Saccharopine Dehydrogenase Activities Research Articles

Two NAD-linked redox shuttles maintain the peroxisomal redox balance in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, peroxisomes are the sole site of fatty acid β-oxidation. During this process, NAD+ is reduced to NADH. When cells are grown on oleate medium, peroxisomal NADH is reoxidised to NAD+ by malate dehydrogenase (Mdh3p) and reduction equivalents are transferred to the cytosol by the malate/oxaloacetate shuttle. The ultimate step in lysine biosynthesis, the NAD+-dependent dehydrogenation of saccharopine to lysine, is another NAD+-dependent reaction performed inside peroxisomes. We have found that in glucose grown cells, both the malate/oxaloacetate shuttle and a glycerol-3-phosphate dehydrogenase 1(Gpd1p)-dependent shuttle are able to maintain the intraperoxisomal redox balance. Single mutants in MDH3 or GPD1 grow on lysine-deficient medium, but an mdh3/gpd1Δ double mutant accumulates saccharopine and displays lysine bradytrophy. Lysine biosynthesis is restored when saccharopine dehydrogenase is mislocalised to the cytosol in mdh3/gpd1Δ cells. We conclude that the availability of intraperoxisomal NAD+ required for saccharopine dehydrogenase activity can be sustained by both shuttles. The extent to which each of these shuttles contributes to the intraperoxisomal redox balance may depend on the growth medium. We propose that the presence of multiple peroxisomal redox shuttles allows eukaryotic cells to maintain the peroxisomal redox status under different metabolic conditions.

Effects of Strain and Production-Cycle on Indices of Lysine Catabolism in Turkeys

In typical turkey production diets, lysine is frequently the second limiting amino acid. Understanding its catabolism could provide opportunities to increase the efficiency of lysine use for protein synthesis. We hypothesize that indices of lysine catabolism in turkey liver vary throughout the production cycle. Two commercial strains of turkey, Strain A and B, were analyzed 8 times over a period of 17 weeks (n = 8 birds/sampling time/strain) for lysine alpha-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) activities and mRNA abundance, in vitro lysine oxidation (LOX), L-amino acid oxidase (LAAO) activity, lysyl oxidase (LO) activity and mRNA expression of cationic amino acid transporters (CAT) 1, 2 and 3. We found differences in hepatic LKR activity (p = 0.007), LKR mRNA (p = 0.0004), SDH activity (p = 0.008), LOX (p<0.0001), lysyl oxidase activity (p<0.01), LAAO (p<0.0001) and CAT1 (p = 0.005) and 2 mRNA abundance (p = 0.0022) throughout the production cycle. We also found an effect of strain on SDH (p = 0.046) and CAT-2 mRNA abundance (p = 0.02); while no age or strain effects were detected for CAT3 mRNA abundance. Interestingly, the average LKR and SDH activities across strains and weeks was 240 and 420 nmol per minute per gram of liver, respectively, as opposed to the average LAAO activity, lysyl oxidase activity and L OX which were 0.70, 0.10 and 13.5 nmol per minute per gram liver, respectively. These data indicate that the saccharopine-dependent pathway is the predominant pathway of lysine degradation in turkey liver and that indices of hepatic lysine catabolism vary throughout the production cycle.

