Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Saccharopine dehydrogenase (N6-(glutar-2-yl)-L-ly-sine:NAD oxidoreductase (L-lysine-forming)) from baker's yeast was purified to homogenicity. The overall purification was about 1,200-fold over the crude extract with a yield of about 24%. The purified enzyme had a sedimentation coefficient (S20,w) of 3.0 S. The molecular weight determinations by sedimentation equilibrium, Sephadex G-100 gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a value of about 39,000 and, therefore, saccharopine dehydrogenase is a single polypeptide chain enzyme. A Stokes radius of 27 A and a diffusion constant of 7.9 X 10(-7) cm2 s-1 were obtained from Sephadex gel filtration chromatography. The enzyme had a high isoelectric pH of 10.1. The NH2-terminal sequence was Ala-Ala----. The enzyme possessed 3 cysteine residues/molecule; no disulfide bond was present. Incubation of saccharopine dehydrogenase with p-chloromercuribenzoate or iodoacetate resulted in complete loss of enzyme activity. Whereas the coenzyme and substrates were ineffective in protecting from inactivation by p-chloromercuribenzoate, iodoacetate inhibition was protected by excess coenzyme.
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PMID:Purification and characterization of saccharopine dehydrogenase from baker's yeast. 41 69

The mammalian aminoadipic semialdehyde synthase is a bifunctional enzyme that catalyzes the first two sequential steps in lysine degradation in the major saccharopine pathway (Markovitz, P. J., Chuang, D. T., and Cox, R. P. (1984) J. Biol. Chem. 259, 11643-11646). We show here that limited proteolysis of the highly purified synthase from bovine liver with elastase, chymotrypsin, and papain resulted in separation of lysine-ketoglutarate reductase and saccharopine dehydrogenase activities as judged by activity stainings of the polyacrylamide gel. Enzyme assays showed no loss of the two activities after digestions with these proteases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis disclosed the presence of two limit polypeptides in the elastolytic digests, i.e. fragment A (Mr = 62,700) and fragment B (Mr = 49,200). These fragments were apparently derived from the same polypeptide (Mr = 115,000) of the parent synthase. The reductase and dehydrogenase activities of the elastase-digested synthase were completely resolved by DEAE-Bio-Gel column chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that fragment A and fragment B were associated with reductase and dehydrogenase activities, respectively. The bovine synthase showed Mr = 420,000 in sedimentation equilibrium, confirming a tetrameric structure for the enzyme. The above results establish that the reductase and dehydrogenase domains of the aminoadipic semialdehyde synthase are separately folded and functionally independent of each other.
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PMID:The bifunctional aminoadipic semialdehyde synthase in lysine degradation. Separation of reductase and dehydrogenase domains by limited proteolysis and column chromatography. 311 Jan 58

Familial hyperlysinemias are autosomal recessive disorders in the oxidative degradation of lysine. Hyperlysinemia type I is associated with a combined deficiency in lysine-ketoglutarate reductase and saccharopine dehydrogenase activities, the first two sequential steps in the lysine degradative pathway. In familial hyperlysinemia type II, only saccharopine dehydrogenase activity is deficient. We report here that these reductase and dehydrogenase activities occur on a single protein based on the following findings. (i) The activity ratio of reductase/dehydrogenase remained constant (close to unity) throughout a 500-fold purification of both enzyme activities from mitochondrial extracts of baboon and bovine livers. The activity profiles of the reductase and the dehydrogenase superimpose on each other as the enzyme was eluted from DEAE-cellulose and Sephacryl S-300 columns. (ii) Activity-staining of the native polyacrylamide gel showed that both activities migrated the same distance toward the anode. (iii) The highly purified enzyme with the reductase and dehydrogenase activities showed a single polypeptide band of Mr = 115,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme from baboon and bovine livers has an apparent Mr of 468,000 (Stokes radius = 69.5 A) as determined by gel filtration, which suggests a tetrameric structure of identical subunits. The presence in mammalian tissues of a single protein catalyzing both the reductase and dehydrogenase reactions explains the combined enzyme deficiency observed in hyperlysinemia type I. We propose that the bifunctional enzyme be called aminoadipic semialdehyde synthase.
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PMID:Familial hyperlysinemias. Purification and characterization of the bifunctional aminoadipic semialdehyde synthase with lysine-ketoglutarate reductase and saccharopine dehydrogenase activities. 643 29

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.
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PMID:The enzymology of lysine catabolism in rice seeds--isolation, characterization, and regulatory properties of a lysine 2-oxoglutarate reductase/saccharopine dehydrogenase bifunctional polypeptide. 924 48

