Lysine aspartate kinase

LT-aspartokinase <10> (<10> lysine-threonine-sensitive isoenzyme [3]) [3] aspartate kinase (phosphorylating) aspartate kinase III <11> [33] aspartic kinase... 

Relton, J.M. Bonner, P.L.R. Wallsgrove, R.M. Lea, P.J. Physical and kinetic properties of lysine-sensitive aspartate kinase purified from carrot cell suspension culture. Biochim. Biophys. Acta, 953, 48-60 (1988)... 

Dotson, S.B. Somers, D.A. Gengenbach, B.G. Purification and characterization of lysine-sensitive aspartate kinase from maize cell cultures. Plant Physiol., 91, 1602-1608 (1989)... 

Kochhar, S. Kochhar, V.K. Sane, P.V. Subunit structure of lysine sensitive aspartate kinase from spinach leaves. Biochem. Mol. Biol. Int., 44, 795-806 (1998)... 

Nomura et al. (1987a) attempted to minimize product inhibitory effect on the aspartate kinase step in lysine biosynthesis and enhance L-lysine production from Brevibacterium Jlavum QL-5 using a combined ED-F system. However, lysine production was not statistically different from that obtained in diffusion dialysis fermentation and about 20% greater than that achieved during conventional fermentation, thus making practically ineffective such a use of ED. 

The L-threonine biosynthetic pathway consists of five enzymatic steps from L-aspartate. E. coli has three aspartate kinase isoenzymes, key enzymes which catalyze the first reaction of the L-threonine biosynthetic pathway. The aspartate kinase isoenzymes I, II, and III encoded by the thrA, metL, and lysC genes, respectively, are affected by feedback inhibition by L-threonine, L-methionine, and L-lysine, respectively. C. glutamicum has only one aspartate kinase encoded by the lysC gene, which is subjected to feedback inhibition by L-lysine and... 

Fig. 10. Schematic representation of the split biosynthetic pathway of L-lysine in wildtype Corynebacterium glutamicum including the branch point of aspartate semialdehyde distribution. The metabolites derived from the aldehyde via the synthase activity are D,L-di-aminopimelate and L-lysine, whereas that resulting from dehydrogenase activity are L-threo-nine, L-methionine, and L-isoleucine. The activity of the dehydrogenase is inhibited at elevated L-threonine concentrations and its synthesis is repressed by L-methionine. Accumulating intracellular lysine causes feedback inhibition of aspartate kinase and activates lysE transcription...

Biosynthesis metabolism Asp is formed from oxaloacetic acid by aspartate aminotransferase (EC 2.6.1.1) and serves as starting material in the biosyntheses of threonine, methionine, and lysine. The first step is catalysed by aspartate kinase (EC 2.7.24) which only occurs in plants and microorganisms. This enzyme exists as 3 isozymes in Escherichia coli and exhibits a typical example of feedback regulation. Asp plays a central role in the biosyntheses of pyrimidines and purines. In the urea cycle Asp condenses with " citrulline to aigininosuccinate, a stimulating neuro-transmitter. ... 

Inhibitor constants (A j) or the concentration of inhibitor required for half-maximal inhibition under specified assay conditions ffo.s) provide an indication of the quantitative sensitivity of an enzyme. Such indicators of plant aspartate kinase sensitivity are consistently below 1.0 mM and in several instances are less than 100 fiM (Table III). Even though the effective concentration of regulatory metabolites in plant cells is difficult to estimate (Section III,C), the demonstrable inhibitory effects of comparatively low concentrations of lysine or threonine on aspartate kinase i/t vitro suggest that enzyme activity is likely to be regulated by these pathway products in vivo. 

