Lysine-ketoglutarate Reductase Research Articles

Effect of Dietary Crude Protein and Apparent Metabolizable Energy Levels on Growth Performance, Nitrogen Utilization, Serum Parameter, Protein Synthesis, and Amino Acid Metabolism of 1- to 10-Day-Old Male Broilers.

This research compared how different levels of dietary crude protein (CP) and apparent metabolizable energy (AME) affect the growth performance, nitrogen utilization, serum parameters, protein synthesis, and amino acid (AA) metabolism in broilers aged 1 to 10 days. In a 4 × 3 factorial experimental design, the broilers were fed four levels of dietary CP (20%, 21%, 22%, and 23%) and three levels of dietary AME (2800 kcal/kg, 2900 kcal/kg, and 3000 kcal/kg). A total of 936 one-day-old male Arbor Acres broilers were randomly allocated to 12 treatments with 6 replications each. Growth performance, nitrogen utilization, serum parameter, gene expression of protein synthesis, and AA metabolism were evaluated at 10 d. The results revealed no interaction between dietary CP and AME levels on growth performance (p > 0.05). However, 22% and 23% CP enhanced body weight gain (BWG), the feed conversion ratio (FCR), total CP intake, and body protein deposition but had a detrimental effect on the protein efficiency ratio (PER) compared to 20% or 21% CP (p < 0.05). Broilers fed diets with 2800 kcal/kg AME showed increased feed intake (FI) and inferior PER (p < 0.05). Broilers fed diets with 3000 kcal/kg AME showed decreased muscle mRNA expression of mammalian target of the rapamycin (mTOR) and Atrogin-1 compared to those fed diets with 2800 kcal/kg and 2900 kcal/kg AME (p < 0.05). Increasing dietary CP level from 20% to 23% decreased muscle mTOR and increased S6K1 mRNA expression, respectively (p < 0.05). The muscle mRNA expression of Atrogin-1 was highest for broilers fed 23% CP diets (p < 0.05). The mRNA expression of betaine homocysteine methyltransferase (BHMT) and Liver alanine aminotransferase of the 22% and 23% CP groups were higher than those of 20% CP (p < 0.05). Significant interactions between dietary CP and AME levels were observed for muscle AMPK and liver lysine-ketoglutarate reductase (LKR) and branched-chain alpha-keto acid dehydrogenase (BCKDH) mRNA expression (p < 0.05). Dietary AME level had no effect on muscle AMPK mRNA expression for broilers fed 21% and 22% CP diets (p > 0.05), whereas increasing dietary AME levels decreased AMPK mRNA expression for broilers fed 23% CP diets (p < 0.05). The mRNA expression of LKR and BCKDH was highest for broilers fed the diet with 2800 kcal/kg AME and 22% CP, while it was lowest for broilers fed the diet with 3000 kcal/kg AME and 20% CP. The findings suggest that inadequate energy density hindered AA utilization for protein synthesis, leading to increased AA catabolism for broilers aged 1 to 10 days, and a dietary CP level of 22% and an AME level of 2900 to 3000 kcal/kg may be recommended based on performance and dietary protein utilization.

LYSINE KETOGLUTARATE REDUCTASE TRANS-SPLICING RELATED 1 is involved in temperature-dependent root growth in rice.

Root length is an important root parameter directly related to the uptake of water and nutrients. However, the molecular mechanisms controlling root length are still not fully understood. Here, we isolated a short-root mutant of rice, dice2 (defective in cell elongation 2). The cell length and meristem size of the roots were decreased in dice2, but the root function in terms of mineral element uptake, root cell width, and root anatomy were hardly altered compared with wild-type (WT) rice. The root growth defect in dice2 could be partially rescued by high temperature. Map-based cloning combined with a complementation test revealed that the short-root phenotype was caused by a nonsense mutation in a gene which was annotated to encode Lysine Ketoglutarate Reductase Trans-Splicing related 1 (OsLKRT1). OsLKRT1, encoding a cytosol-localized protein, was expressed in all cells of the root tip and elongation region as well as the shoot. RNA-seq analysis showed that there was no difference between dice2 and the WT in the expression level of genes involved in root development identified so far. These results indicate that OsLKRT1 is involved in a novel pathway required for root cell elongation in rice, although its exact role remains to be further investigated.