Effect of dietary lysine on hepatic lysine catabolism in broilers ,

Lysine is frequently a first- or second-limiting amino acid in poultry diets. Improving the efficiency of lysine use for protein synthesis would effectively lower the lysine requirement and decrease feed costs. Understanding how lysine is degraded and how the degradation is regulated would identify potential molecular targets for interventions to decrease lysine degradation. To better understand lysine degradation in poultry, 3 experiments were conducted. In experiment 1, one-day-old chicks were fed 1.07, 1.25, 1.73, or 3.28% dietary lysine for 2 wk. In experiments 2 and 3, fourteen-day-old chicks were fed 1.07 or 1.25% dietary lysine for 2 wk. Measures of liver lysine catabolism including lysine α-ketoglutarate reductase (LKR) and lysine oxidation (LOX) were assessed. The α-aminoadipate δ-semialdehyde synthase (AASS) is a bifunctional enzyme composed of both LKR and saccharopine dehydrogenase activities, and the relative abundance of this protein and mRNA were likewise assessed. Moreover, potential alternative pathways of lysine catabolism that depend on l-amino acid oxidase (AAOX) and on lysyl oxidase (LYLOX) were considered. In experiment 1, chicks fed lysine-deficient diets had decreased (P < 0.05) LKR activities compared with chicks fed at or above the requirement. However, the lowered LKR activities were not associated with a decreased (P > 0.05) LOX as measured in vitro. In experiments 2 and 3, chicks 28 d of age did not decrease LKR activity (P > 0.05) in response to a lysine-deficient diet. No changes in AASS protein abundance or mRNA were detected. Likewise, no differences in the mRNA abundances of AAOX or LYLOX were detected. The activity of AAOX did increase (P < 0.05) in birds fed a lysine-adequate diets compared with those fed a lysine-deficient diet. Based on kinetic parameters and assumed concentrations, AAOX could account for about 20% of liver lysine oxidation in avians.

Tissue distribution of indices of lysine catabolism in growing swine1

The primary pathway of lysine degradation in pigs presumably depends on the bifunctional protein α-aminoadipate δ-semialdehyde synthase (AASS), which contains lysine α-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) activities. In liver, AASS is restricted to the mitochondrial matrix and lysine is presumptively transported through the plasma membrane by a cationic AA transporter (CAT1/2) and through the inner mitochondrial membrane by 1 or both mitochondrial ornithine transporters (ORC-1/ORC-2). Lysyl oxidase (LO) may represent an alternative pathway of lysine oxidation. The objective of this experiment was to analyze the distribution of indices of lysine catabolism in various pig tissues. We assessed LKR, SDH, and LO activities, lysine oxidation, mRNA abundance of LKR, CAT1/2, and ORC1/2, and AASS protein abundance (via SDH antibody) in liver, heart, kidney medulla and cortex, triceps, longissimus, whole intestine, enterocytes, and intestine stripped of enterocytes in 10 growing pigs, weighing ∼25 kg. The LKR activity differed across tissues (P<0.001) and was greatest in liver, intestine, and kidney samples, and LKR mRNA abundance (P<0.001) was greatest in liver; although, LKR activity and mRNA abundance were detected in all other tissues. Activity of SDH (P<0.001) and SDH mRNA abundance (P<0.001) were affected by tissue and were greatest in liver compared with all other tissues analyzed. The AASS protein abundance (P<0.001) was greatest in whole intestine and liver. Activity of LO (P<0.0001) was greatest in muscle samples. The abundance of ORC-1 (P<0.001) and ORC-2 mRNA (P<0.001) differed among tissues, and ORC-1 was greatest in liver, kidney, and intestinal preparations, and ORC-2 mRNA abundance was greatest in liver and intestine. Interestingly, LKR activity was correlated with ORC-1 (r=0.32, P<0.05) and ORC-2 (r=0.41, P<0.05) expression. The expression of CAT-1 was uniform in all tissues, whereas CAT-2 (P<0.01) was greatest in liver. In conclusion, these data indicate that extra-hepatic tissues contribute to lysine catabolism as do enzymes other than LKR.

The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH

Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate l-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/Et and V/KEt at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V1/Et and V1/KNADEt at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pKa of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.

