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Karen E Koch - One of the best experts on this subject based on the ideXlab platform.
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sucrose metabolism regulatory mechanisms and pivotal roles in sugar sensing and plant development
Current Opinion in Plant Biology, 2004Co-Authors: Karen E KochAbstract:Sucrose cleavage is vital to multicellular plants, not only for the allocation of crucial carbon resources but also for the initiation of hexose-based sugar signals in importing structures. Only the Invertase and reversible sucrose synthase reactions catalyze known paths of sucrose breakdown in vivo. The regulation of these reactions and its consequences has therefore become a central issue in plant carbon metabolism. Primary mechanisms for this regulation involve the capacity of Invertases to alter sugar signals by producing glucose rather than UDPglucose, and thus also two-fold more hexoses than are produced by sucrose synthase. In addition, vacuolar sites of cleavage by Invertases could allow temporal control via compartmentalization. In addition, members of the gene families encoding either Invertases or sucrose synthases respond at transcriptional and posttranscriptional levels to diverse environmental signals, including endogenous changes that reflect their own action (e.g. hexoses and hexose-responsive hormone systems such as abscisic acid [ABA] signaling). At the enzyme level, sucrose synthases can be regulated by rapid changes in sub-cellular localization, phosphorylation, and carefully modulated protein turnover. In addition to transcriptional control, Invertase action can also be regulated at the enzyme level by highly localized inhibitor proteins and by a system that has the potential to initiate and terminate Invertase activity in vacuoles. The extent, path, and site of sucrose metabolism are thus highly responsive to both internal and external environmental signals and can, in turn, dramatically alter development and stress acclimation.
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Soluble Invertase Expression Is an Early Target of Drought Stress during the Critical, Abortion-Sensitive Phase of Young Ovary Development in Maize
Plant physiology, 2002Co-Authors: Mathias Neumann Andersen, Folkard Asch, Christian R. Jensen, Henrik Næsted, V.o. Mogensen, Karen E KochAbstract:To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid Invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble Invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble Invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these Invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble Invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble Invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal Invertase localization is clearly distinct from the well-characterized association of insoluble Invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble Invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble Invertase gene during the early, abortion-susceptible phase of development.
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rapid repression of maize Invertases by low oxygen Invertase sucrose synthase balance sugar signaling potential and seedling survival
Plant Physiology, 1999Co-Authors: Ying Zeng, Wayne T Avigne, Karen E KochAbstract:We show here that Invertase gene expression and the Invertase-sucrose (Suc) synthase ratio decrease abruptly in response to low oxygen in maize root tips. In addition to aiding in the conservation of carbon and possibly ATP, this response has the potential to directly affect sugar signaling relative to carbon flux. Experiments were motivated by the potential for a reduced Invertase/Suc synthase balance to alter the impact of respiratory and/or membrane carbon flux on sugar signaling. Maize ( Zea mays L.) seedlings with 5-cm primary roots were exposed to anoxic (0% [v/v] O 2 ), hypoxic (3% [v/v] O 2 ), and aerobic conditions. Rapid repression of the Ivr1 and Ivr2 maize Invertases by low oxygen was evident in root tips within 3 h at both the transcript and activity levels. The speed and extent of this response increased with the degree of oxygen deprivation and differed with genotypes. This decrease in expression also contrasted markedly to that of other genes for respiratory Suc metabolism, such as Suc synthases, which typically increased or remained constant. Although previous work showed that the contrasting effects of sugars on Suc synthase genes were reflected in their regulation by hypoxia and anoxia, the same was not observed for the differentially sugar-responsive Invertases. Theoretically advantageous reductions in the Invertase/Suc synthase balance thus resulted. However, where this response was extreme (an Oh43 inbred), total sucrolytic capacity dropped below an apparent minimum and root tip viability was reduced. Paradoxically, only Oh43 seedlings showed survival levels >80% (versus
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a similar dichotomy of sugar modulation and developmental expression affects both paths of sucrose metabolism evidence from a maize Invertase gene family
