Threonine accumulation in the seeds of a barley mutant with an altered aspartate kinase

Barley (Hordeum vulgare L.) mutants altered in the regulation of synthesis of aspartate-derived amino acids were sought by screening embryos for growth on a medium containing lysine plus threonine. One mutant, Rothamsted 2501, was selected with good growth. From the segregation of resistance in the following generations, it was concluded that the resistance was conferred by a dominant gene, Lt1. No homozygous Lt1/Lt1 fertile plants have been recovered. Partially purified aspartate kinase preparations from resistant and sensitive plants were separated on DEAE-cellulose chromatography into three peaks of activity (I, II, III) and the feedback regulatory properties of these peaks determined. These peaks are considered to be three isozymic forms of aspartate kinase, one predominantly sensitive to threonine and two sensitive to lysine or lysine plus S-adenosyl methionine. The feedback characteristics of one of the peaks of aspartate kinase activity from resistant plants were changed such that lysine was half-maximally inhibitory at 10 rather than 0.4mm. Increases in the concentrations of the free pools of threonine (4×) and methionine (2×) were measured in young plants grown on a basal medium. Threonine in the soluble fraction of mature seeds from resistant plants was increased from 0.8 to 9.6% of the total threonine content. The total content of both threonine and methionine of the seeds was increased by 6% compared with grain of similar nitrogen content.S.E.R. acknowledges the receipt of a Council of Europe Scholarship through The British Council. Part of this was also supported by EEC Grant 473.     

In Vivo Regulation of Threonine and Isoleucine Biosynthesis in Lemna paucicostata Hegelm. 6746

Little, if any, regulation of threonine synthesis was observed in Lemna paucicostata Hegelm. 6746 supplemented with concentrations of threonine and/or isoleucine that allow for uptake of these amino acids in amounts sufficient for total plant requirements, and that increase tissue concentrations of soluble threonine manyfold. High tissue concentrations of soluble threonine generated endogenously in isoleucine-supplemented plants were no more effective in regulation than a similar concentration of threonine accumulated from the medium. These studies exclude also major regulation of threonine biosynthesis by bivalent repression by threonine plus isoleucine. Isoleucine biosynthesis was severely inhibited by supplementation with isoleucine, but not with threonine or methionine. The fivefold increase in soluble threonine in isoleucine-supplemented plants suggests that threonine dehydratase is a major locus for feedback regulation of isoleucine synthesis. It is concluded that regulation of threonine biosynthesis differs from that of the other amino acids of the aspartate family (isoleucine, methionine, and lysine), each of which strongly feedback regulates its own synthesis. Methionine supplementation had a negligible effect on the tissue concentration of soluble threonine, indicating that threonine is not important in balancing changes of flux into methionine by equivalent changes of flux through the step catalyzed by aspartokinase.     

Arabidopsis loss-of-function mutant in the lysine pathway points out complex regulation mechanisms

In plants, the amino acids lysine, threonine, methionine and isoleucine have L-aspartate-beta-semialdehyde (ASA) as a common precursor in their biosynthesis pathways. How this ASA precursor is dispersed among the different pathways remains vague knowledge. The proportional balances of free and/or protein-bound lysine, threonine, isoleucine and methionine are a function of protein synthesis, secondary metabolism and plant physiology. Some control points determining the flux through the distinct pathways are known, but an adequate explanation of how the competing pathways share ASA in a fine-tuned amino acid biosynthesis network is yet not available. In this article we discuss the influence of lysine biosynthesis on the adjacent pathways of threonine and methionine. We report the finding of an Arabidopsis thaliana dihydrodipicolinate synthase T-DNA insertion mutant displaying lower lysine synthesis, and, as a result of this, a strongly enhanced synthesis of threonine. Consequences of these cross-pathway regulations are discussed.     

Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum

The regulation of the six enzymes responsible for the conversion of aspartate to lysine, together with homoserine dehydrogenase, was studied in Corynebacterium glutamicum. In addition to aspartate kinase activity, the synthesis of diaminopimelate decarboxylase was also found to be regulated. The specific activity of this enzyme was reduced to one-third in extracts of cells grown in the presence of lysine. Aspartate-semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, and diaminopimelate dehydrogenase were neither influenced in their specific activity, nor inhibited, by any of the aspartate family of amino acids. Homoserine dehydrogenase was repressed by methionine (to 15% of its original activity) and inhibited by threonine (4% remaining activity). Inclusion of leucine in the growth medium resulted in a twofold increase of homoserine dehydrogenase specific activity. The flow of aspartate semialdehyde to either lysine or homoserine was influenced by the activity of homoserine dehydrogenase or dihydrodipicolinate synthase. Thus, the twofold increase in homoserine dehydrogenase activity resulted in a decrease in lysine formation accompanied by the formation of isoleucine. In contrast, repression of homoserine dehydrogenase resulted in increased lysine formation. A similar increase of the flow of aspartate semialdehyde to lysine was found in strains with increased dihydrodipicolinate synthase activity, constructed by introducing the dapA gene of Escherichia coli (coding for the synthase) into C. glutamicum.     

Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): study of enzymes and nitrogen-containing compounds.

Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-l-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound.     

Regulatory mechanisms after short‐ and long‐term perturbed lysine biosynthesis in the aspartate pathway: the need for isogenes in Arabidopsis thaliana

The aspartate‐derived amino acid pathway in plants is an intensively studied metabolic pathway, because of the biosynthesis of the four essential amino acids lysine, threonine, isoleucine and methionine. The pathway is mainly controlled by the key regulatory enzymes aspartate kinase (AK; EC 2.7.2.4), homoserine dehydrogenase (HSDH; EC 1.1.1.3) and 4‐hydroxy‐tetrahydrodipicolinate synthase (EC 4.3.3.7), formerly referred to as dihydrodipicolinate synthase (DHDPS). They are encoded by isoenzyme families and it is not known why such families are evolutionarily maintained. To gain more insight into the specific roles and regulation of the isoenzymes, we inhibited DHDPS in Arabidopsis thaliana with the chemical compound (N,N‐dimethylglycinatoboranyloxycarbonylmethyl)‐dimethylamine‐borane (DDAB) and compared the short‐term effects on the biochemical and biomolecular level to the long‐term adaptations in dhdps knockout mutants. We found that DHDPS2 plays a crucial role in controlling lysine biosynthesis, thereby stabilizing flux through the whole aspartate pathway. Moreover, DHDPS2 was also shown to influence the threonine level to a large extent. In addition, the lysine‐sensitive AKs, AKLYS1 and AKLYS3 control the short‐ and long‐term responses to perturbed lysine biosynthesis in Arabidopsis thaliana.     

Lysine metabolism in higher plants

Summary. The essential amino acid lysine is synthesised in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine. Enzyme kinetic studies and the analysis of mutants and transgenic plants that overaccumulate lysine, have indicated that the major site of the regulation of lysine synthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, there is strong evidence that lysine is also subject to catabolism in plants, specifically in the seed. The two enzymes involved in lysine breakdown, lysine 2-oxoglutarate reductase (also known as lysine α-ketoglutarate reductase) and saccharopine dehydrogenase exist as a single bifunctional protein, with the former activity being regulated by lysine availability, calcium and phosphorylation/dephosphorylation. Received December 21, 1999 Accepted February 7, 2000     

Effects of Orthophosphate and Adenosine 5'-Phosphate on Threonine Synthase and Cystathionine gamma-Synthate of Lemna paucicostata Hegelm. 6746

Inorganic phosphate (Pi) inhibits threonine synthase of Lemna, and cystathionine γ-synthase less strongly. AMP is an extremely potent and structurally specific inhibitor of threonine synthase. Each inhibition progressively decreases with increasing concentrations of O-phosphohomoserine (OPH). To study the in vivo effects of these inhibitions, Lemna was grown with a range of Pi concentrations. A 25,000-fold increase in Pi concentration in the culture medium caused an increase of only 6-fold in total phosphorus of the plants. This is explained by the fact that a high affinity Pi uptake system is selectively down-regulated during growth with high concentrations of Pi. Pi and AMP in plants grown with various Pi concentrations were determined, and concentrations estimated for chloroplasts, the organelle containing threonine synthase and cystathionine γ-synthase. Calculations indicated that for growth at standard external Pi (0.4 millimolar) or above, if total OPH were uniformly distributed within the plants, activities of the two enzymes in question would be severely inhibited, and each would fall two orders of magnitude below the amount required to provide threonine (plus isoleucine) or methionine adequate for growth. If OPH were restricted to chloroplasts, these inhibitions would be much less severe, resulting in enzyme activities approaching the required physiological amounts. Evidence is presented that even up to 50 millimolar external Pi, this ion does not limit production of threonine or methionine sufficiently to retard growth, consistent with the postulated localization of OPH within chloroplasts.     