In plant and mammalian cells, excess lysine is catabolized by a pathway that is initiated by two enzymes, namely, lysine-ketoglutarate reductase and saccharopine dehydrogenase. In this study, we report the cloning of an Arabidopsis cDNA encoding a bifunctional polypeptide that contains both of these enzyme activities linked to each other. RNA gel blot analysis identified two mRNA bands-a large mRNA containing both lysine-ketoglutarate reductase and saccharopine dehydrogenase sequences and a smaller mRNA containing only the saccharopine dehydrogenase sequence. However, DNA gel blot hybridization using either the lysine-ketoglutarate reductase or the saccharopine dehydrogenase cDNA sequence as a probe suggested that the two mRNA populations apparently are encoded by the same gene. To test whether these two mRNAs are functional, protein extracts from Arabidopsis cells were fractionated by anion exchange chromatography. This fractionation revealed two separate peaks-one containing both coeluted lysine-ketoglutarate reductase and saccharopine dehydrogenase activities and the second containing only saccharopine dehydrogenase activity. RNA gel blot analysis and in situ hybridization showed that the gene encoding lysine-ketoglutarate reductase and saccharopine dehydrogenase is significantly upregulated in floral organs and in embryonic tissues of developing seeds. Our results suggest that lysine catabolism is subject to complex developmental and physiological regulation, which may operate at gene expression as well as post-translational levels.
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PMID:Regulation of lysine catabolism through lysine-ketoglutarate reductase and saccharopine dehydrogenase in Arabidopsis. 928 8

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.
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PMID:Structure and regulation of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase in maize. 965 71

We have isolated a cDNA clone, designated ZLKRSDH, encoding the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) from maize. The predicted polypeptide has an N-terminal LKR domain and a C-terminal SDH domain that are similar to the yeast LYS1 and LYS9 monofunctional proteins, respectively. The maize LKR/SDH protein is located in the cytoplasm of subaleurone endosperm cell layers. Transcripts and polypeptides as well as enzyme activities showed an upregulation and downregulation during endosperm development. The developmental expression of ZLKRSDH was examined in normal and opaque2 seeds. In the mutant endosperm, mRNA levels were reduced by >90%, with concomitant reductions in polypeptide levels and LKR/SDH activity. These results suggest that lysine levels in the endosperm are likely to be controlled at the transcriptional level by the Opaque2 transcription factor.
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PMID:The role of opaque2 in the control of lysine-degrading activities in developing maize endosperm. 1052 27

Both in mammals and plants, excess lysine (Lys) is catabolized via saccharopine into alpha-amino adipic semialdehyde and glutamate by two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. To study the control of metabolite flux via this bifunctional enzyme, we have purified it from developing soybean (Glycine max) seeds. LKR activity of the bifunctional LKR/SDH possessed relatively high K(m) for its substrates, Lys and alpha-ketoglutarate, suggesting that this activity may serve as a rate-limiting step in Lys catabolism. Despite their linkage, the LKR and SDH enzymes possessed significantly different pH optima, suggesting that SDH activity of the bifunctional enzyme may also be rate-limiting in vivo. We have previously shown that Arabidopsis plants contain both a bifunctional LKR/SDH and a monofunctional SDH enzymes (G. Tang, D. Miron, J.X. Zhu-Shimoni, G. Galili [1997] Plant Cell 9: 1-13). In the present study, we found no evidence for the presence of such a monofunctional SDH enzyme in soybean seeds. These results may provide a plausible regulatory explanation as to why various plant species accumulate different catabolic products of Lys.
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PMID:Purification and characterization of bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase from developing soybean seeds. 1085 95

Both plants and animals catabolize lysine via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide. We recently demonstrated that Arabidopsis plants possess not only a bifunctional LKR/SDH but in addition a monofunctional SDH enzyme. We also speculated that these two enzymes may be controlled by a single gene (G. Tang et al. Plant Cell, 1997, 9, 1305-1316). By expressing several epitope-tagged and GUS reporter constructs, we demonstrate in the present study that the Arabidopsis monofunctional SDH is encoded by a distinct gene, which is, however, nested entirely within the coding and 3' non-coding regions of the larger bifunctional LKR/SDH gene. The entire open reading frame of the monofunctional SDH gene, as well as some components of its promoter, are also parts of the translated coding sequence of the bifunctional LKR/SDH gene. These special structural characteristics, combined with the fact that the two genes encode simultaneously two metabolically related but distinct enzymes, render the LKR/SDH locus a novel type of a composite locus. Not all plant species possess an active monofunctional SDH gene and the production of this enzyme is correlated with an increased flux of lysine catabolism. Taken together, our results suggest that the composite LKR/SDH locus serves to control an efficient, highly regulated flux of lysine catabolism
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PMID:A novel composite locus of Arabidopsis encoding two polypeptides with metabolically related but distinct functions in lysine catabolism. 1092 13

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.
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PMID:The catabolic function of the alpha-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine-oxoglutarate reductase, but not saccharopine dehydrogenase. 1099 64


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