Effects of Feeding Mochidawara and Koshihikari Rice on Growth Performance, Blood Parameters, DM and CP Digestibility and Liver Lysine-Ketoglutarate Reductase Activity in Weanling Piglets

Effects of Feeding Mochidawara and Koshihikari Rice on Growth Performance, Blood Parameters, DM and CP Digestibility and Liver Lysine-Ketoglutarate Reductase Activity in Weanling Piglets

PSIX-24 Effects of rice feeding on growth performance and protein (amino acids) metabolism in weaning piglets

Abstract This experiment was conducted to clarify the effects of feeding rice on growth performance and protein (amino acids) metabolism in weaning piglets. Sixteen weaning piglets with an average initial weight of 7.5 kg were divided into two groups. One group was fed a corn-soybean meal based diet, and the other was fed a rice-soybean meal diet. Each diet contained about 46% of corn or rice. A 2-week growth trial was conducted and growth performance, dry matter and crude protein digestibility, liver lysine-ketoglutarate reductase activity, plasma urea nitrogen, glucose, insulin, and free amino acids concentrations were measured. The average daily gain (g/d) and feed efficiency (gain/feed) in rice-fed piglets were significantly higher than those in corn-fed piglets. Average feed intake, however, was not significantly different between corn- and rice-fed piglets. Dry matter and and crude protein digestibility were not significantly different between two groups. Liver lysine-ketoglutarate reductase activity tended to be lower (P = 0.073) in rice-fed piglets than that in corn-fed piglets. Plasma urea nitrogen concentration in rice-fed piglets was significantly lower than that in corn-fed piglets. Plasma glucose and insulin concentrations were significantly higher in rice-fed piglets than those in corn-fed piglets. Plasma free valine, isoleucine, tryptophan, and glysine concentrations were significantly higher in rice-fed piglets than those in corn-fed piglets. On the other hand, plasma histidine concentration in rice-fed piglets was significantly lower than that in corn-fed piglets. In conclusion, these results clearly show that rice feeding affects the protein and amino acids metabolism and improves the growth performance in weaning piglets. Rice could be considered to be a good feedstuff for weaning piglets.

Effects of rice feeding on growth performance and protein (amino acids) metabolism in weanling piglets.

We investigated the effects of rice feeding on growth performance and protein (amino acids) metabolism of weanling piglets. In all, 16 weanling piglets with an average initial weight of 7.5kg were divided into two groups. One group was fed a corn-soybean meal-based diet, and the other was fed a rice-soybean meal diet, containing around 46% of corn or rice, respectively. A two-week growth trial was conducted. The average daily gain (p=.025) and feed efficiency (p=.011) in rice-fed piglets were significantly higher than those in corn-fed piglets. Liver lysine-ketoglutarate reductase activity tended to be lower (p=.073) in rice-fed piglets than in corn-fed piglets. Plasma urea nitrogen concentration in rice-fed piglets was significantly lower than that in corn-fed piglets. Plasma glucose and insulin concentrations were significantly higher in rice-fed piglets than in corn-fed piglets. Plasma-free valine, isoleucine, and tryptophan concentrations were significantly higher in rice-fed piglets than in corn-fed piglets. In contrast, plasma histidine concentration was significantly lower in rice-fed piglets than in corn-fed piglets. Overall, these results show that rice feeding improves the growth performance and affects the protein (amino acids) metabolism in weanling piglets.

Lysine catabolism in Haemonchus contortus and Teladorsagia circumcincta

Catabolism of lysine through the pipecolate, saccharopine and cadaverine pathways has been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Both enzymes of the saccharopine pathway (lysine ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH)) were active in L3 and adult worms of both species. All three enzymes which catabolise lysine to α-amino adipic semialdehyde via pipecolate (lysine oxidase (LO), Δ1-piperideine-2-carboxylate reductase (Pip2CR) and pipecolate oxidase (PipO)) were present in adult worms, whereas the pathway was incomplete in L3 of both species; Pip2CR activity was not detected in the L3 of either parasite species. In adult worms, the saccharopine pathway would probably be favoured over the pipecolate pathway as the Km for lysine was lower for LKR than for LO. Neither lysine dehydrogenase nor lysine decarboxylase activity was detected in the two parasite species. Enzyme activities and substrate affinities were higher for all five enzymes in adult worms than in L3. An unexpected finding was that both LKR and SDH were dual co-factor enzymes and not specific for either NAD+ or NADP+, as is the case in other organisms. This novel property of LKR/SDH suggests it could be a good candidate for anthelmintic targeting.