The Plant Cell, 1996Co-Authors: Wayne T Avigne, Donald R Mccarty, Karen E KochAbstract:Invertase and sucrose synthase catalyze the two known paths for the first step in carbon use by sucrose-importing plant cells. The hypothesis that sugar-modulated expression of these genes could provide a means of import adjustment was initially suggested based on data from sucrose synthases alone; however, this hypothesis remained largely conjectural without critical evidence for Invertases. Toward this end, a family of maize Invertases was cloned and characterized. Here, we show that Invertases are indeed sugar modulated and, surprisingly, like the sucrose synthase genes, fall into two classes with contrasting sugar responses. In both families, one class of genes is upregulated by increasing carbohydrate supply (Sucrose synthase1 [Sus1] and Invertase2 [Ivr2]), whereas a second class in the same family is repressed by sugars and upregulated by depletion of this resource (Shrunken1 [Sh1] and Invertase1 [Ivr1]). The two classes also display differential expression during development, with sugar-enhanced genes (Sus1 and Ivr2) expressed in many importing organs and sugar-repressed, starvation-tolerant genes (Sh1 and Ivr1) upregulated primarily during reproductive development. Both the Ivr1 and Ivr2 Invertase mRNAs are abundant in root tips, very young kernels, silk, anthers, and pollen, where a close relationship is evident between changes in message abundance and soluble Invertase activity. During development, patterns of expression shift as assimilate partitioning changes from elongating silks to newly fertilized kernels. Together, the data support a model for integrating expression of genes differentially responsive to carbohydrate availability (i.e., feast and famine conditions) with developmental signals. The demonstration that similar regulatory patterns occur in both paths of sucrose metabolism indicates a potential to influence profoundly the adjustment of carbon resource allocation.
Wim Van Den Ende - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of Arabidopsis thaliana neutral Invertase 2
Acta crystallographica. Section F Structural biology communications, 2020Co-Authors: Łukasz Pawel Tarkowski, Willem Lammens, Wim Van Den Ende, V.g. Tsirkone, Em Osipov, Steven Beelen, Rudy Vergauwen, Sergei V. StrelkovAbstract: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-beta 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 alpha-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 angstrom resolution. The hexameric AtNIN2 structure is highly similar to that of InvA, pointing to high evolutionary conservation of neutral Invertases.
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donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes
FEBS Journal, 2009Co-Authors: Wim Van Den Ende, Willem Lammens, Andre Van Laere, Lindsey Schroeven, Katrien Le RoyAbstract: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.
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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 Physiology, 2007Co-Authors: Katrien Le Roy, Willem Lammens, Maureen Verhaest, Barbara De Coninck, A Rabijns, Andre Van Laere, Wim Van Den EndeAbstract: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.
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structure evolution and expression of the two Invertase gene families of rice
Journal of Molecular Evolution, 2005Co-Authors: Wim Van Den Ende, Andre Van Laere, Shihua Cheng, John BennettAbstract:Invertases catalyze the irreversible hydrolysis of sucrose to glucose and fructose. Plants contain two unrelated families of these enzymes: acid forms that derive from periplasmic Invertases of eubacteria and are found in cell wall and vacuole, and neutral/alkaline forms evolved from the cytosolic Invertases of cyanobacteria. Genomes of rice (Oryza sativa) and thale cress (Arabidopsis thaliana) contain multiple genes encoding these two families. Here for rice we identify the member genes of a cell-wall group (designated OsCIN1–9), a vacuolar group (OsVIN1–2), and two ancient neutral/alkaline groups: α (OsNIN1–4) and β (OsNIN5–8). In Arabidopsis these groups contain six, two, four and five members, respectively. It is believed that the vacuolar group evolved from the cell-wall group. We provide evidence that the N-terminal signal peptide that directs cell-wall Invertases co-translationally into the endoplasmic reticulum for secretion was replaced in the vacuolar group by a sequence similar to the complex N-terminal motif that targets alkaline phosphatase post-translationally to the vacuolar membrane of yeast. Since the last common ancestor of Arabidopsis and rice, the two Invertase families evolved equally rapidly via gene duplication and gene loss, but the acid Invertase family underwent ∼10 events of intron loss compared with a single event of intron gain in the neutral/alkaline Invertase family. Transcripts were detected for all rice Invertase genes except OsCIN9. The acid Invertase genes showed greater spatial and temporal diversity of expression than the neutral/alkaline genes.
