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Gad Galili - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional control of Aspartate Kinase expression during darkness and sugar depletion in Arabidopsis: involvement of bZIP transcription factors.
Planta, 2011Co-Authors: Shai Ufaz, Gad Galili, Vijaya Shukla, Yulia Soloveichik, Yelena Golan, Frank Breuer, Zsuzsa Koncz, Csaba Koncz, Aviah ZilbersteinAbstract:Initial steps of Aspartate-derived biosynthesis pathway (Asp pathway) producing Lys, Thr, Met and Ile are catalyzed by bifunctional (AK/HSD) and monofunctional (AK-lys) Aspartate Kinase (AK) enzymes. Here, we show that transcription of all AK genes is negatively regulated under darkness and low sugar conditions. By using yeast one-hybrid assays and complementary chromatin immunoprecipitation analyses in Arabidopsis cells, the bZIP transcription factors ABI5 and DPBF4 were identified, capable of interacting with the G-box-containing enhancer of AK/HSD1 promoter. Elevated transcript levels of DPBF4 and ABI5 under darkness and low sugar conditions coincide with the repression of AK gene expression. Overexpression of ABI5, but not DPBF4, further increases this AK transcription suppression. Concomitantly, it also increases the expression of asparagines synthetase 1 (ASN1) that shifts Aspartate utilization towards asparagine formation. However, in abi5 or dpbf4 mutant and abi5, dpbf4 double mutant the repression of AK expression is maintained, indicating a functional redundancy with other bZIP-TFs. A dominant-negative version of DPBF4 fused to the SRDX repressor domain of SUPERMAN could counteract the repression and stimulate AK expression under low sugar and darkness in planta. This effect was verified by showing that DPBF4-SRDX fails to recognize the AK/HSD1 enhancer sequence in yeast one-hybrid assays, but increases heterodimmer formation with DPBF4 and ABI5, as estimated by yeast two-hybrid assays. Hence it is likely that heterodimerization with DPBF4-SRDX inhibits the binding of redundantly functioning bZIP-TFs to the promoters of AK genes and thereby releases the repressing effect. These data highlight a novel transcription control of the chloroplast Aspartate pathway that operates under energy limiting conditions.
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Characterization of monofunctional Aspartate Kinase genes in maize and their relationship with free amino acid content in the endosperm
Journal of experimental botany, 2007Co-Authors: Xuelu Wang, Gad Galili, Jose A. Lopez-valenzuela, Bryan C. Gibbon, Bertrand Gakière, Brian A. LarkinsAbstract:A quantitative trait locus has previously been identified in maize (Zea mays L.) that influences the level of free amino acids in the endosperm, especially those from the Aspartate pathway: lysine, threonine, methionine, leucine, and isoleucine. Because this locus occurs in a region of the genome containing ask2, a monofunctional Aspartate Kinase, the nature of the monofunctional Aspartate Kinase genes in the parental inbreds, Oh545o2 and Oh51Ao2, was investigated. Two genes, Ask1 and Ask2 were isolated, and Ask2 was mapped to the ask2 locus. Nucleotide sequence analysis of the Ask2 alleles from Oh545o2 and Oh51Ao2 showed they differ by one amino acid. Both alleles complemented a yeast Aspartate Kinase mutant, hom3, and based on the growth of the yeast mutant it appeared that Ask2Oh545o2 produces an enzyme with greater total activity than that encoded by the Oh51Ao2 allele. The results suggest that the higher level of free amino acids derived from the Aspartate pathway in Oh545o2 endosperm results from a single amino acid change in the ASK2 enzyme that has pleiotropic effects on its activity.
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Additive effects of the feed-back insensitive bacterial Aspartate Kinase and the Brazil nut 2S albumin on the methionine content of transgenic narbon bean (Vicia narbonensis L.)
