Bacterial expression, purification and preliminary X-ray crystallographic characterization of the invertase inhibitor Nt-CIF from tobacco

Hothorn, Michael; Bonneau, Fabien; Stier, Günter; Greiner, Steffen; Scheffzek, Klaus

doi:10.1107/s0907444903021036

Cited by 20 publications

(25 citation statements)

References 13 publications

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“…Subsequently, the NcoI/SalI-excised fragment was ligated into vector pETM20 (27). The thioredoxin fusion protein was expressed in E. coli Rosetta gami DE3 (Novagen, Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for the Redox Control of Plant Glutamate Cysteine Ligase

Hothorn

Wachter

Gromes

et al. 2006

Journal of Biological Chemistry

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104

163

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Glutathione (GSH) plays a crucial role in plant metabolism and stress response. The rate-limiting step in the biosynthesis of GSH is catalyzed by glutamate cysteine ligase (GCL) the activity of which is tightly regulated. The regulation of plant GCLs is poorly understood. The crystal structure of substrate-bound GCL from Brassica juncea at 2.1-Å resolution reveals a plantunique regulatory mechanism based on two intramolecular redox-sensitive disulfide bonds. Reduction of one disulfide bond allows a ␤-hairpin motif to shield the active site of B. juncea GCL, thereby preventing the access of substrates. Reduction of the second disulfide bond reversibly controls dimer to monomer transition of B. juncea GCL that is associated with a significant inactivation of the enzyme. These regulatory events provide a molecular link between high GSH levels in the plant cell and associated down-regulation of its biosynthesis. Furthermore, known mutations in the Arabidopsis GCL gene affect residues in the close proximity of the active site and thus explain the decreased GSH levels in mutant plants. In particular, the mutation in rax1-1 plants causes impaired binding of cysteine.Glutathione (GSH) is the predominant non-protein thiol compound in eukaryotic and prokaryotic cells (1). Through reversible oxidation to glutathione disulfide, it acts as a major cellular redox buffer. In the plant chloroplast GSH can reach millimolar concentrations depending on diverse external stress stimuli and on the day-night transition. Light/dark-induced changes in stromal pH further modulate the redox potential of GSH (2).In plants, GSH detoxifies reactive oxygen species via the ascorbic acid-GSH-cycle (3) and glutathione peroxidases (4). It is involved in the detoxification of xenobiotics via glutathione S-transferases (5), represents the precursor for the heavy metal ions sequestering phytochelatins (6), and serves as a major defense component against a wide range of abiotic and biotic stress factors (7). GSH can post-translationally modify proteins via glutathionylation (8, 9) and may act as a signaling molecule (10, 11). Notably, GSH represents a major storage and transport form of reduced sulfur in plants (12).The biosynthesis of GSH occurs in two sequential ATP-dependent steps. Glutamate cysteine ligase (GCL, 3 EC 6.3.2.2.) produces the reaction intermediate ␥-glutamylcysteine (Scheme 1). Glutathione synthetase (EC 6.3.2.3.) then catalyzes the addition of a glycine residue.Both enzymes are encoded by single genes in Arabidopsis, belonging to the Brassicaceae family (13-15). In this plant family GCL is localized exclusively in the plastids, whereas glutathione synthetase is found both in plastids and in the cytosol (16). Under most conditions, GCL activity is rate-limiting in plant GSH biosynthesis (17). This is highlighted by significantly reduced glutathione levels in AtGCL mutants (18,19).Plant GCL protein sequences diverge from those of nonplant eukaryotic organisms (mammals, Drosophila). Although some bacteria share low sequence homology w...

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“…Subsequently, the NcoI/SalI-excised fragment was ligated into vector pETM20 (27). The thioredoxin fusion protein was expressed in E. coli Rosetta gami DE3 (Novagen, Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for the Redox Control of Plant Glutamate Cysteine Ligase

Hothorn

Wachter

Gromes

et al. 2006

Journal of Biological Chemistry

Self Cite

104

163

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“…Wild-type PMEI and mutant forms have been expressed and purified as described (Wolf et al, 2003) following protocols established for the related invertase inhibitor Nt-CIF (Hothorn et al, 2003). Before crystallization, samples were concentrated to ;5 mg/mL using a Vivapore 10/20 mL concentrator (7.5 kD MWCO; Vivascience, Hannover, Germany) and dialyzed against 100 mM NaCl, 10 mM Hepes, pH 7.0.…”