Several plant aspartate kinases are activated by other amino acids such as valine, alanine, and isoleucine (Table HI). Activation does not appear to be a general effect of hydrophobic amino acids, since neither methionine nor leucine influence the activity of the maize enzyme yet, leucine has been reported to activate the enzyme isolated from Sinapsis alba and inhibit the enzyme from Helianthus annus. Interaction of these secondary effectors with the various aspartate kinases has not been fully explored, but several observations suggest a considerable degree of complexity. Alanine partially relieves threonine inhibition of the enzyme isolated from pea seedlings (Aames and Rognes, 1974). Lysine inhibition of maize aspartokinase is diminished in the presence of isoleucine, alanine, or valine (Bryan e/ al., 1970) and threonine, even though it does not inhibit the maize enzyme, counteracts... 

Fig. 7. Sequential control of the synthesis of the aspartate family of amino adds. Temporal control of the flow of carbon is illustrated by the successive Figs. 1-5. Potential quantitative changes in flow are approximated by the thickness of the solid arrows. As the concentration of an end product is increased (indicated by closed boxes), the pattern of synthesis is altered by utilization of negative (-) or positive (+) regulatory mechanisms as described in the text. The pattern of control which is illustrated assumes that aspartate kinase is sensitive to inhibition only by lysine. Variations of this pattern are discussed in the text.

Aspartate kinase is usually subject to very strong inhibition by lysine and/or threonine (Miflin et ai, 1979). Even if the worker can be certain that the activity that is measured is due to the action of asparagine synthetase, there is still no information on whether glutamine or ammonia is acting as the amino donor. It is for these reasons that the hydroxamate assay is not recommended. 

Lysine plus threonine severely inhibits growth of maize in a synergistic manner. Growth inhibition could result from combined effects of lysine on aspartate kinase and threonine on homoserine dehydrogenase, resulting in starvation for methionine. Growth inhibition by lysine -i- threonine can be overcome by supplying methionine. Bryan [1980) Miflin [1977). 

Fig. 14.1 The biosynthesis pathway of L-threonine. The pathway consists of centeral metabolic pathways and the threonine terminal pathways. The centeral metabolic pathways involve glycolysis, phosphate pentose pathway, TCA cycle and anaplerotic pathways. The threonine terminal pathway consists of five enzymetic steps. The first, third, and fourth reactions are catalyzed by the three key enzymes aspartate kinase, homoserine dehydrogenase, tmd homoserine kinase, respectively. There are four competing pathways that affect the biosynthesis of L-threonine, leading to formation of L-lysine, L-metMonine, L-isoleucdne, and glycine...

E. coli aspartate kinase III is subjected to feed-back inhibition by L-lysine. The inhibition is complete (Chassagnole et al. 2001), and the inhibitory mechanism is... 

Fig. 14.2 L-lysine binding site in E. coli aspartate kinase III. (a) The dimer structure of the regulatory domains is shown. Regulatory domains from two different chains are shown in blue and green, respectively. The bound L-lysine molecules are shown in pink, (b) The bound L-lysine molecule and the amino acid residues involved in its binding are both shown in the manner of sticks. These models are buUt by using the PyMOL software, Protein Data Bank (accession mrmber 2J0X) and the published information by Kotaka et al. (2006)...

Kato C, Kurihara T, Kobashi N, Yamane H, Nishiyama M (2004) Conversion of feedback regulation in aspartate kinase by domain exchange. Biochem Bioph Res Co 316 802-808 Kim YH, Park JS, Cho JY, Cho KM, Park YH, Lee J (2004) Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli. Biochem J 381 823-829 Klaffl S, Eikmanns BJ (2010) Genetic and functional analysis of the soluble oxaloacetate decarboxylase from Corynebacterium glutamicum. J Bacterid 192 2604-2612 Komatsubara S, Kisumi M, Murata K, Chibata 1 (1978) Threonine production by regulatory mutants of Serratia marcescens. Appl Environ Microbiol 35 834-840 Kotaka M, Ren J, Lockyer M, Hawkins AR, Stammers DK (2006) Structures of R- and T-state Escherichia coli aspartokinase 111 mechanisms of the allosteric transition and inhibition by lysine. J Biol Chem 281 31544-31552... 

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