Molecular Breeding, 2003Co-Authors: Dimitri Demidov, Gad Galili, Christian Horstmann, Martin Meixner, Thomas Pickardt, Isolde Saalbach, Klaus MüntzAbstract:So far two different strategies for engineering high methionine (Met) grain legumes were followed separately in several laboratories: a) The transfer of foreign genes encoding Met-rich proteins, and b) the engineering of Met biosynthesis pathways. In some cases a down regulation of the formation of endogenous sulfur-containing compounds was observed due to the expression of Met-rich foreign proteins. Since this might result from competition of the foreign protein with endogenous compounds for limited Met supply both strategies were combined in the present work. Double transformants of narbon bean ( Vicia narbonensis L.) were generated which express seed-specifically the Met-rich Brazil nut 2S albumin (BNA) as well as a feed-back insensitive bacterial Aspartate Kinase (AK) known to stimulate Met biosynthesis in transgenic tobacco seeds. In order to produce double transformants a homozygous transgenic BNA line of narbon bean was either retransformed with the AK gene or crossed with an AK line. For the first time the influence of a deregulated AK on amino acids of the Aspartate pathway was studied in seeds of a transgenic legume. Effects of expressing the foreign genes on inorganic sulphate, free and protein-bound Met and other amino acids of the Aspartate pathway as well as on free sulphhydryl compounds of mature seeds were analysed. AK lines had 10 to 12 percent and the BNA line 80 percent increased Met in mature seeds. Double transformants showed additive but not synergistic effects of the expression of AK and BNA gene on seed Met. In their mature seeds protein-bound Met reached levels 2.0 to 2.4 times higher than in the wildtype. The Met level of best line corresponds approximately to the FAO standard for Met in a nutritionally balanced protein for human food or for feeding monogastric animals.
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Expression of an Arabidopsis Aspartate Kinase/Homoserine Dehydrogenase Gene Is Metabolically Regulated by Photosynthesis-Related Signals but Not by Nitrogenous Compounds
Plant physiology, 1998Co-Authors: Judith X. Zhu-shimoni, Gad GaliliAbstract:Although the control of carbon fixation and nitrogen assimilation has been studied in detail, relatively little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper we report our study of the metabolic regulation of expression of an Arabidopsis Aspartate Kinase/homoserine dehydrogenase (AK/HSD) gene, which encodes two linked key enzymes in the biosynthetic pathway of Aspartate family amino acids. Northern blot analyses, as well as expression of chimeric AK/HSD-β-glucuronidase constructs, have shown that the expression of this gene is regulated by the photosynthesis-related metabolites sucrose and phosphate but not by nitrogenous compounds. In addition, analysis of AK/HSD promoter deletions suggested that a CTTGACTCTA sequence, resembling the binding site for the yeast GCN4 transcription factor, is likely to play a functional role in the expression of this gene. Nevertheless, longer promoter fragments, lacking the GCN4-like element, were still able to confer sugar inducibility, implying that the metabolic regulation of this gene is apparently obtained by multiple and redundant promoter sequences. The present and previous studies suggest that the conversion of Aspartate into either the storage amino acid asparagine or Aspartate family amino acids is subject to a coordinated, reciprocal metabolic control, and this biochemical branch point is a part of a larger, coordinated regulatory mechanism of nitrogen and carbon storage and utilization.
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Expression of an Aspartate Kinase Homoserine Dehydrogenase Gene Is Subject to Specific Spatial and Temporal Regulation in Vegetative Tissues, Flowers, and Developing Seeds
Plant physiology, 1997Co-Authors: Judith X. Zhu-shimoni, Simcha Lev-yadun, B. Matthews, Gad GaliliAbstract:Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes are regulated during plant development. In the present report, we have used the [beta]-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana Aspartate Kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of Aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually dimished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of Aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.
Makoto Nishiyama - One of the best experts on this subject based on the ideXlab platform.
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Aspartate Kinase involved in 4-hydroxy-3-nitrosobenzamide biosynthesis in Streptomyces murayamaensis.
