Biosynthesis and degradation of saccharopine, an intermediate of lysine metabolism

Fellows, Florence C. I.

doi:10.1042/bj1360321

Cited by 24 publications

(13 citation statements)

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“…The high amount isolated from Reseda odorata establishes saccharopine as a constituent of higher plants. Both 2-aminoadipic acid and saccharopine are intermediates of lysine biosynthesis in fungi (25), but are also known as products of lysine catabolism in animals (3,4,13). Lysine biosynthesis in those higher plants hitherto examined proceeds via the 2,6-diaminopimelic acid pathway (2,29,30).…”

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Lysine Catabolism in Barley (Hordeum vulgare L.)

Møller¹

1976

Plant Physiol.

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Lysine catabolism in seedlings of barley (ffordeum vulgare L. var. Emir) was studied by direct injection of the foliowing tracers into the endosperm of the seedlings: aspartic acid-3-14C, 2-aminoadipic acid-l-14C, saccharopine_14C, 2,6-aminopimelic acid-_l(7)_'4C, and lysne-_l '4C. Labeled saccharopine was formed only after the adm tion of either labeled 2,6-diaminopimelic acid or labeled lysine to the seedlings.The metabolic fate of the other tracers administered also supported a catabolic lysine pathway yin saccharopine, and apparently proceeding by a reversal of some of the biosynthetic steps of the 2-aminoadipic acid pathway known from lysine biosynthesis in most fungi. Pipecolic acid seems not to be on the main pathway of L-lysine catabolism in barley seedlings.A considerable diversity of pathways exists not only for the biosynthesis but also for the catabolism of lysine. At least three catabolic routes have been proposed to operate in fungi (17) and animals (4), aerobic bacteria (12,34,36), and anaerobic bacteria (35), respectively. Studies on lysine catabolism in higher plants are scarce and no general pathway has been established.The conversion of lysine to alkaloids occurs in a limited number of plants (10,22). However, the following observations and results on the presence of possible products of lysine catabolism and on catabolic pathways have been reported. 2-Aminoadipic acid is a common constituent of higher plants (11,18). Saccharopine, N6-(2'-glutaryl)lysine, has been isolated from the seed kernels of Fagopyrum esculentum (buckwheat) (32), and recently from the inflorescence of Reseda odorata (H. Sorensen, personal communication). The high amount isolated from Reseda odorata establishes saccharopine as a constituent of higher plants. Both 2-aminoadipic acid and saccharopine are intermediates of lysine biosynthesis in fungi (25), but are also known as products of lysine catabolism in animals (3, 4, 13). Lysine biosynthesis in those higher plants hitherto examined proceeds via the 2,6-diaminopimelic acid pathway (2, 29, 30). The operation of the 2-aminoadipic acid pathway at a very low rate has not been excluded because 2-aminoadipic acid and saccharopine are incorporated to a low extent into lysine (29,31 (26). An amine oxidase in Pisum sativum converting L-lysine to 2-amino-6-oxocaproic acid has also been reported (24). In Acacia phyllodes, 2-aminoadipic acid and pipecolic acid are formed from lysine via 2-amino-6-oxocaproic acid (5). Unfortunately, all these studies are conducted with plants belonging to ,uCi, 10 mCi/mmole) and 50 ,ul of 1 N NaOH. The clear solution was kept at 50 to 52 C for 24 hr. The reaction mixture was lyophilized, dissolved in H20, and applied to a strongly acidic ion exchange resin (Dowex 5OW x 8, 200-400 mesh, H+, 0.2 x 1.5 cm). After washing with 8 ml of H20, labeled saccharopine was eluted with 8 ml of 1 N pyridine. After lyophilization and solution in H20, paper chromatography of the pyridine eluate on Whatman No. 3MM followed by autoradiography showed two st...

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Lysine Catabolism in Barley (Hordeum vulgare L.)

Møller¹

1976

Plant Physiol.

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“…The small amount of residual activity reported in Table 1 is beyond the sensitivity of the analytic technique and it is not certain that any enzymatic activity was detected. Fellows (8) contends that in ox liver this metabolic step is performed by an enzyme distinct from lysine-ketoglutarate reductase and has named the enzyme saccharopine oxidoreductase. Studies of human placenta also suggest that saccharopine oxidoreductase and lysineketoglutarate reductase may be two distinct enzymes (10).…”

Section: Discussionmentioning

confidence: 99%

“…This solution must remain tightly stoppered because the ammonia is volatile at this pH. A final ammonia concentration of 80 mM is essential to the reaction (8).…”

