Lysine 2-oxoglutarate Reductase Research Articles

Characterization and structure of the human lysine-2-oxoglutarate reductase domain, a novel therapeutic target for treatment of glutaric aciduria type 1.

In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyses the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine-2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for inborn errors of metabolism such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR and show that it is activated by alkylation of cysteine 414 by N-ethylmaleimide. We also provide evidence that AASS is rate-limiting upon high lysine exposure of mice. Finally, we present the crystal structure of the human LOR domain. Our combined work should enable future efforts to identify inhibitors of this novel drug target.

X chromosome‐linked inhibitor of apoptosis protein reduces oxidative stress after cerebral irradiation or hypoxia‐ischemia through up‐regulation of mitochondrial antioxidants

We demonstrate that X chromosome-linked inhibitor of apoptosis protein (XIAP) counteracts oxidative stress in two essentially different disease-related models of brain injury, hypoxia-ischemia and irradiation, as judged by lower expression of nitrotyrosine (5-fold) and 4-hydroxy-2-nonenal (10-fold) in XIAP-overexpressing compared with wild-type mice. XIAP overexpression induced up-regulation of at least three antioxidants residing in mitochondria, superoxide dismutase 2, thioredoxin 2 and lysine oxoglutarate reductase. Cytochrome c release from mitochondria was reduced in XIAP-overexpressing mice. Hence, in addition to blocking caspases, XIAP can regulate reactive oxygen species in the brain, at least partly through up-regulation of mitochondrial antioxidants. XIAP-induced prevention of oxidative stress was not secondary to tissue protection because although XIAP overexpression provides tissue protection after hypoxia-ischemia, it does not prevent tissue loss after irradiation. This is a previously unknown role of XIAP and may provide the basis for development of novel protective strategies for both acute and chronic neurodegenerative diseases, where oxidative stress is an integral component of the injury mechanisms involved.

Saccharopine Dehydrogenase Activity in the High-Lysine Opaque and Floury Maize Mutants

Lysine is an essential amino acid normally present in very low concentration in cereal seeds. In previous reports we have studied the metabolism of lysine in several distinct high-lysine maize mutants and observed drastic variations in the activity of saccharopine dehydrogenase (SDH), a key enzyme involved in lysine degradation. We have now analyzed the activity of SDH using non-denaturing polyacrylamide gel electrophoresis (PAGE) to identify possible isoenzymes that could explain the patterns of activity previously observed. The results indicated the presence of at least two SDH isoenzymes, one contributing to approximately 90% of the total enzyme activity and a minor form only present in the wild type lines and the opaque-1 mutant. The results suggest that the differences in total SDH activity among the genotypes tested are due to alterations in the predominant SDH isoenzymic form, which is likely to be the bifunctional polypeptide containing lysine 2-oxoglutarate reductase.

Isolation and Characterization of Enzymes Involved in Lysine Catabolism from Sorghum Seeds

Lysine is an essential amino acid synthesized in plants via the aspartic acid pathway. The catabolism of lysine is performed by the action of two consecutive enzymes, lysine 2-oxoglutarate reductase (LOR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9). The final soluble lysine concentration in cereal seeds is controlled by both synthesis and catabolism rates. The production and characterization of high-lysine plants species depends on knowledge of the regulatory aspects of lysine metabolism and manipulation of the key enzymes. We have for the first time isolated, partially purified, and characterized LOR and SDH from developing sorghum seeds, which exhibited low levels of activity. LOR and SDH were only located in the endosperm and were very unstable during the isolation and purification procedures. LOR and SDH exhibited some distinct properties when compared to the enzymes isolated from other plant species, including a low salt concentration required to elute the enzymes during anion-exchange chromatography and the presence of multimeric forms with distinct molecular masses.

Genetic control of lysine metabolism in maize endosperm mutants.