Methionine-lysine-threonine-isoleucine interrelationships in the amino acid nutrition of rice callus tissue

Growth of rice callus tissue is discouraged when methionineis excluded from CMAA medium. While determining the methionineelimination effect, the amino acid interrelationships amongmethionine, lysine, threonine and isoleucine in the nutritionof the callus tissue were found. Poor growth, found in cultureson methionine deficient media was seen only when the media containedboth threonine and lysine, simultaneously. The substitutionof homoserine for methionine was also observed. Determination of free amino acid composition in tissues revealedthat free methionine was barely detectable in tissues grownwith sufficient amounts of threonine and lysine. When the concentrationof either threonine or lysine was reduced, the free methioninecontent of the tissue increased. When the methionine deficientmedium was supplemented with homoserine, the free methioninein the tissue increased, although the tissue retained a considerableamount of free threonine and lysine. Cultivation of tissue onan isoleucine deficient medium resulted in a significant decreasein free threonine content. These experimental results suggest that the biosynthetic pathwayto methionine is cooperatively inhibited by threonine and lysine,and that threonine decomposition is inhibited by its end productisoleucine. (Received February 19, 1970; )     

Selection and characterization of a feedback-insensitive tissue culture of maize

Tissue culture selection techniques were used to isolate a maize (Zea mays L.) variant D33, in which the aspartate family pathway was less sensitive to feedback inhibition by lysine. D33 was recovered by successively subculturing cultures originally derived from immature embryos on MS medium containing growth-inhibitory levels of lysine+threonine. The ability of D33 to grow vigorously on lysine+ threonine medium was retained after growth for 12 months on nonselection medium. New cultures initiated from shoot tissues of plants regenerated from D33 also were resistant to lysine+threonine inhibition. The K_i of aspartokinase for its feedback inhibitor, lysine, was about 9-fold higher in D33 than for the enzyme from unselected cultures. The free pools of lysine, threonine, isoleucine and methionine were increased 2–9-fold in D33 cultures. This was consistent with the observed change in feedback regulation of aspartokinase, the first enzyme common to the biosynthesis of these amino acids in the aspartate pathway. The accumulated evidence including the stability of resistance in the cultures, the resistance of cultures initiated from regenerated plants, the altered feedback regulation, and the increased free amino acids, indicates a mutational origin for these traits in line D33.Abbreviation LT lysine+threonine in equimolar concentration Paper No. 10880, Scientific Journal Series, Minnesota Agricultural Expertment Station     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

水稻抗氨基酸类似物突变体的筛选——Ⅰ.抗S-(2-氨乙基)L-半胱氨酸(AEC)突变体的筛选

以赖氨酸类似物S-(2-氨乙基)L-半胱氨酸(AEC)为选择剂,从水稻花药培养中筛选出一个抗性突变体(R_(AEC))。突变体愈伤组织经过6个月继代培养后仍保持抗性稳定。R_(AEC)再生植株根尖诱导的愈伤组织经过3个月继代培养也保持稳定的抗性。R_(AEC)细胞内赖氨酸含量提高了近2倍,苏氨酸提高5倍多。其他氨基酸,如蛋氨酸、酪氨酸、丝氨酸等都有较大量的提高。 R_(AEC)愈伤组织对赖氨酸加苏氨酸混合物也具有抗性。突变体植株较原始类型稍矮小,巳正常结实。     

Genetic and amino-acid analysis of two maize threonine-overproducing,lysine-insensitive aspartate kinase mutants