Lysine degradation through the saccharopine pathway in bacteria: LKR and SDH in bacteria and its relationship to the plant and animal enzymes

Lysine degradation through the saccharopine pathway has been shown only in plants and animals. Here, we show that bacteria possess the genes encoding lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH). In Silicibacter, the contiguous lkr and sdh genes are interspersed, in another frame, by a polypeptide of unknown function. The bacterial enzyme does not contain the 110-amino-acid interdomain (ID) that intersperses the LKR and SDH domains of the plant enzyme. The ID was found in Cyanobacteria interspersing polypeptides without similarities and activities of LKR and SDH. The LKR/SDH bifunctional polypeptide of animals and plants may have arisen from a α-proteobacterium with a configuration similar to that of Silicibacter, whereas the ID in the plant enzyme may have been inherited from Cyanobacteria.

Tissue distribution of lysine metabolism in commercial turkeys

The essential amino acid lysine is typically the first or second limiting amino acid in diets fed to growing turkeys. However, there is little information about how and where lysine is degraded. Therefore, an investigation of the tissue distribution of lysine catabolism was performed using liver, kidney, enterocytes, breast, thigh, heart and lung of four and eight week old turkeys. The tissues were assayed for the initial enzyme activity of the primary pathway of lysine oxidation, lysine ketoglutarate reductase (LKR). Additionally, lysine oxidation (LOX) was determined based on the recovery of 14CO2 from radioactive lysine. All tissues assayed had measureable LKR activity except lung. Liver had the highest activity (5.2 mmol/min*g liver). Intestine, kidney and liver oxidized lysine (P&lt;0.05) and liver had the highest lysine oxidation (15.9 μmol/min*g tissue). A significant week * tissue interaction and week effect were detected. A positive correlation (P&lt;0.05) was observed between LKR activity and LOX. In conclusion, it is clear that extra hepatic tissues contribute to whole body lysine metabolism. (Support: HATCH WVA 470, Virginia Poultry Grower Cooperative)

Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme

BackgroundAmong the dietary essential amino acids, the most severely limiting in the cereals is lysine. Since cereals make up half of the human diet, lysine limitation has quality/nutritional consequences. The breakdown of lysine is controlled mainly by the catabolic bifunctional enzyme lysine ketoglutarate reductase - saccharopine dehydrogenase (LKR/SDH). The LKR/SDH gene has been reported to produce transcripts for the bifunctional enzyme and separate monofunctional transcripts. In addition to lysine metabolism, this gene has been implicated in a number of metabolic and developmental pathways, which along with its production of multiple transcript types and complex exon/intron structure suggest an important node in plant metabolism. Understanding more about the LKR/SDH gene is thus interesting both from applied standpoint and for basic plant metabolism.ResultsThe current report describes a wheat genomic fragment containing an LKR/SDH gene and adjacent genes. The wheat LKR/SDH genomic segment was found to originate from the A-genome of wheat, and EST analysis indicates all three LKR/SDH genes in hexaploid wheat are transcriptionally active. A comparison of a set of plant LKR/SDH genes suggests regions of greater sequence conservation likely related to critical enzymatic functions and metabolic controls. Although most plants contain only a single LKR/SDH gene per genome, poplar contains at least two functional bifunctional genes in addition to a monofunctional LKR gene. Analysis of ESTs finds evidence for monofunctional LKR transcripts in switchgrass, and monofunctional SDH transcripts in wheat, Brachypodium, and poplar.ConclusionsThe analysis of a wheat LKR/SDH gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes show lineage-specific differences between monocots and dicots, and findings suggest variation in activity of LKR/SDH genes among plants. Although most plant genomes seem to contain a single conserved LKR/SDH gene per genome, poplar possesses multiple contiguous genes. A preponderance of SDH transcripts suggests the LKR region may be more rate-limiting. Only switchgrass has EST evidence for LKR monofunctional transcripts. Evidence for monofunctional SDH transcripts shows a novel intron in wheat, Brachypodium, and poplar.

LKR/SDH plays important roles throughout the tick life cycle including a long starvation period.

BackgroundLysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in plants and mammals. However, to date, the properties of the lysine degradation pathway and biological functions of LKR/SDH have been very little described in arthropods such as ticks.Methodology/Principal FindingsWe isolated and characterized the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) from a tick, Haemaphysalis longicornis, cDNA library that encodes a bifunctional polypeptide bearing domains similar to the plant and mammalian LKR/SDH enzymes. Expression of LKR/SDH was detected in all developmental stages, indicating an important role throughout the tick life cycle, including a long period of starvation after detachment from the host. The LKR/SDH mRNA transcripts were more abundant in unfed and starved ticks than in fed and engorged ticks, suggesting that tick LKR/SDH are important for the starved tick. Gene silencing of LKR/SDH by RNAi indicated that the tick LKR/SDH plays an integral role in the osmotic regulation of water balance and development of eggs in ovary of engorged females.Conclusions/SignificanceTranscription analysis and gene silencing of LKR/SDH indicated that tick LKR/SDH enzyme plays not only important roles in egg production, reproduction and development of the tick, but also in carbon, nitrogen and water balance, crucial physiological processes for the survival of ticks. This is the first report on the role of LKR/SDH in osmotic regulation in animals including vertebrate and arthropods.

Epistatic Interactions betweenOpaque2Transcriptional Activator and Its Target GeneCyPPDK1Control Kernel Trait Variation in Maize

Association genetics is a powerful method to track gene polymorphisms responsible for phenotypic variation, since it takes advantage of existing collections and historical recombination to study the correlation between large genetic diversity and phenotypic variation. We used a collection of 375 maize (Zea mays ssp. mays) inbred lines representative of tropical, American, and European diversity, previously characterized for genome-wide neutral markers and population structure, to investigate the roles of two functionally related candidate genes, Opaque2 and CyPPDK1, on kernel quality traits. Opaque2 encodes a basic leucine zipper transcriptional activator specifically expressed during endosperm development that controls the transcription of many target genes, including CyPPDK1, which encodes a cytosolic pyruvate orthophosphate dikinase. Using statistical models that correct for population structure and individual kinship, Opaque2 polymorphism was found to be strongly associated with variation of the essential amino acid lysine. This effect could be due to the direct role of Opaque2 on either zein transcription, zeins being major storage proteins devoid of lysine, or lysine degradation through the activation of lysine ketoglutarate reductase. Moreover, we found that a polymorphism in the Opaque2 coding sequence and several polymorphisms in the CyPPDK1 promoter nonadditively interact to modify both lysine content and the protein-versus-starch balance, thus revealing the role in quantitative variation in plants of epistatic interactions between a transcriptional activator and one of its target genes.

DLKR/SDH regulates hormone-mediated histone arginine methylation and transcription of cell death genes

The sequential modifications of histones form the basis of the histone code that translates into either gene activation or repression. Nuclear receptors recruit a cohort of histone-modifying enzymes in response to ligand binding and regulate proliferation, differentiation, and cell death. In Drosophila melanogaster, the steroid hormone ecdysone binds its heterodimeric receptor ecdysone receptor/ultraspiracle to spatiotemporally regulate the transcription of several genes. In this study, we identify a novel cofactor, Drosophila lysine ketoglutarate reductase (dLKR)/saccharopine dehydrogenase (SDH), that is involved in ecdysone-mediated transcription. dLKR/SDH binds histones H3 and H4 and suppresses ecdysone-mediated transcription of cell death genes by inhibiting histone H3R17me2 mediated by the Drosophila arginine methyl transferase CARMER. Our data suggest that the dynamic recruitment of dLKR/SDH to ecdysone-regulated gene promoters controls the timing of hormone-induced gene expression. In the absence of dLKR/SDH, histone methylation occurs prematurely, resulting in enhanced gene activation. Consistent with these observations, the loss of dLKR/SDH in Drosophila enhances hormone-regulated gene expression, affecting the developmental timing of gene activation.

Increasing lysine levels in pigeonpea (Cajanus cajan (L.) Millsp) seeds through genetic engineering