Bioscience biotechnology and biochemistry, 2016Co-Authors: Mai Tsujimoto, Ayako Yoshida, Takeo Tomita, Tomohisa Kuzuyama, T. Shimizu, Yasuo Ohnishi, Makoto NishiyamaAbstract:Streptomyces murayamensis carries two Aspartate Kinase (AK) genes: one for the biosynthesis of lysine, threonine, and methionine, and the other (nspJ) contained in the biosynthetic gene cluster for the secondary metabolite, 4-hydroxy-3-nitrosobenzamide, for catalyzing the first reaction. AKs involved in the biosynthesis of amino acids are often regulated allosterically by the end products. In the present study, we characterized NspJ to investigate whether AKs involved in secondary metabolism were also allosterically regulated. NspJ was in α2β2 and (α2β2)2 heterooligomeric forms, and was insensitive to all the compounds tested including lysine, threonine, and methionine. The reduction in the activity following the removal of ammonium sulfate, which induced subunit dissociation, suggests that the β subunit may be involved in stabilizing the structure of the α subunit in order to exhibit its activity. This study has provided the first example of a feedback-insensitive α2β2-type AK, which is involved in the s...
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Mechanism of Concerted Inhibition of α2β2-type Hetero-oligomeric Aspartate Kinase from Corynebacterium glutamicum
The Journal of biological chemistry, 2010Co-Authors: Ayako Yoshida, Takeo Tomita, Tomohisa Kuzuyama, Makoto NishiyamaAbstract:Aspartate Kinase (AK) is the first and committed enzyme of the biosynthetic pathway producing Aspartate family amino acids, lysine, threonine, and methionine. AK from Corynebacterium glutamicum (CgAK), a bacterium used for industrial fermentation of amino acids, including glutamate and lysine, is inhibited by lysine and threonine in a concerted manner. To elucidate the mechanism of this unique regulation in CgAK, we determined the crystal structures in several forms: an inhibitory form complexed with both lysine and threonine, an active form complexed with only threonine, and a feedback inhibition-resistant mutant (S301F) complexed with both lysine and threonine. CgAK has a characteristic α2β2-type heterotetrameric structure made up of two α subunits and two β subunits. Comparison of the crystal structures between inhibitory and active forms revealed that binding inhibitors causes a conformational change to a closed inhibitory form, and the interaction between the catalytic domain in the α subunit and β subunit (regulatory subunit) is a key event for stabilizing the inhibitory form. This study shows not only the first crystal structures of α2β2-type AK but also the mechanism of concerted inhibition in CgAK.
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Crystal structures of the regulatory subunit of Thr-sensitive Aspartate Kinase from Thermus thermophilus.
The FEBS journal, 2009Co-Authors: Ayako Yoshida, Takeo Tomita, Tomohisa Kuzuyama, Hidetoshi Kono, Shinya Fushinobu, Makoto NishiyamaAbstract:Crystal structures of the regulatory subunit of Thr-sensitive Aspartate Kinase (AK; EC 2.7.2.4) from Thermus thermophilus (TtAKβ) were determined at 2.15 A in the Thr-bound form (TtAKβ-Thr) and at 2.98 A in the Thr-free form (TtAKβ-free). Although both forms are crystallized as dimers, the contact surface area of the dimer interface in TtAKβ-free (3200 A2) is smaller than that of TtAKβ-Thr (3890 A2). Sedimentation equilibrium analyzed by ultracentrifugation revealed that TtAKβ is present in equilibrium between a monomer and dimer, and that Thr binding shifts the equilibrium to dimer formation. In the absence of Thr, an outward shift of β-strands near the Thr-binding site (site 1) and a concomitant loss of the electron density of the loop region between β3 and β4 near the Thr-binding site are observed. The mechanism of regulation by Thr is discussed on the basis of the crystal structures. TtAKβ has higher thermostability than the regulatory subunit of Corynebacterium glutamicum AK, with a difference in denaturation temperature (Tm) of 40 °C. Comparison of the crystal structures of TtAKβ and the regulatory subunit of C. glutamicum AK showed that the well-packed hydrophobic core and high Pro content in loops contribute to the high thermostability of TtAKβ.
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Structural Insight into concerted inhibition of alpha 2 beta 2-type Aspartate Kinase from Corynebacterium glutamicum.
