An alternate sucrose binding mode in the E203Q Arabidopsis invertase mutant: an X-ray crystallography and docking study

In the present study, we report on the X-ray crystallographic structure of a GH32 invertase mutant, (i.e., the Arabidopsis thaliana cell-wall invertase 1-E203Q, AtcwINV1-mutant) in complex with sucrose. This structure was solved to reveal the features of sugar binding in the catalytic pocket. However, as demonstrated by the X-ray structure the sugar binding and the catalytic pocket arrangement is significantly altered as compared with what was expected based on previous X-ray structures on GH-J clan enzymes. We performed a series of docking and molecular dynamics simulations on various derivatives of AtcwINV1 to reveal the reasons behind this modified sugar binding. Our results demonstrate that the E203Q mutation introduced into the catalytic pocket triggers conformational changes that alter the wild type substrate binding. In addition, this study also reveals the putative productive sucrose binding modus in the wild type enzyme.     

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

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.     

Insights into the fine architecture of the active site of chicory fructan 1-exohydrolase: 1-kestose as substrate vs sucrose as inhibitor

* Invertases and fructan exohydrolases (FEHs) fulfil important physiological functions in plants. Sucrose is the typical substrate for invertases and bacterial levansucrases but not for plant FEHs, which are usually inhibited by sucrose. * Here we report on complexes between chicory (Cichorium intybus) 1-FEH IIa with the substrate 1-kestose and the inhibitors sucrose, fructose and 2,5 dideoxy-2,5-imino-D-mannitol. Comparisons with other family GH32 and 68 enzyme-substrate complexes revealed that sucrose can bind as a substrate (invertase/levansucrase) or as an inhibitor (1-FEH IIa). * Sucrose acts as inhibitor because the O2 of the glucose moiety forms an H-linkage with the acid-base catalyst E201, inhibiting catalysis. By contrast, the homologous O3 of the internal fructose in the substrate 1-kestose forms an intramolecular H-linkage and does not interfere with the catalytic process. Mutagenesis showed that W82 and S101 are important for binding sucrose as inhibitor. * The physiological implications of the essential differences in the active sites of FEHs and invertases/levansucrases are discussed. Sucrose-inhibited FEHs show a K(i) (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.     

Insights into the catalytic properties of bamboo vacuolar invertase through mutational analysis of active site residues

Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to family 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Boβfruct3 using Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid residues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L, E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K_m values of the four mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants W159L and E316Q exhibited lower k_cat values than the wild-type enzyme, but an increase in the k_cat value was observed for the mutants W159F and C317A. The results of this study demonstrate that these residues have individual functions in catalyzing sucrose hydrolysis.     

Transforming wheat vacuolar invertase into a high affinity sucrose:sucrose 1-fructosyltransferase

Vacuolar invertases (VIs) degrade sucrose to glucose and fructose. Additionally, the fructan plant wheat (Triticum aestivum) contains different fructosyltransferases (FTs), which have evolved from VIs by developing the capacity to bind sucrose or fructans as acceptor substrates. Modelling studies revealed a hydrogen bonding network in the conserved WMNDPNG motif of VIs, which is absent in FTs. In this study, the hydrogen bonding network of wheat VI was disrupted by site-directed mutagenesis in the 23WMNDPNG29 motif. While the single mutants (W23Y, N25S) showed a moderate increase in 1-kestose production, a synergistic effect was observed for the double mutant (W23Y+N25S), showing a 17-fold increase in transfructosylation capacity, and becoming a real sucrose:sucrose 1-fructosyltransferase. Vacuolar invertases are fully saturable enzymes, contrary to FTs. This is the first report on the development of a fully saturable FT with respect to 1-kestose formation. The superior kinetics (K(m) approximately 43 mM) make the enzyme useful for biotechnological applications. The results indicate that changes in the WMNDPNG motif are necessary to develop transfructosylating capability. The shift towards smaller and/or more hydrophilic residues in this motif might contribute to the formation of a specific acceptor site for binding of sugar, instead of water.     