Section: Methods Expression Crystallization and Data Collectionmentioning

confidence: 99%

Structural Insights into the Target Specificity of Plant Invertase and Pectin Methylesterase Inhibitory Proteins

et al. 2004

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Pectin methylesterase (PME) and invertase are key enzymes in plant carbohydrate metabolism. Inhibitors of both enzymes constitute a sequence family of extracellular proteins. Members of this family are selectively targeted toward either PME or invertase. In a comparative structural approach we have studied how this target specificity is implemented on homologous sequences. By extending crystallographic work on the invertase inhibitor Nt-CIF to a pectin methylesterase inhibitor (PMEI) from Arabidopsis thaliana, we show an a-helical hairpin motif to be an independent and mobile structural entity in PMEI. Removal of this hairpin fully inactivates the inhibitor. A chimera composed of the a-hairpin of PMEI and the four-helix bundle of Nt-CIF is still active against PME. By contrast, combining the corresponding segment of Nt-CIF with the four-helix bundle of PMEI renders the protein inactive toward either PME or invertase. Our experiments provide insight in how these homologous inhibitors can make differential use of similar structural modules to achieve distinct functions. Integrating our results with previous findings, we present a model for the PME-PMEI complex with important implications. INTRODUCTIONAt the posttranslational level, the activity of enzymes is commonly regulated by various mechanisms, including residuedirected protein modifications such as phosphorylation, glycosylation, and interaction with specific inhibitors. The nature of these inhibitors may range from small molecules to entire proteins, as found with the well-studied inhibitors of proteases (Bode and Huber, 1992). In plants, inhibitory proteins are often targeted toward sugar-modifying enzymes that escape cellular control mechanisms upon secretion into the plant cell wall or the vacuole (Juge et al., 2004). We are focusing on the structure-function relationship of an inhibitor family that regulates the activity of plant acid invertase and pectin methylesterase (PME).Invertases convert the transport sugar sucrose into its building blocks, fructose and glucose. In higher plants, invertases exist in compartment specific isoforms, with only extracytosolic species being sensitive to inhibitory proteins. Altered activity of extracellular invertase has been shown to have dramatic effects on growth and development (Cheng et al., 1996;Tang et al., 1999;Goetz et al., 2001). This is consistent with roles of invertase activity in vital cellular processes such as carbohydrate transport (Roitsch et al., 2003), sugar signaling (Koch, 1996(Koch, , 2004Smeekens, 1998;Wobus and Weber, 1999), and stress response (Ehness et al., 1997;Roitsch et al., 2003). Protein inhibitors of invertase (Greiner et al., 1998) affect enzyme activity in a strictly pH-dependent manner (Rausch and Greiner, 2004) and have been proposed as transgenic tools to engineer post-harvest sucrose metabolism in crop plants (Greiner et al., 1999).PMEs catalyze the demethylesterification of the homogalacturonan component of pectins, highly heterogeneous polymers (Vorwerk et al., 2004) that repre...

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“…The amplification products for PTB1 and PTB2 were digested with PciI-NotI, whereas the PTB3 product was digested with NcoI-NotI. Subsequently, the At-PTB-specific inserts were cloned in the NcoI-NotI-digested expression vector pETM-20 (Hothorn et al, 2003), resulting in constructs encoding Thioredoxin A-His6-tagged PTB proteins with a TEV protease cleavage site. For generation of a PIF6-specific antibody, the cds of the transcript variant At3g62090.3 was amplified with primers DNA55 and DNA56.…”

Section: Cloning Proceduresmentioning

confidence: 99%

Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes

et al. 2012

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Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 59 splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid-dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner.

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Bacterial expression, purification and preliminary X-ray crystallographic characterization of the invertase inhibitor Nt-CIF from tobacco

Cited by 20 publications

References 13 publications

Structural Basis for the Redox Control of Plant Glutamate Cysteine Ligase

Structural Basis for the Redox Control of Plant Glutamate Cysteine Ligase

Structural Insights into the Target Specificity of Plant Invertase and Pectin Methylesterase Inhibitory Proteins

Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes

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