Section: Saccharopine Oxidoreductasementioning

confidence: 99%

Multiple Enzyme Defects in Familial Hyperlysinemia

et al. 1976

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“…lysine + a-ketoglutarate + NADPH -* saccharopine + NADP+ The enzyme has been characterized in human and animal tissues (3,7,8) where it is believed to catalyze the first step of lysine catabolism (4). A similar reaction involving NAD is catalyzed by saccharopine dehydrogenase, an enzyme found in yeast and fungi where the reverse of the above reaction is considered to be the final step of lysine biosynthesis (5,6,12,16).…”

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“…The assay mixture contained K-phosphate (pH 7.0; 300 ,umol), L-lysine (50 ,pmol), a-ketoglutarate (25 ,umol), NADPH (300 nmol), and enzyme (0.1-0.2 mg protein) in a total volume of 3 ml. The mixture was incubated at 30°C, and oxidation of NADPH was monitored in a spectrophotometer at 340 nm.Lysine-ketoglutarate reductase catalyzes the following reaction: lysine + a-ketoglutarate + NADPH -* saccharopine + NADP+ The enzyme has been characterized in human and animal tissues (3,7,8) where it is believed to catalyze the first step of lysine catabolism (4). A similar reaction involving NAD is catalyzed by saccharopine dehydrogenase, an enzyme found in yeast and fungi where the reverse of the above reaction is considered to be the final step of lysine biosynthesis (5,6,12,16).…”

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Lysine-Ketoglutarate Reductase Activity in Developing Maize Endosperm

Arruda¹,

Sodek²,

Silva³

1982

Plant Physiol.

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Lysine-ketoglutarate reductase activity was detected and characterzed in the developing endosperm of maize (Zea mays L). The enzyme showed specificity for its substrates: lysine, a-ketoglutarate, and NADPH. Formation of the reaction product saccharopine was demonstrated. The pH optimum of the enzyme was cdose to 7, and the Km for lysine and aketoglutarate were 5.2 and 1.8 millmolar, respectively. between 25 and 70%o saturation was collected and taken up in 2.5 ml of extraction buffer. After desalting on a Sephadex G-25 column (1.5 x 16 cm), the protein fraction was assayed for lysineketoglutarate reductase activity using conditions based on those described by Hutzler and Dancis (7). The assay mixture contained K-phosphate (pH 7.0; 300 ,umol), L-lysine (50 ,pmol), a-ketoglutarate (25 ,umol), NADPH (300 nmol), and enzyme (0.1-0.2 mg protein) in a total volume of 3 ml. The mixture was incubated at 30°C, and oxidation of NADPH was monitored in a spectrophotometer at 340 nm.Lysine-ketoglutarate reductase catalyzes the following reaction: lysine + a-ketoglutarate + NADPH -* saccharopine + NADP+ The enzyme has been characterized in human and animal tissues (3,7,8) where it is believed to catalyze the first step of lysine catabolism (4). A similar reaction involving NAD is catalyzed by saccharopine dehydrogenase, an enzyme found in yeast and fungi where the reverse of the above reaction is considered to be the final step of lysine biosynthesis (5,6,12,16). Thus, in some organisms saccharopine is an intermediate of lysine biosynthesis while in the others it appears to be involved in lysine breakdown. Actually, several pathways for lysine catabolism are known (11), but in higher plants no general pathway has been established. Nevertheless, tracer studies with barley seedlings have provided strong evidence for the pathway involving saccharopine as intermediate (10), suggesting that this route may be important for lysine breakdown in cereals at least. However, we are unaware of any studies with enzymes in higher plants in support of this pathway.In this report, we describe the characterization of lysine-ketoglutarate reductase in developing maize endosperm, a tissue known to degrade lysine extensively (14). MATERIALS AND METHODS RESULTS AND DISCUSSIONThe data in Table I show that partially purified extracts of maize endosperm oxidized NADPH in the presence of lysine and a-ketoglutarate. In the absence of one or both of these substrates, the oxidation of NADPH was minimal. Specificity for the substrates is indicated by the fact that several amino acids, including closely related structures such as L-ornithine and D-lysine, would not substitute for L-lysine, nor would oxaloacetate or pyruvate substitute for a-ketoglutarate. NADH would not replace NADPH as electron donor, as was found for the enzyme from human liver (7).To confirm that the activity being measured was truly lysineketoglutarate reductase, it would be essential to demonstrate the formation of saccharopine in the assay. This was attempted using I4CJlysine...

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Biosynthesis and degradation of saccharopine, an intermediate of lysine metabolism

Cited by 24 publications

References 14 publications

Lysine Catabolism in Barley (Hordeum vulgare L.)

Lysine Catabolism in Barley (Hordeum vulgare L.)

Multiple Enzyme Defects in Familial Hyperlysinemia

Lysine-Ketoglutarate Reductase Activity in Developing Maize Endosperm

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