Aspartate kinase (aspartokinase) (AK) [EC 2.7.2.4] catalyzes the phosphorylation of aspartate to aspartyl-4-phosphate with the accompanying hydrolysis of ATP. Plants generally possess 2 or 3 AK isoenzymes which may differ in sensitivity to feedback inhibition by end-products. In barley one isoenzyme, AK-I, is inhibited by threonine, and the other two, AK-II and AK-III, are inhibited by lysine (Galili, 1995). The lysine-sensitive AKs are synergistically inhibited by lysine and S-adenosylmethionine (SAM) (Zhu-Shimoni et al, 1997).

Some AK isoenzymes in plants appear to be bifunctional proteins that contain both AK and homoserine dehydrogenase (HSD) [EC 1.1.1.3] (e.g. Weisemann and Matthews, 1993). This is similar to the situation in Escherichia coli where two of the AK isoenzymes, AK-I and AK-II, both of which are lysine-insensitive, also exist as bifunctional proteins. Higher plant cDNAs similar to the E. coli AK-I/HSD-I and AK-II/HSD-II (e.g. Weisemann and Matthews, 1993) enzymes have been obtained, but until 1997 it was not known whether plants also contain a lysine-sensitive AK isozyme that lacks HSD activity, as occurs with E. coli AK-III (Galili, 1995). However, in 1997, Frankard et al (1997) isolated a cDNA clone encoding a monofunctional AK from an Arabidopsis thaliana cell suspension cDNA library. This cDNA shows highest identity with the Escherichia coli AKIII enzyme that is feedback-inhibited by lysine (encoded by lysC) (Frankard et al, 1997). It lacks a HSD-encoding sequence at the COOH end of the peptide. Two homologous genes were detected in the A. thaliana genome (Frankard et al, 1997). Simultaneously, Tang et al (1997) also cloned an Arabidopsis cDNA encoding a monofunctional AK homologous to the lysine-sensitive enzyme of E. coli. The implications of these findings are that the lysine-sensitive monofunctional AKs may be specifically involved in lysine biosynthesis in plants.

An AK/HSD gene of Arabidopsis appears to be regulated at the transcriptional level by light and photosynthetic metabolites, sucrose and phosphate, but apparently not by N availability (Zhu-Shimoni and Galili, 1998). Expression of AK/HSD appears to be coordinated reciprocally with asparagine synthetase which is repressed by light and sucrose, and stimulated by dark and nitrogen. This reciprocal control may favor asparagine synthesis at night and efficient conversion of aspartate to aspartate family amino acids during the day (Zhu-Shimoni and Galili, 1998).

HSD catalyzes the first reaction that is uniquely associated with threonine, methionine and isoleucine biosynthesis; the conversion of aspartate-4-semialdehyde to homoserine. Higher plants generally contain at least two forms of HSD, a threonine-sensitive form and a threonine-insensitive form. Carrot HSD can be reversibly interconverted in vitro between a threonine-sensitive trimeric form, and a threonine-insensitive dimeric form, depending on whether the medium contains threonine (Galili, 1995).

Homoserine kinase [EC 2.7.1.39] catalyzes the formation of O-phospho-L-homoserine, an important branch point intermediate in the pathways for methionine and threonine synthesis in plants. The Arabidopsis thaliana homoserine kinase genomic open reading frame (located on the top arm of chromosome II) and a corresponding cDNA have been identified (Lee and Leustek, 1999). The HSK gene has homology with homoserine kinase from bacteria and fungi and contains a conserved motif found in a group of ATP-dependent metabolite kinases and thought to comprise the ATP binding site. The N-terminal 50 amino acids of the HSK protein show features of a transit peptide directing localization to plastids.

A cDNA encoding threonine synthase [EC 4.2.99.2] was isolated from Arabidopsis thaliana by functional complementation of an Escherichia coli mutant devoid of threonine synthase activity (Curien et al, 1996). The expressed protein has an N-terminal transit peptide consistent with plastidic localization. The recombinant protein is allosterically activated by S-adenosylmethionine (activity is increased up to 85-fold by S-adenosylmethionine (SAM) and specifically inhibited by AMP (Laber et al, 1999)). The N-terminal part of the mature enzyme is implicated in determining the sensitivity to S-adenosylmethionine (Curien et al, 1996; 1998; Laber et al, 1999). The Arabidopsis threonine synthase is a dimer, while the E. coli and yeast enzymes are monomers (Laber et al, 1999).