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cloning and functional analysis of chicory root fructan 1 exohydrolase i 1 feh i a vacuolar enzyme derived from a cell wall Invertase ancestor mass fingerprint of the 1 feh i enzyme
Plant Journal, 2000Co-Authors: Wim Van Den Ende, An Michiels, Joke De Roover, Peter Verhaert, Andre Van LaereAbstract:Summary This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT–PCR, 5′ and 3′ RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall Invertases (44–53%) than to vacuolar Invertases (38–41%) and fructosyl transferases (33–38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall Invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar Invertase, 1-FEH I might have evolved from a cell-wall Invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.
Thomas Roitsch - One of the best experts on this subject based on the ideXlab platform.
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extracellular Invertase is involved in the regulation of clubroot disease in arabidopsis thaliana
Molecular Plant Pathology, 2011Co-Authors: Johannes Siemens, Mariacruz Gonzalez, Sebastian Wolf, Christina Hofmann, Steffen Greiner, Thomas Rausch, Thomas Roitsch, Jutta LudwigmullerAbstract:Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of Invertases has been observed in a microarray experiment, and Invertase and Invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that Invertases are important for gall development. Different transgenic lines expressing an Invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of Invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that Invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of Invertase activity could be used as a tool to reduce clubroot symptoms.
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post translational derepression of Invertase activity in source leaves via down regulation of Invertase inhibitor expression is part of the plant defense response
Molecular Plant, 2010Co-Authors: Katharina Bonfig, Andrea Gabler, Uwe K Simon, Nora Luschinebengreuth, Martina Hatz, Susanne Berger, Naseem Muhammad, Jurgen Zeier, Alok Krishna Sinha, Thomas RoitschAbstract:There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound Invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of Invertase genes in response to pathogen infection, the role of post-translational regulation of Invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous Invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in Invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ Invertase activity was detectable only upon infection by P. syringae. In contrast, a high Invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro Invertase activity of control leaves and the high in situ Invertase activity in infected leaves can be explained by the pathogen-dependent repression of Invertase inhibitor expression and a concomitant reduction in Invertase inhibitor activity. The functional importance of the release of Invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical Invertase inhibitor acarbose. Post-translational inhibition of extracellular Invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of Invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports a role for extracellular Invertase in plant defense. The acarbose-mediated increase in susceptibility was also detectable in sid2 and cpr6 mutants and resulted in slightly elevated levels of salicylic acid, demonstrating that the effect is independent of the salicylic acid-regulated defense pathway. These findings provide an explanation for high extractable Invertase activity found in source leaves that is kept inhibited in situ by post-translational interaction between Invertase and the Invertase inhibitor proteins. Upon pathogen infection, the Invertase activity is released by repression of Invertase inhibitor expression, thus linking the local induction of sink strength to the plant defense response.
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Infection with virulent and avirulent P. syringae strains differentially affects photosynthesis and sink metabolism in Arabidopsis leaves
Planta, 2006Co-Authors: Katharina B. Bonfig, Thomas Roitsch, Andrea Gabler, Ulrich Schreiber, Susanne BergerAbstract:Infection of plants with pathogens leads not only to the induction of defence reactions but also to changes in carbohydrate metabolism. In this study, the effects of infection by a virulent and an avirulent strain of P. syringae on spatio-temporal changes in photosynthesis were compared using chlorophyll fluorescence imaging. The maximum PSII quantum yield, effective PSII quantum yield and nonphotochemical quenching were decreased in Arabidopsis leaves infected with either strain. At the same time, the quantum yield of nonregulated energy dissipation was increased. These changes could be detected by chlorophyll fluorescence imaging before symptoms were visible by eye. The effects were restricted to the vicinity of the infection site and did not spread to uninfected areas of the leaf. Qualitatively similar changes in photosynthetic parameters were observed in both interactions. Major differences between the responses to both strains were evident in the onset and time course of changes. A decrease in photosynthesis was detectable already at 3 h only after challenge with the avirulent strain while after 48 h the rate of photosynthesis was lower with the virulent strain. In contrast to photosynthesis, the regulation of marker genes for source/sink relations and the activities of Invertase isoenzymes showed qualitative differences between both interactions. Inoculation of the virulent but not the avirulent strain resulted in downregulation of photosynthetic genes and upregulation of vacuolar Invertases. The activity of vacuolar Invertases transiently increased upon infection with the virulent strain but decreased with the avirulent strain while extracellular Invertase activity was downregulated in both interactions.