Journal of molecular biology, 2007Co-Authors: Ayako Yoshida, Takeo Tomita, Tomohisa Kuzuyama, Takeshi Kurihara, Shinya Fushinobu, Makoto NishiyamaAbstract:Abstract Aspartate Kinase (AK) catalyzes the first step of the biosynthesis of the aspartic acid family amino acids, and is regulated via feedback inhibition by end-products including Thr and Lys. To elucidate the mechanism of this inhibition, we determined the crystal structure of the regulatory subunit of AK from Corynebacterium glutamicum at 1.58 A resolution in the Thr-binding form, the first crystal structure of the regulatory subunit of α 2 β 2 -type AK. The regulatory subunit contains two ACT domain motifs per monomer and is arranged as a dimer. Two non-equivalent ACT domains from different chains form an effector-binding unit that binds a single Thr molecule, and the resulting two effector-binding units of the dimer associate perpendicularly in a face-to-face manner. The regulatory subunit is a monomer in the absence of Thr but becomes a dimer by adding Thr. The dimerization is eliminated in mutant AKs with changes in the Thr-binding region, suggesting that the dimerization induced by Thr binding is a key step in the inhibitory mechanism of AK from C. glutamicum . A putative Lys-binding site and the inhibitory mechanism of CgAK are discussed.
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Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of Aspartate Kinase from Thermus thermophilus.
Acta crystallographica. Section F Structural biology and crystallization communications, 2007Co-Authors: Ayako Yoshida, Takeo Tomita, Tomohisa Kuzuyama, Makoto NishiyamaAbstract:Aspartate Kinase (AK) from Thermus thermophilus, which catalyzes the first step of threonine and methionine biosynthesis, is regulated via feedback inhibition by the end product threonine. To elucidate the mechanism of regulation of AK, the regulatory subunit (the beta subunit of T. thermophilus AK) was crystallized in the presence of the inhibitor threonine. Diffraction data were collected to 2.15 A at a synchrotron source. The crystal belongs to the cubic space group P4(3)32 or P4(1)32, with unit-cell parameters a = b = c = 141.8 A.
Renaud Dumas - One of the best experts on this subject based on the ideXlab platform.
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Allosteric monofunctional Aspartate Kinases from Arabidopsis.
FEBS Journal, 2006Co-Authors: Gilles Curien, Mathieu Laurencin, Mylène Robert-genthon, Renaud DumasAbstract:Plant monofunctional Aspartate Kinase is unique among all Aspartate Kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional Aspartate Kinases. We show that Aspartate Kinase 2 and Aspartate Kinase 3 are inhibited only by lysine, and that Aspartate Kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, Aspartate Kinase 1 displayed low apparent affinity for lysine compared to Aspartate Kinase 2 and Aspartate Kinase 3. In the presence of SAM, the apparent affinity of Aspartate Kinase 1 for lysine increased considerably, with K0.5 values for lysine inhibition similar to those of Aspartate Kinase 2 and Aspartate Kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent Km values for the substrates ATP and Aspartate. The mechanism of Aspartate Kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accordance with equilibrium binding analyses indicating that SAM binding to Aspartate Kinase 1 requires prior binding of lysine. The three-dimensional structure of the Aspartate Kinase 1–Lys–SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell18, 1681–1692]. Taken together, the data suggest that, upon binding to the inactive Aspartate Kinase 1–Lys complex, SAM promotes a slow conformational transition leading to formation of a stable Aspartate Kinase 1–Lys–SAM complex. The increase in Aspartate Kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between Aspartate Kinase 1 and Aspartate Kinase 1–Lys towards the inactive form.
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Identification of six novel allosteric effectors of Arabidopsis thaliana Aspartate Kinase-homoserine dehydrogenase isoforms. Physiological context sets the specificity.
The Journal of biological chemistry, 2005Co-Authors: Gilles Curien, Stéphane Ravanel, Mylène Robert, Renaud DumasAbstract:Abstract The Arabidopsis genome contains two genes predicted to code for bifunctional Aspartate Kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on Aspartate Kinase I activity (4-fold activation). The six effectors of Aspartate Kinase I were all activators of Aspartate Kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that Aspartate Kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the Aspartate-derived amino acid pathway in plant.
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Identification of six novel allosteric effectors of Arabidopsis thaliana Aspartate Kinase-Homoserine dehydrogenase. Physiological context sets the specificity
Journal of Biological Chemistry, 2005Co-Authors: Gilles Curien, Stéphane Ravanel, Mylène Robert, Renaud DumasAbstract:The Arabidopsis genome contains two genes predicted to code for bifunctional Aspartate Kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on Aspartate Kinase I activity (4-fold activation). The six effectors of Aspartate Kinase I were all activators of Aspartate Kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that Aspartate Kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the Aspartate-derived amino acid pathway in plant.
