RESUMO
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.
Assuntos
Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Sacarose/metabolismo , beta-Frutofuranosidase/metabolismo , Sequência de Aminoácidos , Biocatálise , Clonagem Molecular , DNA Complementar/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Pichia/metabolismo , Proteínas de Plantas/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , beta-Frutofuranosidase/químicaRESUMO
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.
Assuntos
Frutanos/biossíntese , Hexosiltransferases/metabolismo , Pachysandra/enzimologia , Proteínas de Plantas/metabolismo , Trissacarídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Domínio Catalítico , Cristalografia por Raios X , Hexosiltransferases/química , Hexosiltransferases/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Pachysandra/genética , Pachysandra/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trissacarídeos/químicaRESUMO
Although fructans play a crucial role in wheat kernel development, their metabolism during kernel maturation is far from being understood. In this study, all major fructan-metabolizing enzymes together with fructan content, fructan degree of polymerization and the presence of fructan oligosaccharides were examined in developing wheat kernels (Triticum aestivum L. var. Homeros) from anthesis until maturity. Fructan accumulation occurred mainly in the first 2 weeks after anthesis, and a maximal fructan concentration of 2.5 ± 0.3 mg fructan per kernel was reached at 16 days after anthesis (DAA). Fructan synthesis was catalyzed by 1-SST (sucrose:sucrose 1-fructosyltransferase) and 6-SFT (sucrose:fructan 6-fructosyltransferase), and to a lesser extent by 1-FFT (fructan:fructan 1-fructosyltransferase). Despite the presence of 6G-kestotriose in wheat kernel extracts, the measured 6G-FFT (fructan:fructan 6G-fructosyltransferase) activity levels were low. During kernel filling, which lasted from 2 to 6 weeks after anthesis, kernel fructan content decreased from 2.5 ± 0.3 to 1.31 ± 0.12 mg fructan per kernel (42 DAA) and the average fructan degree of polymerization decreased from 7.3 ± 0.4 (14 DAA) to 4.4 ± 0.1 (42 DAA). FEH (fructan exohydrolase) reached maximal activity between 20 and 28 DAA. No fructan-metabolizing enzyme activities were registered during the final phase of kernel maturation, and fructan content and structure remained unchanged. This study provides insight into the complex metabolism of fructans during wheat kernel development and relates fructan turnover to the general phases of kernel development.
Assuntos
Frutanos/metabolismo , Triticum/crescimento & desenvolvimento , Triticum/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologiaRESUMO
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.
Assuntos
Adaptação Fisiológica , Congelamento , Frutanos/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/isolamento & purificação , Pachysandra/enzimologia , Sequência de Aminoácidos , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , Hexosiltransferases/química , Hexosiltransferases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Peso Molecular , Pachysandra/genética , Mapeamento de Peptídeos , Filogenia , Pichia/metabolismo , Alinhamento de SequênciaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Estresse Oxidativo , Peroxidases/metabolismo , Folhas de Planta/metabolismo , beta-Frutofuranosidase/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Ascorbato Peroxidases , Citosol/enzimologia , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/genética , Peroxidases/genética , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Protoplastos/enzimologia , Protoplastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , beta-Frutofuranosidase/genéticaRESUMO
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.
Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Domínio Catalítico , Sequência Conservada , Especificidade por Substrato , TriptofanoRESUMO
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.
Assuntos
Sacarose/metabolismo , beta-Frutofuranosidase/metabolismo , Substituição de Aminoácidos , Arabidopsis/enzimologia , Simulação por Computador , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , beta-Frutofuranosidase/química , beta-Frutofuranosidase/genéticaRESUMO
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.
Assuntos
Beta vulgaris/enzimologia , Cichorium intybus/enzimologia , Glicosídeo Hidrolases/genética , Sacarose/química , Sacarose/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Engenharia Genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
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.
Assuntos
Arabidopsis/citologia , Arabidopsis/enzimologia , Parede Celular/enzimologia , beta-Frutofuranosidase/química , Cristalização , Difração de Raios XRESUMO
Fructans are important vacuolar reserve carbohydrates with drought, cold, ROS and general abiotic stress mediating properties. They occur in 15% of all flowering plants and are believed to display health benefits as a prebiotic and dietary fiber. Fructans are synthesized by specific fructosyltransferases and classified based on the linkage type between fructosyl units. Inulins, one of these fructan types with ß(2-1) linkages, are elongated by fructan:fructan 1-fructosyltransferases (1-FFT) using a fructosyl unit from a donor inulin to elongate the acceptor inulin molecule. The sequence identity of the 1-FFT of Viguiera discolor (Vd) and Helianthus tuberosus (Ht) is 91% although these enzymes produce distinct fructans. The Vd 1-FFT produces high degree of polymerization (DP) inulins by preferring the elongation of long chain inulins, in contrast to the Ht 1-FFT which prefers small molecules (DP3 or 4) as acceptor. Since higher DP inulins have interesting properties for industrial, food and medical applications, we report here on the influence of two amino acids on the high DP inulin production capacity of the Vd 1-FFT. Introducing the M19F and H308T mutations in the active site of the Vd 1-FFT greatly reduces its capacity to produce high DP inulin molecules. Both amino acids can be considered important to this capacity, although the double mutation had a much higher impact than the single mutations.