The capacity of three maize endosperm opaque mutants (o10, o11 and o13) to accumulate soluble lysine in the seed in relation to their wildtype counterpart, W22+, was investigated. The W22o13 and W22o11 mutants exhibited 278% and 186% increases in soluble lysine, respectively, while for W22o10, a 36% decrease was observed, compared with the wildtype. A quantitative and qualitative study of the N constituents of the endosperm has been conducted and data obtained for the total protein, non-protein N, soluble amino acids, albumins / globulins, zeins and glutelins present in the seed of the mutants. Following 2D-PAGE, a total of 38 different forms of zein polypeptides were detected and considerable differences were noted between the three mutant lines. The metabolism of lysine was also studied by analysis of the enzymes aspartate kinase, homoserine dehydrogenase, lysine 2-oxoglutarate reductase and saccharopine dehydrogenase, which exhibited major changes in activity, depending on the genotype, suggesting that the mutant genes may have distinct regulatory activities.

Lysine catabolism: flow, metabolic role and regulation

Lysine is an essential amino acid, synthesized in plants in the aspartic acid pathway. The lysine catabolism is performed by the action of two consecutive enzymes, lysine 2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase (SDH). The steady state of lysine is controlled by both, synthesis and catabolism rates, with the final soluble lysine concentration in cereal seeds a direct result of these processes. In the last 40 years, the enzymes involved in lysine biosynthesis have been purified and characterized from some plant species such as carrot, maize, barley, rice, and coix. Recent reports have revealed that lysine degradation might be related to various physiological processes, for instance growth, development and response to environmental changes and stress. The understanding of the regulatory aspects of the lysine biosynthetic and catabolic pathways and manipulation of related enzymes is important for the production of high-lysine plants.

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.

Lysine metabolism in higher plants.

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 a-ketoglutarate reductase) and saccharopine dehydrogenase exist as a single bifunctional protein, with the former activity being regulated by lysine availability, calcium and phosphorylation/ dephosphorylation.

Degradation of lysine in rice seeds: Effect of calcium, ionic strength, S‐adenosylmethionine and S‐2‐aminoethyl‐ l‐cysteine on the lysine 2‐oxoglutarate reductase‐saccharopine dehydrogenase bifunctional enzyme

Lysine biosynthesis has been extensively studied and the regulatory enzymes characterized in some of the most important crop plants, however, much less is known about the lysine degradation pathway. Lysine 2‐oxoglutarate reductase (LOR) and saccharopine dehydrogenase (SDH) have recently been partially purified and characterized from plants, and have been shown to exist as a single bifunctional polypeptide. We have further characterized these enzymes from rice endosperm in relation to Ca2+ and ionic strength modulation. Optimum pH values of 7.0 and 8.0 were obtained for LOR and SDH, respectively. The LOR domain of the polypeptide was modulated by Ca2+ and ionic strength, whereas the SDH domain was not. It would appear that the modulation by Ca2+ and ionic strength of LOR is a common feature among plant LOR enzymes. S‐adenosylmethionine (SAM) did not produce any significant effect on either enzyme activity, indicating that it only plays a role in the regulation of lysine biosynthesis. The effect of S‐2‐aminoethyl‐ l‐cysteine (AEC) as both a substrate and an inhibitor of LOR activity was also tested. AEC was shown to partially substitute for lysine as a substrate for LOR, but was also able to inhibit LOR activity, possibly competing with lysine at the active site. The higher Km for AEC compared to lysine may reflect a lower binding affinity for AEC.

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.

Lysine degradation through the saccharopine pathway in mammals: involvement of both bifunctional and monofunctional lysine-degrading enzymes in mouse

Lysine-oxoglutarate reductase and saccharopine dehydrogenase are enzymic activities that catalyse the first two steps of lysine degradation through the saccharopine pathway in upper eukaryotes. This paper describes the isolation and characterization of a cDNA clone encoding a bifunctional enzyme bearing domains corresponding to these two enzymic activities. We partly purified those activities from mouse liver and showed for the first time that both a bifunctional lysine-oxoglutarate reductase/saccharopine dehydrogenase and a monofunctional saccharopine dehydrogenase are likely to be present in this organ. Northern analyses indicate the existence of two mRNA species in liver and kidney. The longest molecule, 3.4 kb in size, corresponds to the isolated cDNA and encodes the bifunctional enzyme. The 2.4 kb short transcript probably codes for the monofunctional dehydrogenase. Sequence analyses show that the bifunctional enzyme is likely to be a mitochondrial protein. Furthermore, enzymic and expression analyses suggest that lysine-oxoglutarate reductase/saccharopine dehydrogenase levels increase in livers of mice under starvation. Lysine-injected mice also show an increase in lysine-oxoglutarate reductase and saccharopine dehydrogenase levels.