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Mechanism of control of Arabidopsis thaliana Aspartate Kinase-homoserine dehydrogenase by threonine.
The Journal of biological chemistry, 2002Co-Authors: Stéphane Paris, Gilles Curien, Claire Viemon, Renaud DumasAbstract:Abstract The regulatory domain of the bifunctional threonine-sensitive Aspartate Kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-αhelix-loop-β strand-loop-β strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites ofArabidopsis thaliana Aspartate Kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wild-type and mutant enzymes demonstrated that each regulatory domain of the monomers of Aspartate Kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln443 and Gln524. Our results also demonstrated that threonine interaction with Gln443 leads to inhibition of Aspartate Kinase activity and facilitates the binding of a second threonine on Gln524. Interaction of this second threonine with Gln524 leads to inhibition of homoserine dehydrogenase activity.
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Overproduction, purification, and characterization of recombinant bifunctional threonine-sensitive Aspartate Kinase-homoserine dehydrogenase from Arabidopsis thaliana.
Protein expression and purification, 2002Co-Authors: Stéphane Paris, Peter M. Wessel, Renaud DumasAbstract:Abstract In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, Aspartate Kinase-homoserine dehydrogenase (AK-HSDH). In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from plants ( Arabidopsis thaliana ). The specific activities corresponding to the forward reaction of AK and reverse reaction of HSDH of AK-HSDH were 5.4 μmol of aspartyl phosphate produced min −1 mg −1 of protein and 18.8 μmol of NADPH formed min −1 mg −1 of protein, respectively. These values are 200-fold higher than those reported previously for partially purified plant enzymes. AK-HSDH exhibited hyperbolic kinetics for Aspartate, ATP, homoserine, and NADP with K M values of 11.6 mM, 5.5 mM, 5.2 mM, and 166 μM, respectively. Threonine was found to inhibit both AK and HSDH activities by decreasing the affinity of the enzyme for its substrates and cofactors. In the absence of threonine, AK-HSDH behaved as an oligomer of 470 kDa. Addition of the effector converted the enzyme into a tetrameric form of 320 kDa.
M Jacobs - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional Aspartate Kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant
Plant molecular biology, 2003Co-Authors: Sven Erik Rognes, M Jacobs, Eric Dewaele, Sten Freddy Aas, Valerie FrankardAbstract:An Aspartate Kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast Aspartate Kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.
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A Mutant of Arabidopsis thaliana lpar;L.) Heynh. with Modified Control of Aspartate Kinase by Threonine
Biochemical Genetics, 1997Co-Authors: B Heremans, M JacobsAbstract:Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grow on 2 m M lysine has been used to isolate mutants that may contain an Aspartate Kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an Aspartate Kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of Aspartate Kinase, were crossed to assess the effects of the simultaneous presence of these different Aspartate Kinase forms. A double mutant (RLT40 × RL4) was isolated and characterized by two feedback-desensitized isozymes of Aspartate Kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double Aspartate Kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1 and ak2) were found to be unlinked.
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A mutant of Arabidopsis thaliana (L.) Heynh. with modified control of Aspartate Kinase by threonine.
Biochemical genetics, 1997Co-Authors: B Heremans, M JacobsAbstract:Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grown on 2 mM lysine has been used to isolate mutants that may contain an Aspartate Kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an Aspartate Kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of Aspartate Kinase, were crossed to assess the effects of the simultaneous presence of these different Aspartate Kinase forms. A double mutant (RLT40 x RL4) was isolated and characterized by two feedback-desensitized isozymes of Aspartate Kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double Aspartate Kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1 and ak2) were found to be unlinked.