Roles of cell-wall invertases and monosaccharide transporters in the growth and development of Arabidopsis

The hydrolysis of sucrose by cell-wall invertases (cwINV) and the subsequent import of hexoses into target cells appears to be crucial for appropriate metabolism, growth and differentiation in plants. Hexose uptake from the apoplast is catalysed by monosaccharide/H+ symporters (Sugar Transport Proteins or STPs), which have the potential to sense sugars. Import of extracellular hexoses may generate signals to orchestrate cellular activities, or simply feed metabolic pathways distinct from those fed by sucrose. It is predicted that Arabidopsis has six cwINV genes and at least 14 STP genes. These genes show different spatial and temporal patterns of expression, and several knock-out mutants have been isolated for analysis. AtSTP1 transports glucose, galactose, xylose, and mannose, but not fructose. It accounts for the majority of the AtSTP activity in vegetative tissues and its activity is markedly repressed by treatment with exogenous sugars. These observations are consistent with a role in the retrieval of cell-wall-derived sugars, for example, during carbohydrate limitation or cell expansion. The AtSTP1 gene is also expressed in developing seeds, where it might be responsible for the uptake of glucose derived from imported sucrose. The large number of AtcwINV and AtSTP genes, together with complex patterns of expression for each, and the possibility that each protein may have more than one physiological function, provides the plant with the potential for a multiplicity of patterns of monosaccharide utilization to direct growth and differentiation or to respond flexibly to changing environmental conditions.     

Acid and Neutral Invertases in the Mesocarp of Developing Muskmelon (Cucumis melo L. cv Prince) Fruit

Acid and neutral invertases were found in the mesocarp of developing muskmelon (Cucumis melo L. cv Prince) fruit and the activities of these enzymes declined with maturation of the fruit, concomitantly with the accumulation of sucrose. Neutral invertase was only present in the soluble fraction and acid invertase was present in both the soluble and cell-wall fractions. The cell-wall fraction contained three types of acid invertase: a NaCl-released invertase; an EDTA-released invertase, and a tightly bound invertase that still remained on the cell wall after treatment with NaCl and EDTA. The soluble acid and neutral invertases could be separated from one another by chromatography on DEAE-cellulose and they exhibited clear differences in their properties, namely, in their pH optima, substrate specificity, K_m values for sucrose, and inhibition by metal ions. The EDTA-released invertase and the soluble acid invertase were similar with regard to their chromatographic behavior on DEAE-cellulose, but the NaCl-released invertase was different because it was adsorbed to a column of CM-cellulose. The soluble acid invertase and two cell-wall bound invertases had very similar characteristics with regard to optimal pH and temperature, K_m value for sucrose, and substrate specificity.     

Relative substrate affinities of wild-type and mutant forms of the Escherichia coli sugar transporter GalP determined by solid-state NMR

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine the relative substrate-binding affinities of mutant forms of the Escherichia coli sugar transporter GalP in membrane preparations. The SSNMR method of (13)C cross-polarization magic-angle spinning (CP-MAS) is applied to five site-specific mutants (W56F, W239F, R316W, T336Y and W434F), which have a range of different sugar-transport activities compared to the wild-type protein. It is shown that binding of the substrate D-glucose can be detected independently of sugar transport activity using SSNMR, and that the NMR peak intensities for uniformly (13)C-labelled glucose are consistent with wild-type GalP and the mutants having different affinities for the substrate. The W239F and W434F mutants showed binding affinities similar to that of the wild-type protein, whereas the affinity of glucose-binding to the W56F mutant was reduced. The R316W mutant showed no detectable binding; this position corresponds to the second basic residue in the highly conserved (R/K)XGR(R/K) motif in the major facilitator superfamily of transport proteins and to a mutation in human GLUT1 found in individuals with GLUT1-deficiency syndrome. The T336Y mutant also showed no detectable binding; this mutation is likely to have perturbed helix structure or packing to an extent that conformational changes in the protein are hindered. The effects of the mutations on substrate-binding are discussed with reference to the putative positions of the residues in a 3D homology model of GalP based on the X-ray crystal structure of the E. coli glycerol-3-phosphate transporter GlpT.     