The aspartate-derived amino-acid pathway leads to the production of the essential amino-acids lysine, methionine, threonine and isoleucine. Aspartate kinase (AK) is the first enzyme in this pathway and exists in isoforms that are feedback inhibited by lysine and threonine. Two maize (Zea mays L.) threonine-overproducing, lysine-insensitive AK mutants (Ask1-LT19 and Ask2-LT20) were previously isolated. The present study was conducted to determine the map location of Ask2 and to examine the amino-acid profiles of the Ask mutants. The threonine-overproducing trait conferred by Ask2-LT20 was mapped to the long arm of chromosome 2. Both mutants exhibited increased free threonine concentrations (nmol/mg dry weight) over wild-type. The percent free threonine increased from approximately 2% in wild-type kernels to 37–54% of the total free amino-acid pool in homozygous mutant kernels. Free methionine concentrations also increased significantly in homozygous mutants. Free lysine concentrations were increased but to a much lesser extent than threonine or methionine. In contrast to previous studies, free aspartate concentrations were observed to decrease, indicating a possible limiting factor in threonine synthesis. Total (free plus protein-bound) amino-acid analyses demonstrated a consistent, significant increase in threonine, methionine and lysine concentrations in the homozygous mutants. Significant increases in protein-bound (total minus free) threonine, methionine and lysine were observed in the Ask mutants, indicating adequate protein sinks to incorporate the increased free amino-acid concentrations. Total amino-acid contents (nmol/kernel) were approximately the same for mutant and wild-type kernels. In five inbred lines both Ask mutations conferred the threonine-overproducing phenotype, indicating high expressivity in different genetic backgrounds. These analyses are discussed in the context of the regulation of the aspartate-derived amino-acid pathway.     

Aspartate metabolism in Mycobacterium avium grown in host tissue and axenically and in Mycobacterium leprae

Aspartokinase activity was detected in extracts from Mycobacterium leprae (recovered from armadillo liver) and in Mycobacterium avium grown axenically and in vivo. Homoserine dehydrogenase activity was only detected in M. leprae and in M. avium grown axenically. Activities, when detected, were 50 to 70% lower in M. leprae or M. avium grown in vivo than in axenically grown M. avium. In these two pathogenic mycobacteria, aspartokinase and homoserine dehydrogenase are subject to feedback inhibition by methionine - an additional regulator over those observed for the enzymes from Mycobacterium smegmatis. Intact mycobacterium incorporated carbon from [U-14C]aspartate into the aspartate family of amino acids (threonine, isoleucine, methionine and lysine) though the rate of incorporation in M. avium grown in vivo was about half that in M. avium grown axenically.     

The effect of aspartate-derived amino acids (lysine,threonine, methionine) on the growth of excised embryos of wheat and barley

Excised wheat (Triticum aestivum L. var. Maris Freeman) and barley (Hordeum vulgare L. var. Maris Mink) embryos were grown on medium containing both nitrate and ammonium ions. Addition of lysine (1 mM) plus threonine (1 mM) caused a synergistic inhibition of growth measured by length of first leaf or dry weight. The inhibition was specifically relieved by methionine, homocysteine and homoserine. Threonine at 0.2–0.3 mM caused half-maximal inhibition of growth at all lysine concentrations whereas lysine increased the synergistic inhibition up to 3 mM. The inhibition is explained by a model in which lysine acts as a feedback inhibitor of aspartate kinase and threonine of homoserine dehydrogenase. This is compatible with published studies of the enzymes involved. The implications of these findings for using lysine plus threonine as a selection system for lysine-overproducing cereals are discussed.Abbreviations Lys Lysine - Thr Threonine - Met Methionine - Hser Homoserine - Hcys Homocysteine     

Enzymes of lysine metabolism from Coix lacryma-jobi seeds

Lysine, threonine, methionine and isoleucine are synthesized through the aspartate metabolic pathway. The concentrations of soluble lysine and threonine in cereal seeds are very low. Coix lacryma-jobi (coix) is a maize-related grass and the enzymological aspects of the aspartate metabolic pathway are completely unknown. In order to obtain information on lysine metabolism in this plant species, two enzymes involved in the biosynthesis of these amino acids (aspartate kinase 〚AK, EC 2.7.2.4〛 and homoserine dehydrogenase 〚HSDH, EC 1.1.1.3〛) and two enzymes involved in lysine degradation (lysine 2-oxoglutarate reductase 〚LOR, EC 1.5.1.8〛 and saccharopine dehydrogenase 〚SDH, EC 1.5.1.9〛) were isolated and partially characterized in coix seeds. AK activity was inhibited by threonine and lysine separately, suggesting the presence of two isoenzymes, one sensitive to lysine and the other sensitive to threonine, with the latter corresponding to approximately 60% of the total AK activity. In contrast to previous results from other plant species, the threonine-sensitive AK eluted from an ion exchange chromatography column at higher KCl concentration than the lysine-sensitive form. The HSDH activity extracted from the seeds was partially inhibited by threonine, indicating the presence of threonine-sensitive and threonine-resistant isoenzymes. LOR and SDH activities were detected only in the endosperm tissue and co-purified on an anion exchange chromatography column, suggesting that the two activities may be linked on a single bifunctional polypeptide, as observed for other plant species. One single SDH activity band was observed on non-denaturing PAGE gels. The K_m for saccharopine of SDH was determined as 0.143 mM and the K_m for NAD as 0.531 mM. Although SDH activity was shown to be stable, LOR, AK and HSDH were extremely unstable, under all buffer systems tested.     