The Arabidopsis thaliana threonine synthase has recently been crystallized and its structure solved at 2.25 A (Thomazeau et al, 2001). The structure reveals a four-domain dimer with a two-stranded beta-sheet arm protruding from one monomer onto the other. This protrusion could form a lever through which the allosteric effect of S-adenosylmethionine is transmitted. The enzyme shows functional domains typical of pyridoxal-P-dependent enzymes, and also has similarities with SAM-dependent methyltransferases (Thomazeau et al, 2001).

It is believed that the enzymes threonine synthase (TS) and cystathionine gamma-synthase (CGS) actively compete for the O-phospho-L-homoserine (OPH) substrate for threonine and methionine biosynthesis, respectively (Bartlem et al, 2000). Consistent with this, an Arabidopsis mutant carrying a single base pair mutation within the gene encoding TS (mto2-1), over-accumulates soluble methionine 22-fold and contains markedly reduced levels of soluble threonine in young rosettes (Bartlem et al, 2000). Thus, the mutation appears to result in decreased threonine biosynthesis and a channeling of OPH to methionine biosynthesis in young rosettes. This further suggests that the feedback regulation of CGS is not sufficient alone for the control of Met biosynthesis in young rosettes and is dependent on TS activity (Bartlem et al, 2000).

Many of the enzymes involved in lysine and threonine synthesis have been localized to plastids. Analyses of cloned DNA sequences confirm that these enzymes are synthesized with transit peptides that direct them into the plastid. However, note that enzymes involved in methionine and SAM (AdoMet) synthesis have been localized to the cytosol (Galili, 1995; Hanson and Roje, 2001).

Bartlem D, Lambein I, Okamoto T, Itaya A, Uda Y, Kijima F, Tamaki Y, Nambara E, Naito S 2000 Mutation in the threonine synthase gene results in an over-accumulation of soluble methionine in Arabidopsis. Plant Physiol. 123: 101-110.

Curien G, Dumas R, Ravanel S, Douce R 1996 Characterization of an Arabidopsis thaliana cDNA encoding an S-adenosylmethionine-sensitive threonine synthase. Threonine synthase from higher plants. FEBS Lett. 390: 85-90.

Curien G, Job D, Douce R, Dumas R 1998 Allosteric activation of Arabidopsis threonine synthase by S-adenosylmethionine. Biochemistry 37: 13212-13221.

Frankard V, Vauterin M, Jacobs M 1997 Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional aspartate kinase. Plant. Mol. Biol. 34: 233-242.

Galili G 1995 Regulation of lysine and threonine synthesis. Plant Cell 7: 899-906.

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Laber B, Maurer W, Hanke C, Grafe S, Ehlert S, Messerschmidt A, Clausen T 1999 Characterization of recombinant Arabidopsis thaliana threonine synthase. Eur. J. Biochem. 263: 212-221.

Lee M, Leustek T 1999 Identification of the gene encoding homoserine kinase from Arabidopsis thaliana and characterization of the recombinant enzyme derived from the gene. Arch. Biochem. Biophys. 372: 135-142.

Tang G, Zhu-Shimoni JX, Amir R, Zchori IB, Galili G 1997 Cloning and expression of an Arabidopsis thaliana cDNA encoding a monofunctional aspartate kinase homologous to the lysine-sensitive enzyme of Escherichia coli. Plant. Mol. Biol. 34: 287-293.

Thomazeau K, Curien G, Dumas R, Biou V 2001 Crystal structure of threonine synthase from Arabidopsis thaliana. Protein Sci. 10: 638-648.

Weisemann JM, Matthews BF 1993 Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase. Plant Mol. Biol. 22: 301-312.

Zhu-Shimoni JX, Galili G 1998 Expression of an Arabidopsis aspartate kinase/homoserine dehydrogenase gene is metabolically regulated by photosynthesis-related signals but not by nitrogenous compounds. Plant Physiol. 116: 1023-1028.

Zhu-Shimoni JX, Lev-Yadun S, Matthews B, Galili G 1997 Expression of an aspartate kinase homoserine dehydrogenase gene is subject to specific spatial and temporal regulation in vegetative tissues, flowers, and developing seeds. Plant Physiol. 113: 695-706.