Lysine degradation through the saccharopine pathway in mammals: involvement of both bifunctional and monofunctional lysine-degrading enzymes in mouse

Lysine-oxoglutarate reductase and saccharopine dehydrogenase are enzymic activities that catalyse the first two steps of lysine degradation through the saccharopine pathway in upper eukaryotes. This paper describes the isolation and characterization of a cDNA clone encoding a bifunctional enzyme bearing domains corresponding to these two enzymic activities. We partly purified those activities from mouse liver and showed for the first time that both a bifunctional lysine-oxoglutarate reductase/saccharopine dehydrogenase and a monofunctional saccharopine dehydrogenase are likely to be present in this organ. Northern analyses indicate the existence of two mRNA species in liver and kidney. The longest molecule, 3.4 kb in size, corresponds to the isolated cDNA and encodes the bifunctional enzyme. The 2.4 kb short transcript probably codes for the monofunctional dehydrogenase. Sequence analyses show that the bifunctional enzyme is likely to be a mitochondrial protein. Furthermore, enzymic and expression analyses suggest that lysine-oxoglutarate reductase/saccharopine dehydrogenase levels increase in livers of mice under starvation. Lysine-injected mice also show an increase in lysine-oxoglutarate reductase and saccharopine dehydrogenase levels.

Structure and regulation of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase in maize.

The lysine-oxoglutarate reductase (LOR) domain of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase (LOR/SDH) from maize endosperm was shown to be activated by Ca2+, high salt concentration, organic solvents and Mg2+. The Ca2+-dependent enhancement of LOR activity was inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and calmidazolium. Limited proteolysis was used to assess the structure/function relationship of the enzyme. Digestion with elastase separated the bifunctional 125-kDa polypeptide into two polypeptides of 65 kDa and 57 kDa, containing the functional domains of LOR and SDH, respectively. Proteolysis did not affect SDH activity, while LOR showed a time-dependent and protease-concentration-dependent inactivation followed by reactivation. Prolonged digestion or increasing amounts of elastase produced a complex pattern of limit polypeptides derived from additional cleavage sites within the 65-kDa (LOR) and 57-kDa (SDH) domains. The SDH-containing polypeptides inhibited the enzymatic activity of LOR-containing polypeptides. When separated from the SDH domain by limited proteolysis and ion-exchange chromatography, the LOR domain retained its Ca2+ activation property, but was no longer activated by high salt concentrations. These results suggest that the LOR activity of the native enzyme is normally inhibited such that after modulation, the enzyme undergoes a conformational alteration to expose the catalytic domain for substrate binding.

The enzymology of lysine catabolism in rice seeds--isolation, characterization, and regulatory properties of a lysine 2-oxoglutarate reductase/saccharopine dehydrogenase bifunctional polypeptide.

