Understanding the role of defective invertases in plants: tobacco Nin88 fails to degrade sucrose.

Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development, and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their highly attenuated sucrose (Suc)-hydrolyzing capacity is due to the absence of aspartate-239 (Asp-239) and tryptophan-47 (Trp-47) homologs, crucial players for stable binding in the active site and subsequent hydrolysis. However, so far, the precise roles of such defective cwINVs remain unclear. In this paper, we report on the functional characterization of tobacco (Nicotiana tabacum) Nin88, a presumed fully active cwINV playing a crucial role during pollen development. It is demonstrated here that Nin88, lacking both Asp-239 and Trp-47 homologs, has no invertase activity. This was further supported by modeling studies and site-directed mutagenesis experiments, introducing both Asp-239 and Trp-47 homologs, leading to an enzyme with a distinct Suc-hydrolyzing capacity. In vitro experiments suggest that the addition of Nin88 counteracts the unproductive and rather aspecific binding of tobacco cwINV1 to the wall, leading to higher activities in the presence of Suc and a more efficient interaction with its cell wall inhibitor. A working model is presented based on these findings, allowing speculation on the putative role of Nin88 in muro. The results presented in this work are an important first step toward unraveling the specific roles of plant defective cwINVs.

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 ß(2-6) fructosyl linkage on 6-kestose to create 6,6-nystose, a mechanism that differs from the creation of a ß(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.

Expansive evolution of the trehalose-6-phosphate phosphatase gene family in Arabidopsis.

Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDP-glucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS- and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from whole-genome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-ß-glucuronidase/green fluorescent protein reporter lines further uncovered cell- and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.

Structural insights into the pH-controlled targeting of plant cell-wall invertase by a specific inhibitor protein.

Invertases are highly regulated enzymes with essential functions in carbohydrate partitioning, sugar signaling, and plant development. Here we present the 2.6 Å crystal structure of Arabidopsis cell-wall invertase 1 (INV1) in complex with a protein inhibitor (CIF, or cell-wall inhibitor of ß-fructosidase) from tobacco. The structure identifies a small amino acid motif in CIF that directly targets the invertase active site. The activity of INV1 and its interaction with CIF are strictly pH-dependent with a maximum at about pH 4.5. At this pH, isothermal titration calorimetry reveals that CIF tightly binds its target with nanomolar affinity. CIF competes with sucrose (Suc) for the same binding site, suggesting that both the extracellular Suc concentration and the pH changes regulate association of the complex. A conserved glutamate residue in the complex interface was previously identified as an important quantitative trait locus affecting fruit quality, which implicates the invertase-inhibitor complex as a main regulator of carbon partitioning in plants. Comparison of the CIF/INV1 structure with the complex between the structurally CIF-related pectin methylesterase inhibitor (PMEI) and pectin methylesterase indicates a common targeting mechanism in PMEI and CIF. However, CIF and PMEI use distinct surface areas to selectively inhibit very different enzymatic scaffolds.

Unexpected presence of graminan- and levan-type fructans in the evergreen frost-hardy eudicot Pachysandra terminalis (Buxaceae): purification, cloning, and functional analysis of a 6-SST/6-SFT enzyme.

About 15% of flowering plants accumulate fructans. Inulin-type fructans with ß(2,1) fructosyl linkages typically accumulate in the core eudicot families (e.g. Asteraceae), while levan-type fructans with ß(2,6) linkages and branched, graminan-type fructans with mixed linkages predominate in monocot families. Here, we describe the unexpected finding that graminan- and levan-type fructans, as typically occurring in wheat (Triticum aestivum) and barley (Hordeum vulgare), also accumulate in Pachysandra terminalis, an evergreen, frost-hardy basal eudicot species. Part of the complex graminan- and levan-type fructans as accumulating in vivo can be produced in vitro by a sucrose:fructan 6-fructosyltransferase (6-SFT) enzyme with inherent sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan 6-exohydrolase side activities. This enzyme produces a series of cereal-like graminan- and levan-type fructans from sucrose as a single substrate. The 6-SST/6-SFT enzyme was fully purified by classic column chromatography. In-gel trypsin digestion led to reverse transcription-polymerase chain reaction-based cDNA cloning. The functionality of the 6-SST/6-SFT cDNA was demonstrated after heterologous expression in Pichia pastoris. Both the recombinant and native enzymes showed rather similar substrate specificity characteristics, including peculiar temperature-dependent inherent 1-SST and fructan 6-exohydrolase side activities. The finding that cereal-type fructans accumulate in a basal eudicot species further confirms the polyphyletic origin of fructan biosynthesis in nature. Our data suggest that the fructan syndrome in P. terminalis can be considered as a recent evolutionary event. Putative connections between abiotic stress and fructans are discussed.

Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana.