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novel mode of hormone induction of tandem tomato Invertase genes in floral tissues
Plant Molecular Biology, 2003Co-Authors: Reinhard K Proels, Susanne Berger, Bettina Hause, Thomas RoitschAbstract:The genomic organization of two extracellular Invertase genes from tomato (Lin5 and Lin7), which are linked in a direct tandem repeat, and their tissue-specific and hormone-inducible expression are shown. Transient expression analysis ofLin5 promoter sequences fused to the β-glucuronidase (GUS) reporter gene (uidA) demonstrates a specific expression of Lin5during tomato fruit development. A Lin5 promoter fragment was fused to the truncated nos promoter to analyse hormone induction via GUS reporter gene activity in transiently transformed tobacco leaves. A specific up-regulation of GUS activity conferred by this Lin5 promoter fragment in response to gibberellic acid (GA), auxin and abscisic acid (ABA) treatment was observed, indicating a critical role of the regulation of Lin5 by phytohormones in tomato flower and fruit development. In situ hybridization analysis of Lin7 shows a high tissue-specific expression in tapetum and pollen. These results support an important role for Lin5 and Lin7 extracellular Invertases in the development of reproductive organs in tomato and contribute to unravel the underlying regulatory mechanisms.
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regulation and tissue specific distribution of mrnas for three extracellular Invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism
Plant Physiology, 1997Co-Authors: Dietmute E Godt, Thomas RoitschAbstract:The aim of the present study was to gain insight into the contribution of extracellular Invertases for sink metabolism in tomato (Lycopersicon esculentum L.). The present study shows that extracellular Invertase isoenzymes are encoded by a gene family comprising four members: Lin5, Lin6, Lin7, and Lin8. The regulation of mRNA levels by internal and external signals and the distribution in sink and source tissues has been determined and compared with mRNA levels of the intracellular sucrose (Suc)-cleaving enzymes Suc synthase and vacuolar Invertase. The specific regulation of Lin5, Lin6, and Lin7 suggests an important function of apoplastic cleavage of Suc by cell wall-bound Invertase in establishing and maintaining sink metabolism. Lin6 is expressed under conditions that require a high carbohydrate supply. The corresponding mRNA shows a sink tissue-specific distribution and the concentration is elevated by stress-related stimuli, by the growth-promoting phytohormone zeatin, and in response to the induction of heterotrophic metabolism. The expression of Lin5 and Lin7 in gynoecia and stamens, respectively, suggests an important function in supplying carbohydrates to these flower organs, whereas the Lin7 mRNA was found to be present exclusively in this specific sink organ.
Andre Van Laere - One of the best experts on this subject based on the ideXlab platform.
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donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes
FEBS Journal, 2009Co-Authors: Wim Van Den Ende, Willem Lammens, Andre Van Laere, Lindsey Schroeven, Katrien Le RoyAbstract: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.
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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 Physiology, 2007Co-Authors: Katrien Le Roy, Willem Lammens, Maureen Verhaest, Barbara De Coninck, A Rabijns, Andre Van Laere, Wim Van Den EndeAbstract: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.