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Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional Aspartate Kinase
Plant molecular biology, 1997Co-Authors: Valerie Frankard, Marc Vauterin, M JacobsAbstract:A cDNA clone encoding a monofunctional Aspartate Kinase (AK, ATP:L-Aspartate 4-phosphotransferase, EC 2.7.2.4) has been isolated from an Arabidopsis thaliana cell suspension cDNA library using a homologous PCR fragment as hybridizing probe. Amplification of the PCR fragment was done using a degenerate primer designed from a conserved region between bacterial monofunctional AK sequences and a primer identical to a region of the A. thaliana bifunctional Aspartate Kinase-homoserine dehydrogenase (AK-HSDH). By comparing the deduced amino acid sequence of the fragment with the bacterial and yeast corresponding gene products, the highest identity score was found with the Escherichia coli AKIII enzyme that is feedback-inhibited by lysine (encoded by lysC). The absence of HSDH-encoding sequence at the COOH end of the peptide further implies that this new cDNA is a plant lysC homologue. The presence of two homologous genes in A. thaliana is supported by PCR product sequences, Southern blot analysis and by the independent cloning of the corresponding second cDNA (see Tang et al., Plant Molecular Biology 34, pp. 287–294 [this issue]). This work is the first report of cloning a plant putative lysine-sensitive monofunctional AK cDNA. The presence of at least two genes is discussed in relation to possible different physiological roles of their respective product.
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Threonine Accumulation in a Mutant of Arabidopsis thaliana (L.) Heynh. with an Altered Aspartate Kinase
Journal of Plant Physiology, 1995Co-Authors: B Heremans, M JacobsAbstract:Summary After mutagenesis, M 2 seedlings of Arabidopsis thaliana were grown on a selective medium containing toxic concentrations of lysine and threonine. One of the LT-resistant mutants (RLT 40) was studied at the biochemical and genetical levels. A six-fold increase in free threonine content was found in 8-day-old mutant plantlets compared with the wild type. The total amino acid content of the mutant was also remarkably increased, essentially due to increased levels of Aspartate, threonine, methionine, isoleucine and lysine. As one of the possible reasons for the LT-resistance resides in a change of the regulatory properties of the first enzyme of the Aspartate pathway, Aspartate Kinase (EC 2.7.2.4), the feedback-inhibition pattern of Aspartate Kinase was examined in the mutant and the wild type. In Arabidopsis , after ion-exchange chromatography of whole plant extracts, three peaks of activity were detected corresponding respectively to a threonine-sensitive isoform, to a lysine-sensitive form and a form insensitive to both inhibitors. The threonine accumulation in RLT 40 could be related to a partial insensitivity of the lysinesensitive form of Aspartate Kinase. Genetical analysis showed that the resistance gene behaved as a dominant, monogenic nuclear trait. Linkage analysis, performed with a multiple marker line, indicated that the mutation is located on chromosome 2, 36.0 cM from the er locus and 19.8 cM from the py locus on chromosome 2. This partially lysine-insensitive mutant of Aspartate Kinase offers ways to a map-based approach for cloning the gene coding for the corresponding isoform in plants.
Ricardo Antunes Azevedo - One of the best experts on this subject based on the ideXlab platform.
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Lysine and threonine biosynthesis in sorghum seeds: characterisation of Aspartate Kinase and homoserine dehydrogenase isoenzymes
Annals of Applied Biology, 2006Co-Authors: Renato Rodrigues Ferreira, Lyndel W. Meinhardt, Ricardo Antunes AzevedoAbstract:Aspartate Kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion-exchange chromatography [diethylaminoethyl (DEAE)-Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S-200), one peak of AK activity co-eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine-sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid, calmodulin, S-adenosylmethionine (SAM), S-2-aminoethyl-l-cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine-sensitive isoenzyme and a second predominant threonine-insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.
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Determination of Aspartate Kinase in maize tissues.
2005Co-Authors: Renato Rodrigues Ferreira, Peter J. Lea, Ariane Vendemiatti, Priscila Lupino Gratão, Ricardo Antunes AzevedoAbstract:Lysine, threonine, methionine and isoleucine are synthesized from Aspartate in a branched pathway in higher plants. Aspartate Kinase plays a key role in the control of the Aspartate pathway. The enzyme is very sensitive to manipulation and storage and the hydroxamate assay normally used to determine Aspartate Kinase activity has to be altered according to the plant species and tissue to be analyzed. We have optimized the assay for the determination of Aspartate Kinase in maize plants callus cell cultures. Among all the assay parameters tested, the concentration of ATP/Mg and temperature were critical for enzyme activity. In the case of temperature, 35°C was shown to be the optimum temperature for Aspartate Kinase activity.