In higher plants the essential amino acids lysine, threonine, methionine and isoleucine are synthesised through a branched pathway starting from aspartate. The key enzyme of lysine biosynthesis in this pathway—dihydrodipicolinate synthase (DHDPS)—is feedback-inhibited by lysine. The dhdps-r1 gene from a mutant Nicotiana sylvestris, which encodes a DHDPS enzyme insensitive to feedback inhibition, was used to improve the lysine content in pigeonpea seeds. The dhdps-r1 coding region driven by a phaseolin or an Arabidopsis 2S2 promoter was successfully overexpressed in the seeds of pigeonpea by using Agrobacterium transformation and particle bombardment. In 11 lines analysed, a 2- to 6-fold enhanced DHDPS activity in immature seeds at a late stage of maturation was found in comparison to wild type. The overexpression of dhdps-r1 led to an enhanced content of free lysine in the seeds of pigeonpea from 1.6 to 8.5 times compared with wild type. However, this was not reflected in an increase in total seed lysine content. This might be explained by a temporal discrepancy between maximal expression of dhdps-r1 and the rate of amino acid incorporation into storage proteins. Assays of the lysine degradative enzyme lysine-ketoglutarate reductase in these seeds showed no co-ordinated regulation of lysine biosynthesis and catabolism during seed maturation. All transgenic plants were fertile and produced morphologically normal seeds.

The lysine-ketoglutarate reductase–saccharopine dehydrogenase is involved in the osmo-induced synthesis of pipecolic acid in rapeseed leaf tissues

Higher plant responses to abiotic stresses are associated with physiological and biochemical changes triggering a number of metabolic adjustments. We focused on l-lysine catabolism, and have previously demonstrated that degradation of this amino acid is osmo-regulated at the level of lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) in Brassica napus. LKR and SDH activities are enhanced by decreasing osmotic potential and decrease when the upshock osmotic treatment is followed by a downshock osmotic one. Moreover we have shown that the B. napus LKR/SDH gene is up-regulated in osmotically-stressed tissues. The LKR/SDH activity produces α-aminoadipate semialdehyde which could be further converted into α-aminoadipate and acetyl CoA. Alternatively α-aminoadipate could behave as a precursor for pipecolic acid. Pipecolic acid is described as an osmoprotectant in bacteria and is co-accumulated with proline in halophytic plants. We suggest that osmo-induction of the LKR/SDH activity could be partly responsible for pipecolic acid accumulation. This proposal has been assessed in this study through pipecolic acid amounts determination in rape leaf discs subjected to various upshift and downshift osmotic treatments. Changes in pipecolic acid level actually behave as those observed for LKR and SDH activities, since it increases or decreases in rape leaf discs treated under hyper- or hypo-osmotic conditions, respectively. In addition we show that pipecolic acid level is positively correlated with the external osmotic potential as well as with the duration of the applied treatment. On the other hand pipecolic acid level is related to the availability of l-lysine and not to that of d-lysine. Collectively the results obtained demonstrate that lysine catabolism through LKR/SDH activity is involved in osmo-induced synthesis of pipecolic acid.

Lysine catabolism in chickens fed at or below their lysine requirement

In chickens, the activity of lysine ketoglutarate reductase (LKR) is reported to be proportional to dietary lysine (Wang et al, 1973). LKR activity resides on the bifunctional aminoadipate semialdehyde synthase enzyme (AASS). To investigate regulation of lysine catabolism, chickens (2wk old) were individually caged and fed either a 1.18% (adequate lysine, N=8) or 0.73% (low lysine, N=8) lysine diet. After 8 days, livers were removed and a portion of each liver was quick frozen in liquid nitrogen to evaluate mRNA expression, and the remaining portion was used for protein assays. Hepatic LKR activity was measured spectrophotometrically at 340nm. AASS and lysyl oxidase (an alternative route of lysine catabolism-LOX) mRNA expression were measured by real-time PCR. AASS protein abundance was measured by western blotting. Surprisingly, chickens fed a low lysine diet gained more weight (P<0.05) than chickens fed the lysine adequate diet, 157.6±13.7 and 113.1±13.7 g respectively. Chickens fed the low lysine diet had a smaller (P<0.05) relative liver size then chickens fed the lysine adequate diet, 3.16±0.23 and 4.23±0.23%, respectively. No significant differences in liver weight, feed consumption, feed efficiency, LKR activity, AASS protein abundance, AASS or LOX mRNA were detected. The results of this study show that 1) chickens can be resistant to short term deficiencies of dietary lysine and 2) alterations in lysine catabolism could be mediated by pathways other then those depending on LOX or AASS. Support: WV Ag. For. Expt. Sta., WVU Res. Corp. and the USDA NRI-2004-35206-14160 (KPB).

The bifunctional LKR/SDH locus of plants also encodes a highly active monofunctional lysine-ketoglutarate reductase using a polyadenylation signal located within an intron.