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structure evolution and expression of the two Invertase gene families of rice
Journal of Molecular Evolution, 2005Co-Authors: Wim Van Den Ende, Andre Van Laere, Shihua Cheng, John BennettAbstract:Invertases catalyze the irreversible hydrolysis of sucrose to glucose and fructose. Plants contain two unrelated families of these enzymes: acid forms that derive from periplasmic Invertases of eubacteria and are found in cell wall and vacuole, and neutral/alkaline forms evolved from the cytosolic Invertases of cyanobacteria. Genomes of rice (Oryza sativa) and thale cress (Arabidopsis thaliana) contain multiple genes encoding these two families. Here for rice we identify the member genes of a cell-wall group (designated OsCIN1–9), a vacuolar group (OsVIN1–2), and two ancient neutral/alkaline groups: α (OsNIN1–4) and β (OsNIN5–8). In Arabidopsis these groups contain six, two, four and five members, respectively. It is believed that the vacuolar group evolved from the cell-wall group. We provide evidence that the N-terminal signal peptide that directs cell-wall Invertases co-translationally into the endoplasmic reticulum for secretion was replaced in the vacuolar group by a sequence similar to the complex N-terminal motif that targets alkaline phosphatase post-translationally to the vacuolar membrane of yeast. Since the last common ancestor of Arabidopsis and rice, the two Invertase families evolved equally rapidly via gene duplication and gene loss, but the acid Invertase family underwent ∼10 events of intron loss compared with a single event of intron gain in the neutral/alkaline Invertase family. Transcripts were detected for all rice Invertase genes except OsCIN9. The acid Invertase genes showed greater spatial and temporal diversity of expression than the neutral/alkaline genes.
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cloning and functional analysis of chicory root fructan 1 exohydrolase i 1 feh i a vacuolar enzyme derived from a cell wall Invertase ancestor mass fingerprint of the 1 feh i enzyme
Plant Journal, 2000Co-Authors: Wim Van Den Ende, An Michiels, Joke De Roover, Peter Verhaert, Andre Van LaereAbstract:Summary This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT–PCR, 5′ and 3′ RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall Invertases (44–53%) than to vacuolar Invertases (38–41%) and fructosyl transferases (33–38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall Invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar Invertase, 1-FEH I might have evolved from a cell-wall Invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.
Herve Seligmann - One of the best experts on this subject based on the ideXlab platform.
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overlapping genes coded in the 3 to 5 direction in mitochondrial genes and 3 to 5 polymerization of non complementary rna by an Invertase
Journal of Theoretical Biology, 2012Co-Authors: Herve SeligmannAbstract:Suppressor tRNAs induce expression of additional (off-frame) genes coded by stopless genetic codes without lengthening genomes, decreasing DNA replication costs. RNA 3′-to-5′ polymerization by tRNAHis guanylyltransferase suggests further cryptic code: hypothetical ‘Invertases’ polymerizing in the 3′-to-5′ direction, advancing in the 5′-to-3′ direction would produce non-complementary RNA templated by regular genes, with different coding properties. Assuming ‘Invertase’ activity, BLAST analyses detect GenBank-stored RNA ESTs and proteins (some potentially coding for the hypothesized Invertase) for human mitochondrial genes. These peptides’ predicted secondary structures resemble their GenBank homologues’. 3′-to-5′ EST lengths increase with their self-hybridization potential: Single-stranded RNA degradation perhaps limits 3′-to-5′ elongation. Independent methods confirm predicted 3′-to-5′ overlapping genes: (a) Presumed 3′-to-5′ overlapping genes avoid codons belonging to circular codes; (b) Spontaneous replicational deamination (mutation) gradients occur at 3rd codon positions, unless these are involved in overlap coding, because mutations are counter selected in overlapping genes. Tests a and b converge on predicted 3′-to-5′ gene expression levels. Highly expressed ones include also fewer stops, and mitochondrial genomes (in Primates and Drosophila) adapt to avoid dependence of 3′-to-5′ coding upon antitermination tRNA activity. Secondary structure, circular code, gradient and coevolution analyses yield each clear positive results independently confirming each other. These positive results (including physical evidence for 3′-to-5′ ESTs) indicate that 3′-to-5′ coding and Invertase activity is an a priori improbable working hypothesis that cannot be dismissed. Note that RNAs produced by Invertases potentially produce triple-stranded DNA:RNA helices by antiparallel Hoogsteen pairings at physiological pH, as previously observed for mitochondrial genomes.
