Overproduction, Purification, and Characterization of Recombinant Bifunctional Threonine-Sensitive Aspartate Kinase-Homoserine Dehydrogenase from Arabidopsis thaliana

Paris, Stéphane; Wessel, Peter M.; Dumas, Renaud

doi:10.1006/prep.2001.1539

Cited by 28 publications

(21 citation statements)

References 30 publications

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“…Although Asd, the corresponding Escherichia coli enzyme, has been studied more extensively and the crystal structure has been determined (Hadfield et al, 1999), relatively little is known about the specific properties of plant enzyme. In vitro overproduction and analysis of the A. thaliana At1g14810 confirmed the predicted aspartate semialdehyde dehydrogenase activity (Paris et al, 2002). Unlike in the case of aspartate kinase, there is no confirmed allosteric regulation of aspartate semialdehyde dehydrogenase in A. thaliana.…”

Section: Biosynthesis Of Aspartate Semialdehyde From Aspartatementioning

confidence: 79%

“…Three of the A. thaliana aspartate kinase genes, At5g13280 (AK1), At5g14060 (AK2) and At3g02020 (AK3), encode monofunctional enzymes (Frankard et al, 1997;Tang et al, 1997;Yoshioka et al, 2001;Curien et al, 2007). The other two, At1g31230 (AK-HSDH1) and At4g19710 (AK-HSDH2), encode bifunctional enzymes with not only aspartate kinase, but also homoserine dehydrogenase (EC 1.1.1.3) activity (Ghislain et al, 1994;Paris et al, 2002;Rognes et al, 2002). Thus, these two bifunctional enzymes catalyze both the first and third steps in the biosynthesis of methionine, threonine, and isoleucine ( Figure 2).…”

Section: Biosynthesis Of Aspartate Semialdehyde From Aspartatementioning

confidence: 99%

“…In vitro assays with cloned enzymes show that their activity is inhibited by threonine and leucine and activated by alanine, cysteine, isoleucine, serine, and valine (Paris et al, 2002;Rognes et al, 2002;Paris et al, 2003;Curien et al, 2005). Based on the concentrations of these amino acids in plant tissue, threonine and alanine are probably the most physiologically relevant effectors (Curien et al, 2005).…”

Section: Biosynthesis Of Aspartate Semialdehyde From Aspartatementioning

confidence: 99%

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Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

Jander

Joshi

2009

The Arabidopsis Book

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The aspartate-derived amino acid pathway in plants leads to the biosynthesis of lysine, methionine, threonine, and isoleucine. These four amino acids are essential in the diets of humans and other animals, but are present in growthlimiting quantities in some of the world's major food crops. Genetic and biochemical approaches have been used for the functional analysis of almost all Arabidopsis thaliana enzymes involved in aspartate-derived amino acid biosynthesis. The branch-point enzymes aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase, cystathionine gamma synthase, threonine synthase, and threonine deaminase contain well-studied sites for allosteric regulation by pathway products and other plant metabolites. In contrast, relatively little is known about the transcriptional regulation of amino acid biosynthesis and the mechanisms that are used to balance aspartatederived amino acid biosynthesis with other plant metabolic needs. The aspartate-derived amino acid pathway provides excellent examples of basic research conducted with A. thaliana that has been used to improve the nutritional quality of crop plants, in particular to increase the accumulation of lysine in maize and methionine in potatoes.

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Section: Biosynthesis Of Aspartate Semialdehyde From Aspartatementioning

confidence: 79%

Section: Biosynthesis Of Aspartate Semialdehyde From Aspartatementioning

confidence: 99%

Section: Biosynthesis Of Aspartate Semialdehyde From Aspartatementioning

confidence: 99%

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Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

Jander

Joshi

2009

The Arabidopsis Book

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“…Aspartate kinase (AK) catalyzes the first step of the aspartate-derived amino acid pathway, and the corresponding gene was found in the QTL of kernel methionine level ( Figure 3, Table 3). In Arabidopsis, two AK genes encode bifunctional enzymes with both aspartate kinase and homoserine dehydrogenase activity (Paris et al, 2002;Rognes et al, 2003). They catalyze both the first and third steps in the biosynthesis of methionine, threonine, and isoleucine.…”

Section: A Maize Primary Metabolic Network Involving Key Genes and Mementioning

confidence: 99%

Genetic Determinants of the Network of Primary Metabolism and Their Relationships to Plant Performance in a Maize Recombinant Inbred Line Population

et al. 2015

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Deciphering the influence of genetics on primary metabolism in plants will provide insights useful for genetic improvement and enhance our fundamental understanding of plant growth and development. Although maize (Zea mays) is a major crop for food and feed worldwide, the genetic architecture of its primary metabolism is largely unknown. Here, we use high-density linkage mapping to dissect large-scale metabolic traits measured in three different tissues (leaf at seedling stage, leaf at reproductive stage, and kernel at 15 d after pollination [DAP]) of a maize recombinant inbred line population. We identify 297 quantitative trait loci (QTLs) with moderate (86.2% of the mapped QTL, R 2 = 2.4 to 15%) to major effects (13.8% of the mapped QTL, R 2 >15%) for 79 primary metabolites across three tissues. Pairwise epistatic interactions between these identified loci are detected for more than 25.9% metabolites explaining 6.6% of the phenotypic variance on average (ranging between 1.7 and 16.6%), which implies that epistasis may play an important role for some metabolites. Key candidate genes are highlighted and mapped to carbohydrate metabolism, the tricarboxylic acid cycle, and several important amino acid biosynthetic and catabolic pathways, with two of them being further validated using candidate gene association and expression profiling analysis. Our results reveal a metabolite-metabolite-agronomic trait network that, together with the genetic determinants of maize primary metabolism identified herein, promotes efficient utilization of metabolites in maize improvement.

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“…Previously, the E. coli HSD was only analyzed in the forward direction with NADPH, and the yeast enzyme was analyzed using NAD(H) in each direction (10 -11). Likewise, kinetic studies of Arabidopsis AK-HSD have primarily relied on using the reverse reaction substrates Hse and NADP ϩ (21,29). Based on comparison of k cat /K m values for GmHSD, the forward reaction is favored by 50 -200-fold over the reverse reaction (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Threonine-insensitive Homoserine Dehydrogenase from Soybean

Schroeder

Zhu

Yanamadala

et al. 2010

Journal of Biological Chemistry

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Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K i ‫؍‬ 160 -240 mM) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K i ϳ150 M). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.

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Overproduction, Purification, and Characterization of Recombinant Bifunctional Threonine-Sensitive Aspartate Kinase-Homoserine Dehydrogenase from Arabidopsis thaliana

Cited by 28 publications

References 30 publications

Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

Aspartate-Derived Amino Acid Biosynthesis inArabidopsis thaliana

Genetic Determinants of the Network of Primary Metabolism and Their Relationships to Plant Performance in a Maize Recombinant Inbred Line Population

Threonine-insensitive Homoserine Dehydrogenase from Soybean

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