Aspartokinase regulation

S Additional information <9, 15, 17, 18, 21, 24, 30, 33, 35> (<18> activity is regulated by light [28] <30> D-aspartate, L-glutamate and -alanine are inactive as substitutes for L-aspartate in the forward reaction, in the reverse reaction ADP cannot be replaced by AMP, UDP, GDP or IDP [1] <17> aspartokinase III, o-isomers of the derivatives of aspartic acid, including D-aspartate cr-benzyl ester and o-aspartate /)-hydroxamate are not substrates regardless of whether the a- or the -carboxyl group is derivatized, L-cysteine sulfinate and 2-methyl-DL-aspartate are no substrates... 

King, N.D. O Brian, M.R. Evidence for direct interaction between enzyme INtr and aspartokinase to regulate bacterial oligopeptide transport. J. Biol. Chem., 276, 21311-21316 (2001)... 

Removal of negative regulations can also enhance L-threonine production. By removing L-lysine-mediated feedback inhibition of aspartokinase III encoded by the lysC gene, L-threonine production could be increased by 30.9% (11.0-14.4 g L 1) in E. coli mutant strain [57]. 

Sophisticated regulation can also evolve by duplication of the genes encoding the biosynthetic enzymes. For example, the phosphorylation of aspartate is the committed step in the biosynthesis of threonine, methionine, and lysine. Three distinct aspartokinases catalyze this reaction in E. coli, an example of a regulatory mechanism called enzyme multiplicity. (Figure 24.24). The catalytic domains of these enzymes show approximately 30% sequence identity. Although the mechanisms of catalysis are essentially identical, their activities are regulated differently one enzyme is not subject to feedback inhibition, another is inhibited by threonine, and the third is inhibited by lysine. 

Aspartokinase is a major point of regulation of the biosynthetic pathways leading to threonine, lysine, and methionine. In bacteria, there are three isoenzymes of aspartokinase. Activity of one form is inhibited specifically by threonine and that of another form by lysine. Synthesis of the third form is inhibited by methionine. 

Isoenzymes. In this case, multiple enzymes are made each carries out the same reaction but is regulated by a different end product. This mechanism is used in both feedback inhibition and feedback repression. A well known example of such control is the aspartic acid family in E. coli where the three aspartokinases are regulated by lysine, threonine and methionine respectively (Stadtman, 1968). 

Methionine biosynthesis is controlled by the metabolic regulation of enzymes in the pathway. Species of Corynebacterium and Brevibacterium have much simpler regulatory mechanisms than E. coli for methionine biosynthesis and are the preferred microbes for overproduction. Aspartokinase is a major enzyme that catalyzes the phosphorylation of aspartic acid and redirects the flux to aspartate family of amino acids. 

In E. coli there are three aspartokinases I (coded by thrA), II (coded by met L), and III (coded by lysC) and two homoserine dehydrogenases I and II that are inhibited or repressed by only one or two amino acids of the aspartate family which ensures that pathway is not shut down with the excess of one product. Each amino acid regulates the first enzyme in its branch to maintain the proper ratio of the amino acids. In Corynebacterium, the regulation is much simpler with only one aspartokinase and here the amino acid biosynthesis is controlled by the synergistic action of the end products. 

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... 

Fine Regulation by Isoenzymes. We have just mentioned the end product inhibition of threonine deaminase by isoleucine. Isoleucine belongs, together with threonine, methionine, and lysine, to the aspartate family of amino acids (Fig. 118). The synthetic pathway to these amino acids starts with the conversion of aspartate into aspartyl phosphate which is catalyzed by aspartokinase. Later the initially undivided synthetic pathway branches out to lead to the amino acids mentioned (Fig. 157). This branching presents intracellular regulation with a serious problem. For example, more than sufficient threonine might be present in the cells. 

The further supply of threonine could then be easily stopped by end product repression or end product inhibition of aspartokinase. However, the synthesis of methionine and lysine would also be stopped, although both of these amino acids might be urgently needed. The solution to the problem lies in the fact that three isoenzymes of aspartokinase have been found in E. coli, one of which can be blocked by threonine and a second by lysine, by end product repression or inhibition. The activity of the third isoenzyme of aspartokinase is also subject to appropriate regulation somewhat later in the synthetic pathway. Although these findings were made in bacteria there are various indications that the situation in higher plants is likely to be quite similar. 

The reactions catalyzed by aspartokinase (1) and aspartate semialdehyde dehydrogenase (2) are utilized for the synthesis of all pathway products, including threonine. Regulation of aspartokinase activity is, therefore, considered to be of central importance in the overall control of the pathway. Two classes of differentially regulated isozymes of aspartokinase have been isolated from plants. One class is comprised of enzymes subject to feedback inhibition by threonine the other encompasses those inhibited by lysine, or by lysine and 5 -adenosylmethionine. Examples of each class have been isolated from several... 

---