In plant, the catabolism of lysine has only been studied in some detail in maize. The enzymes lysine 2-oxoglutarate reductase (also known as lysine alpha-ketoglutarate reductase; LOR) and saccharopine dehydrogenase (SDH), which convert lysine into saccharopine, and saccharopine into glutamic acid and 2-aminoadipate 6-semialdehyde, respectively, were isolated from immature rice seeds and partially purified through a three-step purification procedure involving ammonium sulphate precipitation, and anion-exchange and gel-filtration chromatographies, leading to a final yield of 30% for LOR and 24% for SDH. The molecular masses estimated by gel-filtration chromatography on a Sephacryl S200 column and by native non-denaturing PAGE using Ferguson plots were 203 kDa for both enzymes by gel-filtration and 202 kDa for both enzymes by native non-denaturing PAGE. A second band of LOR and SDH activities on native gels was observed for both enzymes with an estimated molecular mass of 396 kDa, which indicated a multimeric structure. Kinetic studies were consistent with an ordered sequence mechanism for LOR, where 2-oxoglutarate is the first substrate and saccharopine is the last product. The results observed for the LOR/SDH activity ratios during purification, the copurification in all three steps, the molecular masses, the relative mobilities on native non-denaturing gels and the pI estimated for LOR and SDH suggest the existence of a bifunctional polypeptide containing LOR and SDH activities.

Enzyme studies in a patient with saccharopinuria: a defect of lysine metabolism.

Extract: Elevated levels of saecharopine, lysine, and citrulline in urine and plasma were observed in a patient suffering from saccharopinuria. Using radioisotope methods the lysine-degradative enzymes, lysine-oxoglutarate reductase and saecharopine dehyrogenase, were studied in skin fibroblasts grown from this patient and from healthy subjects. The results show that, in contrast to healthy individuals (range 177–320 pmol formed/min/mg protein), the paitent's fibroblasts are completely lacking in saecharopine dehydrogenase activity, which accounts for the presence of the high levels of saccharopine. The patient also has a reduced level of lysine-oxoglutarate reductase activity (333 pmol saccharopine formed/min/mg protein; range 550–1,570 nmol), which may in part explain the hyperlysinemia. A further enzyme saecharopine oxidoreductase which metabolizes saecharopine to lysine was found to be present in the patient's fibroblasts (63 pmol lysine formed/min/mg protein) but absent from those of healthy control subjects. This indicated induction of this enzyme by the patient in an attempt to reduce the high levels of saecharopine in her tissues and body fluid. Speculation: There are two interesting features of this patient with saccharopinuria which the present studies have not clearly defined, namely the presence of high levels of lysine and citrulline found in plasma and urine. There are three possible explanations for the hyperlysinemia which may be operating singly or in combination. Depression in activity of lysine-oxoglutarate reductase (the patient possesses 37% of the normal activity). Inhibitory effect of saecharopine on lysine-oxoglutarate reductase (in vitro studies). Induction by the patient of saccharopine oxidoreductase which metabolizes saccharopine to lysine. No explanation has yet been found for the high levels of citrulline in plasma and urine.

Biosynthesis and degradation of saccharopine, an intermediate of lysine metabolism.

Lysine-2-oxoglutarate reductase was prepared from ox liver and its characteristics were examined. Its activity was totally inhibited in the presence of NH(4)Cl. Under conditions that inhibit saccharopine formation, and in the presence of NADP(+), ox liver mitochondria were found to catalyse the hydrolysis of saccharopine to lysine and alpha-oxoglutarate. The enzyme involved was named saccharopine oxidoreductase. It was partially purified and separated from lysine-oxoglutarate reductase. Comparison of the properties of these two enzymes showed that saccharopine degradation was stimulated under conditions that inhibit its formation. The effect of pH, various cofactors and stability during incubation confirm that saccharopine biosynthesis from, and degradation to, lysine are catalysed by two distinct enzymes.

Lysine metabolism in mammals.

The enzymes involved in the initial degradative steps of lysine metabolism, lysine-2-oxoglutarate reductase and saccharopine dehydrogenase, were studied and their activities in different mammals compared. Values obtained in human, rat, pig, dog, cat, ox and sheep liver indicated that in vitro, appreciable degradation of lysine to saccharopine (4-6nmol/min per mg of protein) occurred. The specific activity of saccharopine dehydrogenase in most species studied was higher than that of lysine-oxoglutarate reductase. The rate of production of glutamate from saccharopine in each animal species was investigated and related to the rate of production of alpha-aminoadipate. The rate of formation of lysine from saccharopine, catalysed by saccharopine oxidoreductase, was examined and correlated with the dietary intake of lysine in each species studied.