RESUMO
Although a lot of vacuolar invertase (EC 3.2.1.26) cDNAs are available from a diversity of plant species, up to now no sequence information is available on invertases from any dicot fructan-containing species. Therefore, we describe the cloning of vacuolar acid invertase cDNA from etiolated Belgian endive leaves (Cichorium intybus L. var. foliosum cv. Flash), formed throughout the forcing process of the witloof chicory roots. Full-length cDNA was obtained by a combination of RT-PCR, PCR and 5'- and 3' RACE RT-PCR, starting with primers based on conserved amino acid sequences. The cloned chicory acid invertase groups together with vacuolar type invertases and fructan biosynthetic enzymes. A putative role for vacuolar type invertases in fructan synthesizing plants is discussed.
RESUMO
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.
Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Aminoácidos/química , Catálise , Frutanos/química , Frutanos/metabolismo , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Cinética , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Especificidade por Substrato , Sacarose/química , Sacarose/metabolismoRESUMO
Higher plants typically do not produce trehalose in large amounts, but their genome sequences reveal large families of putative trehalose metabolism enzymes. An important regulatory role in plant growth and development is also emerging for the metabolic intermediate trehalose-6-P (T6P). Here, we present an update on Arabidopsis trehalose metabolism and a resource for further detailed analyses. In addition, we provide evidence that Arabidopsis encodes a single trehalose-6-P synthase (TPS) next to a family of catalytically inactive TPS-like proteins that might fulfill specific regulatory functions in actively growing tissues.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/metabolismo , Glucosiltransferases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Western Blotting , Glucosiltransferases/química , Glucosiltransferases/genética , Modelos Biológicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Trealose/metabolismoRESUMO
Sugars play important roles as both nutrients and regulatory molecules throughout plant life. Sugar metabolism and signalling function in an intricate network with numerous hormones and reactive oxygen species (ROS) production, signalling and scavenging systems. Although hexokinase is well known to fulfil a crucial role in glucose sensing processes, a scenario is emerging in which the catalytic activity of mitochondria-associated hexokinase regulates glucose-6-phosphate and ROS levels, stimulating antioxidant defence mechanisms and the synthesis of phenolic compounds. As a new concept, it can be hypothesized that the synergistic interaction of sugars (or sugar-like compounds) and phenolic compounds forms part of an integrated redox system, quenching ROS and contributing to stress tolerance, especially in tissues or organelles with high soluble sugar concentrations.
Assuntos
Antioxidantes/metabolismo , Metabolismo dos Carboidratos , Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Animais , Humanos , Estresse FisiológicoRESUMO
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.
Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Glicosídeo Hidrolases/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Sacarose/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismoRESUMO
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.
Assuntos
Hexosiltransferases/metabolismo , Lolium/enzimologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Hexosiltransferases/genética , Cinética , Lolium/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Sacarose/química , Sacarose/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cristalografia por Raios X , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/classificação , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Sacarose/farmacologia , beta-Frutofuranosidase/genéticaRESUMO
Recently, the three-dimensional structure of chicory (Cichorium intybus) fructan 1-exohydrolase (1-FEH IIa) in complex with its preferential substrate, 1-kestose, was determined. Unfortunately, no such data could be generated with high degree of polymerization (DP) inulin, despite several soaking and cocrystallization attempts. Here, site-directed mutagenesis data are presented, supporting the presence of an inulin-binding cleft between the N- and C-terminal domains of 1-FEH IIa. In general, enzymes that are unable to degrade high DP inulins contain an N-glycosylation site probably blocking the cleft. By contrast, inulin-degrading enzymes have an open cleft configuration. An 1-FEH IIa P294N mutant, introducing an N-glycosylation site near the cleft, showed highly decreased activity against higher DP inulin. The introduction of a glycosyl chain most probably blocks the cleft and prevents inulin binding and degradation. Besides cell wall invertases, fructan 6-exohydrolases (6-FEHs) also contain a glycosyl chain most probably blocking the cleft. Removal of this glycosyl chain by site-directed mutagenesis in Arabidopsis thaliana cell wall invertase 1 and Beta vulgaris 6-FEH resulted in a strong decrease of enzymatic activities of the mutant proteins. By analogy, glycosylation of 1-FEH IIa affected overall enzyme activity. These data strongly suggest that the presence or absence of a glycosyl chain in the cleft is important for the enzyme's stability and optimal conformation.
Assuntos
Cichorium intybus/enzimologia , Glicosídeo Hidrolases/metabolismo , Inulina/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Beta vulgaris/enzimologia , Sítios de Ligação , Parede Celular/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosilação , Inulina/química , Inulina/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , beta-Frutofuranosidase/química , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismoRESUMO
Fructans, typically reserve carbohydrates, may also fulfil other more specific roles in plants. It has been convincingly demonstrated that fructan hydrolysis contributes to osmoregulation during flower opening in the monocot species Hemerocallis. We report that a massive breakdown of inulin-type fructans in the petals of Campanula rapunculoides L. (Campanulaceae), associated with flower opening, is accompanied by a strong increase in fructan 1-exohydrolase (1-FEH; EC 3.2.1.153) activity and a decrease in sucrose : sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) activity. The data strongly suggest that the drastic change in the 1-FEH/1-SST activity ratio causes the degradation of inulin, contributing to the osmotic driving force involved in flower opening. All characterised plant FEHs are believed to be derived from tissues that store fructans as a reserve carbohydrate either temporarily (grasses and cereals) or over a longer term (dicot roots and tubers). Here, we focussed on a physiologically distinct tissue and used a reverse transcriptase-polymerase chain reaction based strategy to clone the 1-FEH cDNA from the Campanula petals. The translated cDNA sequence groups along with other dicot FEHs and heterologous expression revealed that the cDNA encodes a 1-FEH without invertase activity. 1-FEH expression analysis in petals correlates well with 1-FEH activity and inulin degradation patterns in vivo, suggesting that this enzyme fulfils an important role during flower opening.
RESUMO
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.