Structural determinant for cold inactivation of rodent L-xylulose reductase

L-Xylulose reductase (XR) is a homotetramer belonging to the short-chain dehydrogenase/reductase family. Human XR is stable at low temperature, whereas the enzymes of mouse, rat, guinea pig, and hamster are rapidly dissociated into their inactive dimeric forms. In order to identify amino acid residues that cause cold inactivation of the rodent XRs, we have here selected Asp238, Leu242, and Thr244 in the C-terminal regions of rodent XRs and performed site-directed mutagenesis of the residues of mouse XR to the corresponding residues (Glu, Trp, and Cys) of the human enzyme. Cold inactivation was prevented partially by the single mutation of L242W and the double mutation of L242W/T244C, and completely by the double mutation of D238E/L242W. The L242W and L242W/T244C mutants existed in both tetrameric and dimeric forms at low temperature and the D238E/L242W mutant retained its tetrameric structure. No preventive effect was exerted by the mutations of D238E and T244C, which were dissociated into their dimeric forms upon cooling. Crystallographic analysis of human XR revealed that Glu238 and Trp242 contribute to proper orientation of the guanidino group of Arg203 of the same subunit to the C-terminal carboxylate group of Cys244 of another subunit through the neighboring residues, Gln137 and Phe241. Thus, the determinants for cold inactivation of rodent XRs are Asp238 and Leu242 with small side chains, which weaken the salt bridges between Arg203 and the C-terminal carboxylate group, and lead to cold inactivation.     

Relative substrate affinities of wild-type and mutant forms of the Escherichia coli sugar transporter GalP determined by solid-state NMR

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine the relative substrate-binding affinities of mutant forms of the Escherichia coli sugar transporter GalP in membrane preparations. The SSNMR method of ¹³C cross-polarization magic-angle spinning (CP-MAS) is applied to five site-specific mutants (W56F, W239F, R316W, T336Y and W434F), which have a range of different sugar-transport activities compared to the wild-type protein. It is shown that binding of the substrate D-glucose can be detected independently of sugar transport activity using SSNMR, and that the NMR peak intensities for uniformly ¹³C-labelled glucose are consistent with wild-type GalP and the mutants having different affinities for the substrate. The W239F and W434F mutants showed binding affinities similar to that of the wild-type protein, whereas the affinity of glucose-binding to the W56F mutant was reduced. The R316W mutant showed no detectable binding; this position corresponds to the second basic residue in the highly conserved (R/K)XGR(R/K) motif in the major facilitator superfamily of transport proteins and to a mutation in human GLUT1 found in individuals with GLUT1-deficiency syndrome. The T336Y mutant also showed no detectable binding; this mutation is likely to have perturbed helix structure or packing to an extent that conformational changes in the protein are hindered. The effects of the mutations on substrate-binding are discussed with reference to the putative positions of the residues in a 3D homology model of GalP based on the X-ray crystal structure of the E. coli glycerol-3-phosphate transporter GlpT.     

Structural and Functional Analyses of β-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3

Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized—that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable β-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 Å. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 Å resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (α/β)₈ domain similar to a triose phosphate isomerase barrel, a five-stranded α/β sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second β-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the − 1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the − 1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.     

A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains

Sucrose has economic and environmental advantages over glucose as a feedstock for bioprocesses. E. coli is widely used in industry, but the majority of current industrial E. coli strains cannot utilize sucrose. Previous attempts to transfer sucrose catabolic capabilities into non-sucrose-utilizing strains have met with limited success due to low growth rates on sucrose and phenotypic instability of the engineered strains. To address these problems, we developed a transferrable sucrose utilization cassette which confers efficient sucrose catabolism when integrated onto the E. coli chromosome. The cassette was based on the csc genes from E. coli W, a strain which grows very quickly on sucrose. Both plasmid-borne expression and chromosomal integration of a repressor-less sucrose utilizing cassette were investigated in E. coli strains K-12, B and C. In contrast to previous studies, strains harboring chromosomal cassettes could grow at the same rate as they do on glucose. Interestingly, we also discovered that spontaneous chromosomal integration of the csc genes was required to allow efficient growth from plasmid-transformed strains. The ability to engineer industrial strains for efficient sucrose utilization will allow substitution of sucrose for glucose in industrial fermentations. This will encourage the use of sucrose as a carbon source and assist in transition of our petrochemical-based economy to a bio-based economy.     