Methionine Biosynthesis in Lemna: STUDIES ON THE REGULATION OF CYSTATHIONINE gamma-SYNTHASE, O-PHOSPHOHOMOSERINE SULFHYDRYLASE, AND O-ACETYLSERINE SULFHYDRYLASE

Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine gamma-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine gamma-synthase, it was observed that (a) adding external methionine (2 mum) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 muml-aminoethoxyvinylglycine or with 36 mum lysine plus 4 mum threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine gamma-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase.The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine gamma-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine gamma-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of O-acetylserine sulfhydrylase provides an explanation in molecular terms for transsulfuration, and not direct sulfhydration, being the dominant pathway for homocysteine biosynthesis.     

芦笋抗S—（2—氨乙基）—L—半胱氨酸（AEC）变异体的筛选

S-(2-氨乙基)-L-半胱氨酸(AEC)可抑制芦笋愈伤组织的生长,此抑制作用可被赖氨酸或甲硫氨酸部分解除。用0.5mmol/L的AEC进行筛选,得到抗性愈伤组织AR10并再生植株。AR10愈伤组织经一年多的继代培养,在离开选择剂组培继代两代后仍保持对AEC的抗性。抗性系愈伤组织还表现出对2mmol/L的半胱氨酸具交叉抗性,对1mmol/L的赖氨酸加苏氨酸表现部分交叉抗性。AR10再生植株一部分保持对AEC的抗性,而一部分则无抗性。对抗性愈伤组织及其再生植株的氨基酸分析表明,愈伤组织内游离赖氨酸、苏氨酸、甲硫氨酸都有增加,而在再生植株内却发现半胱氨酸和赖氨酸的特异性增加,分别是对照植株的5.4和4.6倍。     

Aspartokinase from wheat germ: isolation, characterization, and regulation

Aspartokinase has been isolated from wheat germ and a preliminary survey made of its properties in a partially purified extract. The enzyme has an absolute requirement for ATP and a divalent metal ion. The phosphate donor can be either ATP or GTP, but other nucleotides are ineffective. Both magnesium and manganese will activate the enzyme, whereas calcium shows a trace amount of activity. The enzyme has a Km of 16.7 mm for aspartate, 1.2 mm for ATP, and 3.3 mm for MgCl(2). Lysine inhibits the reaction at fairly low concentrations, and threonine inhibits at high concentrations. Other amino acids which are derived from aspartate (methionine, homoserine, threonine, and isoleucine) have little effect. When lysine and threonine are added together, they show a concerted inhibition of the reaction. The enzyme is also stabilized against heat inactivation by lysine and threonine together but not by either when added separately. It is suggested that aspartokinase from plants is a regulatory enzyme and exhibits a concerted feedback mechanism.     

Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase

The essential amino acids lysine and threonine are synthesized in higher plants by two separate branches of a common pathway. This pathway is primarily regulated by three key enzymes, namely aspartate kinase (AK), dihydrodipicolinate synthase (DHPS) and homoserine dehydrogenase (HSD), but how these enzymes operate in concert is as yet unknown. Addressing this issue, we have expressed in transgenic tobacco plants high levels of bacterial AK and DHPS, which are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Such expression of the bacterial DHPS by itself resulted in a substantial overproduction of lysine, whereas plants expressing only the bacterial AK overproduced threonine. When both bacterial enzymes were expressed in the same plant, the level of free lysine exceeded by far the level obtained by the bacterial DHPS alone. This increase, however, was accompanied by a significant reduction in threonine accumulation compared to plants expressing the bacterial AK alone. Our results suggested that in tobacco plants the synthesis of both lysine and threonine is under a concerted regulation exerted by AK, DHPS, and possibly also by HSD. We propose that the balance between lysine and threonine synthesis is determined by competition between DHPS and HSD on limiting amounts of their common substrate 3-aspartic semialdehyde, whose level, in turn, is determined primarily by the activity of AK. The potential of this molecular approach to increase the nutritional quality of plants is discussed.