Production‐cycle‐related changes in indices of lysine catabolism in turkey liver

In typical turkey production diets, lysine is frequently the second limiting amino acid. Understanding its catabolism could provide opportunities to increase the efficiency of lysine use for protein synthesis. We hypothesize that indices of lysine catabolism in turkey liver vary throughout the production cycle. Two commercial strains of turkey, Hybrid and Nicolas, were analyzed 8 times over a period of 17 weeks (n=8 birds/sampling time/strain) for lysine α‐ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) activity and mRNA abundance, in vitro lysine oxidation (LOX) and amino acid oxidase (AAOX) activity. We found differences in LKR mRNA (P&lt;0.01), SDH activity (P&lt;0.05) and LOX (P&lt;0.0001) and a trend for differences in LKR activity (P=0.09), SDH mRNA (P=0.07) and AAOX activity (P=0.06) throughout the production cycle. No consistent strain differences were detected for any variable. Interestingly, the average LKR and SDH activities across strains and weeks was 230 and 400 nmol per minute per gram of liver, respectively, as opposed to the average AAOX activity and LOX was 1.25 and 13.6 nmol per minute per gram liver, respectively. These data indicate that the saccharopine‐dependent pathway is the predominant pathway of lysine degradation in turkey liver and that indices of hepatic lysine catabolism vary throughout the production cycle. (Support; HATCH WVA 470, Virginia Poultry Growers Cooperative)

Lysine catabolism in pig tissues

The primary pathway of lysine degradation in pigs presumably depends on the bifunctional protein α‐aminoadipate δ‐semialdehyde synthase (AASS) which contains lysine α‐ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) activities. In liver, AASS is restricted to the mitochondrial matrix and lysine is transported, presumably, by one or both mitochondrial ornithine transporters (MOT1/MOT2). Lysyl oxidase (LylOx) may represent a minor pathway of lysine oxidation. We assessed LKR, SDH and LylOx activities, lysine oxidation, mRNA abundance of LKR and MOT1/2 and AASS protein abundance (via SDH antibody) in liver, heart, kidney medulla and cortex, enterocytes, triceps and longissimus. In growing pigs (n=5), tissues oxidized lysine at varying rates. LKR activity was highest in liver (P&lt;0.05). LKR mRNA abundance in liver was 6 to 130‐fold greater than other tissues (P&lt;0.05). No differences in SDH activity across tissues were detected. No AASS protein could be detected in muscle. LylOx activity was highest in muscle. MOT1 and 2 were detected in every tissue, although no differences were detected as a function of tissue; interestingly there was a significant correlation between LKR activity and MOT1 mRNA abundance (P&lt;0.05, R2= 0.48). These data indicate that extra‐hepatic tissues contribute to lysine oxidation as do enzymes other than LKR. (Support; HATCH WVA 470)

α-Aminoadipate δ-Semialdehyde Synthase mRNA Knockdown Reduces the Lysine Requirement of a Mouse Hepatic Cell Line

α-Aminoadipate δ-semialdehyde synthase (AASS) is the bifunctional enzyme containing the lysine α-ketoglutarate reductase (LKR) and saccharopine dehydrogenase activities responsible for the first 2 steps in the irreversible catabolism of lysine. A rare disease in humans, familial hyperlysinemia, can be caused by very low LKR activity and, as expected, reduces the lysine “requirement” of the individual. This concept was applied to a murine hepatic cell line (ATCC, FL83B) utilizing RNA interference (RNAi) to achieve AASS mRNA knockdown. Cells were antibiotic selected for stable transfection of 2 plasmids that express different short hairpin RNA sequences for AASS knockdown. Compared with the wild-type cell line, AASS mRNA abundance was reduced 79.0 ± 6.4% (P < 0.05), resulting in a 29.8 ± 5.2% (P < 0.05) reduction in AASS protein abundance, 41.3 ± 10.0% (P < 0.05) less LKR activity, and a reduction in lysine oxidation by 50.7 ± 11.8%. To determine the effect of AASS knockdown on the lysine requirement, cells were grown in media containing 12.5, 25.0, 50.0, 100, or 200 μmol/L lysine. Using a segmented model approach for growth rate analysis, the lysine requirement of the cell line with AASS silencing was 43.4 ± 1.7 μmol/L, ∼26% lower (P < 0.05), than the lysine requirement of the wild-type cell line. These results indicate AASS knockdown decreases the lysine requirement of the cell via a reduction of lysine catabolism through the saccharopine pathway, providing the initial proof in principle that RNAi can be used to reduce the nutrient requirement of a system.