Both plants and animals catabolize lysine (Lys) via two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide encoded by a single LKR/SDH gene. We have previously shown that the Arabidopsis LKR/SDH gene also encodes a monofunctional SDH that is transcribed from an internal promoter. In the present report, we have identified two cDNAs derived from cotton (Gossypium hirsutum) boll abscission zone that encode a novel enzymatic form of Lys catabolism, i.e. a catabolic monofunctional LKR. The monofunctional LKR mRNA is also encoded by the LKR/SDH gene, using two weak polyadenylation sites located within an intron. In situ mRNA hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses also suggest that the cotton monofunctional LKR is relatively abundantly expressed in parenchyma cells of the abscission zone. DNA sequence analysis of the LKR/SDH genes of Arabidopsis, maize (Zea mays), and tomato (Lycopersicon esculentum) suggests that these genes can also encode a monofunctional LKR mRNA by a similar mechanism. To test whether the LKR/SDH and monofunctional LKR enzymes possess different biochemical properties, we used recombinant Arabidopsis LKR/SDH and monofunctional LKR enzymes expressed in yeast (Saccharomyces cerevisiae) cells. The K(m) of the monofunctional LKR to Lys was nearly 10-fold lower than its counterpart that is linked to SDH. Taken together, our results suggest that the LKR/SDH locus of plants is a super-composite locus that can encode three related but distinct enzymes of Lys catabolism. These three enzymes apparently operate in concert to finely regulate Lys catabolism during plant development.

Molecular genetic dissection and potential manipulation of lysine metabolism in seeds

Summary Lysine is among the most important essential amino acids in the diet of human and livestock because it is presented in severely limiting amounts in cereals and other important crops. Attempts to increase lysine levels in plants were made by reducing the sensitivity of the key enzyme in lysine biosynthesis, namely dihydrodipicolinate synthase, to feedback inhibition by lysine. However, these studies showed that in plant seeds, lysine accumulation is determined not only by the rate of its synthesis, but also by the rate of its catabolism via the α-amino adipic acid pathway. Our laboratory is currently studying the regulation of lysine catabolism in plants in order to explore potentials of reducing lysine catabolic fluxes in transgenic plants. In plants, like animals, lysine is catabolized via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. Yet SDH activity of the LKR/SDH polypeptide may be limiting in vivo because of its non-physiological pH optimum of activity. In some plants, like Arabidopsis and canola, this is overcome by the presence of an additional monofunctional SDH enzyme, which is encoded by the same locus that encodes the bifunctional LKR/SDH enzyme. Results from our, and other laboratories imply that attempts to generate high-lysine crop plants should take into account a seed-specific reduction of lysine catabolism.

Constitutive and seed-specific expression of a maize lysine-feedback-insensitive dihydrodipicolinate synthase gene leads to increased free lysine levels in rice seeds

In order to improve the nutritional value of rice, we prepared transgenic rice plants with a lysine-feedback-insensitive maize dhps gene under the control of CaMV 35S and the rice glutelin GluB-1 promoter for over-expression and seed-specific expression. The transgenic plants were fertile and expressed the dhps gene abundantly or specifically in rice seeds. The transgenic lines (TC lines) containing mutated dhps controlled by CaMV 35S promoter possessed higher mutated DHPS transcript levels and in vitro DHPS activities in seeds than those of TS lines containing the mutated dhps gene driven by a seed-specific promoter, GluB-1. The content of free lysine in immature seeds of both TC and TS lines was higher than that of wild-type plants. The content of free lysine in mature seeds of TC lines was still higher than, but that of TS lines was similar to, that of wild-type plants. From a comparison of DHPS and lysine-ketoglutarate reductase (LKR) expression levels we conclude that the presence of the foreign dhps gene leads to an increase of LKR activity, resulting in enhanced lysine catabolism. However, over-expression of the mutant dhps gene in a constitutive manner overcomes lysine catabolism and sustains a high lysine level in mature rice seeds.

The catabolic function of the α-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine–oxoglutarate reductase, but not saccharopine dehydrogenase

Whereas plants and animals use the α-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine–oxoglutarate reductase (lysine–ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the α-amino adipic acid pathway in plants.

The catabolic function of the alpha-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine-oxoglutarate reductase, but not saccharopine dehydrogenase.

Whereas plants and animals use the alpha-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine-oxoglutarate reductase (lysine-ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the alpha-amino adipic acid pathway in plants.