The control of lysine biosynthesis in maize

Aspartate kinase (EC 2.7.2.4) has been purified 8-fold and characterized from germinating barley (Iordeum vulgare) seedlings. The enzyme is inhibited 50% by 0.7 mM L-lysine and almost completely at 5 mM. LMethionine does not affect the enzyme on its own, but at low concentrations (0.1-1 mM) increases the inhibition in the presence of lysine, dicatig that the two amino acds act as cooperative feedback regulators.Aspartate kinase (ATP: L-aspartate 4-phosphotransferase, EC 2.7.2.4) catalyzes the ATP-dependent conversion of aspartate to f-aspartylphosphate, the first reaction in the biosynthesis of the aspartate family of amino acids -lysine, methionine, threonine, and isoleucine (16).The enzyme is subject to feedback control by its amino acid end products, and a variety of regulatory patterns have been demonstrated for the enzyme from different plant tissues. The enzyme from maize is sensitive to L-lysine (5, 6, 14), whereas in Pisum sativum, the main effector is L-threonine and lysine is inactive (3). In contrast, Wong and Dennis (22,23) suggested that the enzymes from wheat germ and Lemna minor were inhibited cooperatively by lysine and threonine. In both cases, however, their experiments were carried out with very low activity preparations, and high (1-3 mM) concentrations of threonine were required to cause appreciable inhibition. The wheat enzyme was inhibited 60% by 1 mm lysine on its own. Recently, Aarnes (2) studied the enzyme from four green plants and two algae and demonstrated that all were inhibited to varying extends by lysine and/or threonine.In the present paper, we describe the partial purification and properties of an enzyme from barley which is sensitive to inhibition by lysine and to cooperative inhibition by lysine and methionine. Methionine has not previously been demonstrated to have a role in the regulation of aspartate kinase in higher plants.MATERIALS AND METHODS Chemicals. Amino acids and cofactors were purchased from Sigma. Amino acid analogues were purchased from Sigma, Calbiochem, Aldrich Chemical Co. Inc., and R. N. Emanuel Ltd. Those not commercially available were generous gifts from L. 0.1 M KCI, 1 mm EDTA, 10 mm 2-mercaptoethanol, and 30% (v/v) glycerol. After filtration through gauze, the extract was centrifuged at 2,500g for 45 min. Solid (NH4)2SO4 was added to the extract to 60% saturation and the precipitate was collected by centrifugation. This was dissolved in 0.05 M K-phosphate buffer (pH 7.5) with 1 mm EDTA, 5 mm 2-mercaptoethanol, and 20% (v/v) glycerol, and dialyzed overnight against the same buffer. The dialyzed extract was shaken with DEAE-cellulose which was then eluted batchwise with phosphate buffer + 0.2 M NaCl (4 ml/g DEAE-cellulose). The active eluate was concentrated by (NH4)2SO4 precipitation (60%), and the precipitate was redissolved in phosphate buffer and dialyzed overnight. The extract was finally centrifuged at 70,000g for 60 min. The preparation was stable at 5 C for several days and at -15 C for at least a week.Enzyme Assays. The hydroxamate assay...

supporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Properties and Regulation of Aspartate Kinase from Barley Seedlings (Hordeum vulgare L.)

Shewry¹,

Miflin²

1977

“…DHDPS (EC 4.2.1.52), the first enzyme uniquely associated with lysine biosynthesis, catalyzes the condensation of L-ASA and pyruvate. This enzyme activity has been detected in several plants (6,(13)(14)(15)(16)25), and has been found to be localized in the chloroplasts (24). DHDPS from higher plants is feedback-inhibited by low concentrations of lysine (15,16,25) for the enzyme from Escherichia coli (28).…”

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confidence: 99%

Purification and Characterization of Dihydrodipicolinate Synthase from Wheat Suspension Cultures

Kumpaisal¹,

Hashimoto²,

Yamada³

1987

Dihydrodipicolinate synthase, the first enzyme unque to lysine biosynthesis in higher plants, was The biosynthesis of lysine first utilizes the common pathway for the aspartic-family amino acids including lysine, threonine, and methionine, followed by a pathway specific to lysine biosynthesis (5). Since many, though not all, of the enzymes involved in the lysine-specific pathway have been isolated, higher plants are supposed to synthesize lysine via the diaminopimelate pathway (5). In our effort to clarify the regulation of lysine biosynthesis in wheat, we first reported the effects of lysine analogs and aspartate-derived amino acids on growth and AK' (EC. 2.7.2.4) activity in wheat cell suspension cultures (26). The fact that lysine inhibits wheat AK, the first enzyme in the common pathway for aspartate-family amino acids, but not growth of wheat cells, indicates that there should be a more potent site of feedback-inhibition by lysine in the lysine-specific pathway. DHDPS (EC 4.2.1.52), the first enzyme uniquely associated with lysine biosynthesis, catalyzes the condensation of L-ASA and pyruvate. This enzyme activity has been detected in several plants (6,(13)(14)(15)(16)25), and has been found to be localized in the chloroplasts (24). DHDPS from higher plants is feedback-inhibited by low concentrations of lysine (15,16,25) for the enzyme from Escherichia coli (28). For the E. coli enzyme, it is also reported that an enzyme-pyruvate complex exists by Schiff base formation (21) and that the inhibition by lysine is competitive with respect to ASA (20). The kinetic mechanism and the mode of inhibition, however, have not been explored in detail. In view of the potential importance of DHDPS in the regulation of lysine biosynthesis, we studied its properties and kinetics using highly purified enzyme preparations from wheat suspension cultures. MATERILS AND METHODSPlant Material. Suspension cultures of wheat (Triticum aestivum var Chinese Spring), established as described previously (26), were grown on a gyrotory shaker at 100 rpm, 25°C, in the dark. Liquid LS medium (12) supplemented with 10 Mm, 2,4-D, in a total volume of 75 ml, were used in 300-ml flasks. Inoculum weight was 2.0 to 2.5 g fresh weight per flask. Subcultures were carried out at 3-week intervals.Chemicals. Lysine analogs were obtained from the following sources: AEC, DHL, Na-acetyl-L-lysine, Nf-acetyl-L-lysine, and L-lysine hydroxamate were from Sigma Chemical Co.; Ne methyl-L-lysine from Aldrich Chemical Co.; THL from Calbiochem-Behring Corp; and cis and trans isomers of DL-lysyne (A4-lysine) dihydrochloride, DL-lysyne dihydrochloride (26), DL-4-oxalysine dihydrochloride, and DL-homolysine monohydrochloride were synthesized as described previously (26).Butyl-Toyopearl 650 M was obtained from Toyo Soda Manufacturing Co., Tokyo. Sephadex G-25 M, PD-I0 columns, DEAESepharose CL-6B, Phenyl-Superose HR 10/10, Mono Q HR 5/ 5, Superose-12 HR 10/30, and a FPLC system were from Pharmacia, Inc. Protein standards for HPLC were obtained from Oriental...

“…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).…”

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confidence: 99%

Lysine Catabolism in Barley (Hordeum vulgare L.)

Møller¹

1976

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...