Crystal structure of 6-SST/6-SFT from Pachysandra terminalis, a plant fructan biosynthesizing enzyme in complex with its acceptor substrate 6-kestose

Fructans play important roles as reserve carbohydrates and stress protectants in plants, and additionally serve as prebiotics with emerging antioxidant properties. Various fructan types are synthesized by an array of plant fructosyltransferases belonging to family 32 of the glycoside hydrolases (GH32), clustering together with GH68 in Clan-J. Here, the 3D structure of a plant fructosyltransferase from a native source, the Pachysandra terminalis 6-SST/6-SFT (Pt6-SST/6-SFT), is reported. In addition to its 1-SST (1-kestose-forming) and hydrolytic side activities, the enzyme uses sucrose to create graminan- and levan-type fructans, which are probably associated with cold tolerance in this species. Furthermore, a Pt6-SST/6-SFT complex with 6-kestose was generated, representing a genuine acceptor binding modus at the +1, +2 and +3 subsites in the active site. The enzyme shows a unique configuration in the vicinity of its active site, including a unique D/Q couple located at the +1 subsite that plays a dual role in donor and acceptor substrate binding. Furthermore, it shows a unique orientation of some hydrophobic residues, probably contributing to its specific functionality. A model is presented showing formation of a β(2-6) fructosyl linkage on 6-kestose to create 6,6-nystose, a mechanism that differs from the creation of a β(2-1) fructosyl linkage on sucrose to produce 1-kestose. The structures shed light on the evolution of plant fructosyltransferases from their vacuolar invertase ancestors, and contribute to further understanding of the complex structure-function relationships within plant GH32 members.     

Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass into a sucrose:sucrose 1-fructosyltransferase

Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose:sucrose 1-fructosyltransferase [1-SST], sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase [1-FFT], fructan:fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale.     

The role of Asp-295 in the catalytic mechanism of Leuconostoc mesenteroides sucrose phosphorylase probed with site-directed mutagenesis

Replacements of Asp-295 by Asn (D295N) and Glu (D295E) decreased the catalytic center activity of Leuconostoc mesenteroides sucrose phosphorylase to about 0.01% of the wild-type level (k(cat)=200s(-1)). Glucosylation and deglucosylation steps of D295N were affected uniformly, approximately 10(4.3)-fold, and independently of leaving group ability and nucleophilic reactivity of the substrate, respectively. pH dependences of the catalytic steps were similar for D295N and wild-type. The 10(5)-fold preference of the wild-type for glucosyl transfer compared with mannosyl transfer from phosphate to fructose was lost in D295N and D295E. Selective disruption of catalysis to glucosyl but not mannosyl transfer in the two mutants suggests that the side chain of Asp-295, through a strong hydrogen bond with the equatorial sugar 2-hydroxyl, stabilizes the transition states flanking the beta-glucosyl enzyme intermediate by > or = 23kJ/mol.     

Mutagenic analysis of conserved arginine residues in and around the novel sulfate binding pocket of the human Theta class glutathione transferase T2-2.