Over the past decades, considerable advances have been made in understanding the crucial role and the regulation of sucrose metabolism in plants. Among the various sucrose-catabolizing enzymes, alkaline/neutral invertases (A/N-Invs) have long remained poorly studied. However, recent findings have demonstrated the presence of A/N-Invs in various organelles in addition to the cytosol, and their importance for plant development and stress tolerance. A cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) member of the A/N-Invs have been analysed in more detail in Arabidopsis and it was found that At-A/N-InvA knockout plants show an even more severe growth phenotype than At-A/N-InvG knockout plants. The absence of either A/N-Inv was associated with higher oxidative stress defence gene expression, while transient overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts down-regulated the oxidative stress-responsive ascorbate peroxidase 2 (APX2) promoter. Moreover, up-regulation of the APX2 promoter by hydrogen peroxide or abscisic acid could be blocked by adding metabolizable sugars or ascorbate. A hypothetical model is proposed in which both mitochondrial and cytosolic A/N-Invs can generate glucose as a substrate for mitochondria-associated hexokinase, contributing to mitochondrial reactive oxygen species homeostasis.

Freezing tolerance by vesicle-mediated fructan transport.

Fructans are fructose-based polymers associated with freezing tolerance. They might act directly via membrane stabilization or indirectly by stimulating alternative cryoprotectants. Fructans and fructan biosynthetic enzymes, in general, are believed to be present in the vacuole. This paper draws particular attention to the surprising presence of fructans and fructan exohydrolase activity in the apoplast of cold-stressed plants. This observation raises questions concerning the origin of apoplastic fructans and suggests that fructans are transported to the apoplast by post-synthesis mechanisms, perhaps induced by cold. We propose a conceptual vesicle-mediated transport model for the movement of vacuolar fructans to the apoplast, where they could assist in stabilizing the plasma membrane.

Crystal structure of Arabidopsis thaliana neutral invertase 2.

The metabolism of sucrose is of crucial importance for life on Earth. In plants, enzymes called invertases split sucrose into glucose and fructose, contributing to the regulation of metabolic fluxes. Invertases differ in their localization and pH optimum. Acidic invertases present in plant cell walls and vacuoles belong to glycoside hydrolase family 32 (GH32) and have an all-ß structure. In contrast, neutral invertases are located in the cytosol and organelles such as chloroplasts and mitochondria. These poorly understood enzymes are classified into a separate GH100 family. Recent crystal structures of the closely related neutral invertases InvA and InvB from the cyanobacterium Anabaena revealed a predominantly α-helical fold with unique features compared with other sucrose-metabolizing enzymes. Here, a neutral invertase (AtNIN2) from the model plant Arabidopsis thaliana was heterologously expressed, purified and crystallized. As a result, the first neutral invertase structure from a higher plant has been obtained at 3.4 Šresolution. The hexameric AtNIN2 structure is highly similar to that of InvA, pointing to high evolutionary conservation of neutral invertases.

Structural insights into glycoside hydrolase family 32 and 68 enzymes: functional implications.

Glycoside hydrolases (GH) have been shown to play unique roles in various biological processes like the biosynthesis of glycans, cell wall metabolism, plant defence, signalling, and the mobilization of storage reserves. To date, GH are divided into more than 100 families based upon their overall structure. GH32 and GH68 are combined in clan GH-J, not only harbouring typical hydrolases but also non-Leloir type transferases (fructosyltransferases), involved in fructan biosynthesis. This review summarizes the recent structure-function research progress on plant GH32 enzymes, and highlights the similarities and differences compared with the microbial GH32 and GH68 enzymes. A profound analysis of ligand-bound structures and site-directed mutagenesis experiments identified key residues in substrate (or inhibitor) binding and recognition. In particular, sucrose can bind as inhibitor in Cichorium intybus 1-FEH IIa, whereas it binds as substrate in Bacillus subtilis levansucrase and Arabidopsis thaliana cell wall invertase (AtcwINV1). In plant GH32, a single residue, the equivalent of Asp239 in AtcwINV1, appears to be important for sucrose stabilization in the active site and essential in determining sucrose donor specificity.

Creating S-type characteristics in the F-type enzyme fructan:fructan 1-fructosyltransferase of Triticum aestivum L.

Invertases cleave sucrose in glucose and fructose, using water as an acceptor. Fructosyltransferases catalyse the transfer of a fructosyl residue between sucrose and/or fructan molecules. Plant fructosyltransferases (FTs) evolved from vacuolar invertases by small mutational changes, leading to differences in substrate specificity. The S-type of enzymes (invertases, sucrose:sucrose 1-fructosyltransferases or 1-SSTs, and sucrose:fructan 6-fructosyltransferases or 6-SFTs) prefer sucrose as the donor substrate while F-type enzymes (fructan:fructan 1-fructosyltransferases or 1-FFTs and fructan:fructan 6(G)-fructosyltransferases or 6(G)-FFTs) preferentially use fructan as the donor substrate. Recently, a functional Asp/Arg or Asp/Lys couple in the Hypervariable Loop (HVL) was suggested to be essential to keep Asp in a favourable orientation for binding sucrose as the donor substrate in S-type enzymes. However, the F-type enzyme 1-FFT of Triticum aestivum (Ta1-FFT) also contains the Asp/Arg couple in the HVL, although it prefers fructan as the donor substrate. In this paper, mutagenesis studies on Ta1-FFT are presented. In Ta1-SST, Tyr282 (the Asp281 homologue) seems to be essential in creating a tight H-bond Network (HBN) in which the Arg-residue of the Asp/Arg couple is held in a fixed position. This tight HBN is disrupted in Ta1-FFT, leading to a more flexible Arg-residue and a dysfunctional Asp/Arg couple. A single D281Y mutation in Ta1-FFT restored the tight HBN and introduced typical S-type characteristics. Conclusively, in wheat FTs Asp281 (and its homologues) is involved in donor substrate specificity.

