertase 3), AB029310 (Arabidopsis 6-FEH [ancient cell wall invertase 3]), BAB01929 (Arabidopsis cell wall invertase 5), AJ508534 (Beta vulgaris 6-FEH), AJ242538 (chicory 1-FEH), AJ295034 (chicory 1-FEH IIb), AJ509808 (Campanula rapunculoides 1-FEH), AY060533 (Arabidopsis 6&1-FEH [ancient cell wall invertase 6]), AJ516025 (wheat 1-FEH w1), AJ508387 (wheat 1-FEH w2), AB089269 (wheat 6&1-FEH), AJ605333 (Hordeum vulgare 1-FEH), AB089271 (wheat 6-KEH w1), AB089270 (wheat 1-FEH w2), DQ073968 (Lolium perenne 1-FEH), and AM075205 (wheat 6-FEH).

	ACKNOWLEDGMENTS

 
We thank Rudy Vergauwen for his outstanding technical assistance. We are also very grateful to Ed Etxeberria and Marc De Maeyer for critical reading of the manuscript.

Received July 3, 2007; accepted September 7, 2007; published September 14, 2007.

	FOOTNOTES

 
¹ This work was supported by the Fund for Scientific Research (grants to W.V.d.E. and A.R.) and the Institute for the Promotion of Innovation through Science and Technology in Flanders (grant to B.D.C.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wim Van den Ende (wim.vandenende{at}bio.kuleuven.be).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.105049

^* Corresponding author; e-mail wim.vandenende{at}bio.kuleuven.be.

	LITERATURE CITED

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
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