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Determination of Aspartate Kinase activity in maize tissues
Scientia Agricola, 2005Co-Authors: Renato Rodrigues Ferreira, Peter J. Lea, Ariane Vendemiatti, Priscila Lupino Gratão, Ricardo Antunes AzevedoAbstract:Lysine, threonine, methionine and isoleucine are synthesized from Aspartate in a branched pathway in higher plants. Aspartate Kinase plays a key role in the control of the Aspartate pathway. The enzyme is very sensitive to manipulation and storage and the hydroxamate assay normally used to determine Aspartate Kinase activity has to be altered according to the plant species and tissue to be analyzed. We have optimized the assay for the determination of Aspartate Kinase in maize plants callus cell cultures. Among all the assay parameters tested, the concentration of ATP/Mg and temperature were critical for enzyme activity. In the case of temperature, 35°C was shown to be the optimum temperature for Aspartate Kinase activity.
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Effects of calcium, S-adenosylmethionine, S-(2-aminoethyl)-l-cysteine, methionine, valine and salt concentration on rice Aspartate Kinase isoenzymes
Plant Science, 2000Co-Authors: Juverlande Lugli, Salete Aparecida Gaziola, Ricardo Antunes AzevedoAbstract:Abstract The activities of two Aspartate Kinase (EC 2.7.2.4) isoenzymes that have been partially purified from developing rice seeds, were studied in the presence of calcium, calmodulin inhibitors, S -adenosylmethionine, S -(2-aminoetyl)- l -cysteine, methionine, valine and increased salt concentrations. None of the compounds tested was able to produce any significant alteration in threonine-sensitive Aspartate Kinase activity. On the other hand, the activity of the lysine-sensitive Aspartate Kinase was slightly increased by calcium. The increase in activity was not observed when EGTA was added in combination with calcium. S -adenosylmethionine alone inhibited the activity by 12% and intensified the inhibition caused by lysine. S -(2-aminoethyl)- l -cysteine also inhibited the activity of Aspartate Kinase, but not to the same extent of lysine. Methionine and valine stimulated slight increases in activity, whereas KCl up to 500 mM did not cause any change in Aspartate Kinase activity. These results with rice Aspartate Kinase indicate that lysine-sensitive Aspartate Kinase is also synergistically inhibited by S -adenosylmethionine, as observed for other plants species. Although some increase in Aspartate Kinase activity was observed in the presence of calcium, the magnitude of the alterations was not sufficient to indicate a regulatory role of calcium on Aspartate Kinase.
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Isolation, partial purification and characterization of isoenzymes of Aspartate Kinase from rice seeds
Journal of Plant Physiology, 1998Co-Authors: Cristiana M.g. Teixeira, Salete Aparecida Gaziola, Juverlande Lugli, Ricardo Antunes AzevedoAbstract:Summary Aspartate Kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been isolated and partially purified from immature rice (Oriza sativa L.) seeds. Three methods of protein separation (ammonium sulphate precipitation, anion-exchange chromatography and gel filtration chromatography) were used in order to purify and identify the isoenzymes of AK and HSDH. Two peaks of AK activity were eluted from the anion-exchange chromatography column (FFQ-Sepharose) with 230 and 286 mmol/L KCl. The first peak in elution order was inhibited by threonine, while the second peak was strongly inhibited by lysine. An optimum pH of 7.4 was determined for total AK activity. The optimum temperature for AK action was about 35 °C, whereas a combination of 12.5 mmol/L magnesium sulphate and 20 mmol/L ATP produced the highest level of activity. After the ammonium sulphate step, only one form of HSDH that was completely inhibited by threonine, was observed. The threonine-sensitive forms of HSDH and AK co-purified, independent of the protein purification method used exhibiting a molecular mass of 186 kDa. The lysine-sensitive AK had a molecular mass estimated to be 167 kDa. These results confirm the hypothesis that in higher plants, threonine and lysine-sensitive forms of AK are present and suggest that a bifunctional polypetide containing threonine-sensitive AK and HSDH is present is rice seeds.