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.

Influencing the binding configuration of sucrose in the active sites of chicory fructan 1-exohydrolase and sugar beet fructan 6-exohydrolase.

The hydrolytic plant enzymes of family 32 of glycoside hydrolases (GH32), including acid cell wall type invertases (EC 3.2.1.26), fructan 1-exohydrolases (1-FEH; EC 3.2.1.153) and fructan 6-exohydrolases (6-FEH; EC 3.2.1.154), are very similar at the molecular and structural levels, but are clearly functionally different. The work presented here aims at understanding the evolution of enzyme specificity and functional diversity in this family by means of site-directed mutagenesis. It is demonstrated for the first time that invertase activity can be introduced in an S101L mutant of chicory (Cichorium intybus) 1-FEH IIa by influencing the orientation of Trp 82. At high sucrose and enzyme concentrations, a shift is proposed from a stable inhibitor configuration to an unstable substrate configuration. In the same way, invertase activity was introduced in Beta vulgaris 6-FEH by introducing an acidic amino acid in the vicinity of the acid-base catalyst (F233D mutant), creating a beta-fructofuranosidase type of enzyme with dual activity against sucrose and levan. As single amino acid substitutions can influence the donor substrate specificity of FEHs, it is predicted that plant invertases and FEHs may have diversified by introduction of a very limited number of mutations in the common ancestor.

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.

Crystallization and preliminary X-ray diffraction study of a cell-wall invertase from Arabidopsis thaliana.

Cell-wall invertase 1 (AtcwINV1), a plant protein from Arabidopsis thaliana which is involved in the breakdown of sucrose, has been crystallized in two different crystal forms. Crystal form I grows in space group P3(1) or P3(2), whereas crystal form II grows in space group C222(1). Data sets were collected for crystal forms I and II to resolution limits of 2.40 and 2.15 A, respectively.

pKa modulation of the acid/base catalyst within GH32 and GH68: a role in substrate/inhibitor specificity?

Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst.

Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes.

Plant family 32 glycoside hydrolase enzymes include hydrolases (cell wall invertases, fructan exohydrolases, vacuolar invertases) and fructosyltransferases. These enzymes are very similar at the molecular and structural levels but are functionally different. Understanding the basis of the functional diversity in this family is a challenging task. By combining structural and site-directed mutagenesis data, Asp239 in AtcwINV1 was identified as an amino acid critical for binding and stabilizing sucrose. Plant fructan exohydrolases lack such an Asp239 equivalent. Substitution of Asp239 led to the loss of invertase activity, while its introduction in fructan exohydrolases increased invertase activity. Some fructan exohydrolases are inhibited by sucrose. The difference between the inhibitor (fructan exohydrolase) and the substrate (invertase) binding configurations of sucrose can be explained by the different orientation of Trp82. Furthermore, the evolutionary hydrolase/transferase transition could be mimicked and the difference between S-type fructosyltransferases (sucrose as donor) and F-type fructosyltransferases (fructan as donor) could be unravelled.

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.

Crystal structures of Arabidopsis thaliana cell-wall invertase mutants in complex with sucrose.

In plants, cell-wall invertases fulfil important roles in carbohydrate partitioning, growth, development and crop yield. In this study, we report on different X-ray crystal structures of Arabidopsis thaliana cell-wall invertase 1 (AtcwINV1) mutants with sucrose. These structures reveal a detailed view of sucrose binding in the active site of the wild-type AtcwINV1. Compared to related enzyme-sucrose complexes, important differences in the orientation of the glucose subunit could be observed. The structure of the E203Q AtcwINV1 mutant showed a complete new binding modus, whereas the D23A, E203A and D239A structures most likely represent the productive binding modus. Together with a hydrophobic zone formed by the conserved W20, W47 and W82, the residues N22, D23, R148, E203, D149 and D239 are necessary to create the ideal sucrose-binding pocket. D239 can interact directly with the glucose moiety of sucrose, whereas K242 has an indirect role in substrate stabilization. Most probably, K242 keeps D239 in a favourable position upon substrate binding. Unravelling the exact position of sucrose in plant cell-wall invertases is a necessary step towards the rational design of superior invertases to further increase crop yield and biomass production.

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.