The human Theta class glutathione transferase GSTT2-2 has a novel sulfatase activity that is not dependent on the presence of a conserved hydrogen bond donor in the active site. Initial homology modeling and the crystallographic studies have identified three conserved Arg residues that contribute to the formation of (Arg107 and Arg239), and entry to (Arg242), a sulfate binding pocket. These residues have been individually mutated to Ala to investigate their potential role in substrate binding and catalysis. The mutation of Arg107 had a significant detrimental effect on the sulfatase reaction, while the Arg242 mutation caused only a small reduction in sulfatase activity. Surprisingly, the Arg239 had an increased activity resulting from a reduction in stability. Thus, Arg239 appears to play a role in maintaining the architecture of the active site. Electrostatic calculations performed on the wild-type and mutant forms of the enzyme are in good agreement with the experimental results. These findings, along with docking studies, suggest that prior to conjugation, the location of 1-menaphthyl sulfate, a model substrate for the sulfatase reaction, is approximately midway between the position ultimately occupied by the naphthalene ring of 1-menaphthylglutathione and the free sulfate. It is further proposed that the Arg residues in and around the sulfate binding pocket have a role in electrostatic substrate recognition.     

α-Galactosidase/sucrose kinase (AgaSK), a novel bifunctional enzyme from the human microbiome coupling galactosidase and kinase activities

α-Galactosides are non-digestible carbohydrates widely distributed in plants. They are a potential source of energy in our daily food, and their assimilation by microbiota may play a role in obesity. In the intestinal tract, they are degraded by microbial glycosidases, which are often modular enzymes with catalytic domains linked to carbohydrate-binding modules. Here we introduce a bifunctional enzyme from the human intestinal bacterium Ruminococcus gnavus E1, α-galactosidase/sucrose kinase (AgaSK). Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide-binding motif. Its biochemical characterization showed that AgaSK is able to hydrolyze melibiose and raffinose to galactose and either glucose or sucrose, respectively, and to specifically phosphorylate sucrose on the C6 position of glucose in the presence of ATP. The production of sucrose-6-P directly from raffinose points toward a glycolytic pathway in bacteria, not described so far. The crystal structures of the galactosidase domain in the apo form and in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding and suggesting a cross-talk between the galactose and kinase domains.     

Arabidopsis AtcwINV3 and 6 are not invertases but are fructan exohydrolases (FEHs) with different substrate specificities

The genome of Arabidopsis thaliana contains six putative cell-wall type invertase genes (AtcwINV1-6). Heterologous expression of AtcwINV1, 3 and 6 cDNAs in Pichia pastoris revealed that the enzymes encoded by AtcwINV3 and 6 did not show invertase activity. Instead, AtcwINV3 is a 6-FEH and AtcwINV6 is a fructan exohydrolase (FEH) that can degrade both inulin and levan-type fructans. For AtcwINV6 it is proposed to use the term (6&1) FEH. In contrast, AtcwINV1 is a typical invertase. FEH activity was also detected in crude extracts of different parts of Arabidopsis. To verify that the FEH activity of AtcwINV3 and 6 were not artefacts of the heterologous expression system, the protein corresponding to AtcwINV3 was isolated from whole Arabidopsis plants and indeed showed only 6-FEH activity and no invertase activity. Although no fructans can be detected in Arabidopsis plants, it is shown that kestoses (trimers) can be synthesized in crude leaf extracts. The putative physiological significance of FEH in so-called non-fructan plants is discussed.     

Characterization of two members of the maize gene family, Incw3 and Incw4, encoding cell-wall invertases

Two maize putative cell-wall invertase genes (Incw3 and Incw4) have been isolated by screening a genomic DNA library (Zea mays L. W22) using the cDNA probes encoding the two maize cell-wall invertases Incw1 and Incw2. The Incw3 and Incw4 genes contain six exons/five introns and five exons/four introns, respectively. The protein sequences deduced from both genes revealed a beta-fructosidase motif and a cysteine catalytic site known to be conserved in invertase genes. A detailed analysis of the protein and nucleotide sequences provides evidence that the Incw3 and the Incw4 genes encode putative cell-wall invertases. Furthermore, the isoelectric point deduced from the INCW4 protein sequence suggested that the Incw4 gene may encode a unique type of cell-wall invertase unbound in the apoplast. Gene expression studies using RT-PCR and in-situ RT-PCR hybridization showed that the Incw3 expression is organ/tissue-specific and developmentally regulated. In contrast, the Incw4 gene is constitutively expressed in all vegetative and reproductive tissues tested.