α‐Aminoadipate δ‐semialdehyde synthase mRNA knockdown reduces the lysine requirement of a murine hepatic cell line

α-Aminoadipate δ-semialdehyde synthase (AASS) is the bifunctional enzyme containing the lysine α-ketoglutarate reductase (LKR) and saccharopine dehydrogenase activities responsible for the first tw...

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.

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.

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.

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.

Purification and characterization of bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase from developing soybean seeds.

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.

Mitochondrial Lysine Uptake Limits Hepatic Lysine Oxidation in Rats Fed Diets Containing 5, 20 or 60% Casein

Sixty male Sprague-Dawley rats were randomly allotted to receive diets containing 5, 20 or 60% casein. Rats had access to the diet only during the initial 8 h of the daily 12-h dark period. Hepatic mitochondrial lysine uptake, lysine α-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SacD) activities, and in vitro lysine oxidation (LOX) were measured 0, 6, 12, 18 and 24 h after the start of the dark period. Diurnal variation of mitochondrial lysine uptake was not detected (P > 0.10) although uptake varied 3-fold over 24 h. Mitochondrial lysine uptake was greater (P < 0.05) for rats fed diets containing 60% casein than for rats fed diets containing 5% casein. Diurnal variation of LKR was detected (P < 0.05) in rats fed diets containing 20 and 60% casein. Diurnal variation of SacD was detected (P < 0.05) in rats fed diets containing 60% casein. Increased casein consumption resulted in increased LKR and SacD activities (4- to 5-fold; P < 0.05). Diurnal variation of LOX was detected in rats fed diets containing 20 and 60% casein (P < 0.05). Increasing the casein concentration in the diet from 5 to 60% resulted in a 7-fold increase in LOX (P < 0.05). To make rate comparisons, LKR and SacD activities and LOX were predicted from a range of substrate concentrations (0.1 to 5.0 mmol/L). Overall, LKR and SacD were 6–107 times that of LOX, suggesting that, in liver, mitochondrial lysine uptake limits LOX.

Regulation of lysine catabolism through lysine-ketoglutarate reductase and saccharopine dehydrogenase in Arabidopsis.

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.

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.

Molecular and functional analysis of the LYS1 gene of Candida albicans

The LYS1 gene of Candida albicans has been localized to a 1.8-kb DNA fragment present on the plasmid YpBRG2. YpBRG2 has been shown to complement the saccharopine dehydrogenase mutant Stx4-4A of Saccharomyces cerevisiae. Transformants of S. cerevisiae Stx4-4A exhibited significant saccharopine dehydrogenase activity, and cells that had lost YpBRG2 after nonselective growth had no enzyme activity. The DNA sequence of the LYS1 gene has been determined. The LYS1 DNA contains typical yeast upstream regulatory sequences, including the GCN4 motif and candidate sequences responsible for transcription termination within the 3' noncoding region. The fragment contained an open reading frame of 1,146 nucleotides coding for a putative protein of 382 amino acids. The open reading frame has 60% identity at the nucleotide level and 71% similarity at the amino acid level to the LYS5 gene of Yarrowia lipolytica, which is believed to code for saccharopine dehydrogenase. A peptide of 11 amino acids has been found, which is present in S. cerevisiae, Y. lipolytica, and C. albicans. This peptide can be expanded to 16 amino acids when the sequences from Y. lipolytica and C. albicans are compared. A motif responsible for the binding of the adenosine residue of NADH has been described previously and is very similar to this peptide, which may be the site of NADH binding in the saccharopine dehydrogenase of C. albicans.

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.

Evidence that Thraustochytrium is unable to synthesize lysine

When sporangia of Thraustochytrium aureum were grown in the presence of l -[4- 14 C]aspartic acid and [2- 14 C]acetate virtually no radioactivity was found in the lysine either free or bound in protein. No activity of saccharopine dehydrogenase (NAD + , lysine requiring) could be detected in extracts of the sporangia. These results indicate that neither the aminoadipic acid nor the diaminopimelic acid pathway are present in this organism. Attempts to confirm these findings by other growth studies using T. aureum and T. roseum were inconclusive. The results confirm previous investigations which appear to point to the conclusion that Thraustochytrium and related organisms are not fungi.