Willem Lammens

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.

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.

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.

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 β-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.

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-β-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.

X-ray diffraction structure of a cell-wall invertase from Arabidopsis thaliana

Cell-wall invertases play crucial roles during plant development. They hydrolyse sucrose into its fructose and glucose subunits by cleavage of the alpha1-beta2 glycosidic bond. Here, the structure of the Arabidopsis thaliana cell-wall invertase 1 (AtcwINV1; gene accession code At3g13790) is described at a resolution of 2.15 A. The structure comprises an N-terminal fivefold beta-propeller domain followed by a C-terminal domain formed by two beta-sheets. The active site is positioned in the fivefold beta-propeller domain, containing the nucleophile Asp23 and the acid/base catalyst Glu203 of the double-displacement enzymatic reaction. The function of the C-terminal domain remains unknown. Unlike in other GH 32 family enzyme structures known to date, in AtcwINV1 the cleft formed between both domains is blocked by Asn299-linked carbohydrates. A preliminary site-directed mutagenesis experiment (Asn299Asp) removed the glycosyl chain but did not alter the activity profile of the enzyme.

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.

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.

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.