27.01. Synthesis of threonine from aspartate
The first enzyme in the pathway of threonine biosynthsis is Aspartate kinase (aspartokinase) (AK) [EC 2.7.2.4]. This enzyme catalyzes the phosphorylation of aspartate to aspartyl-4-phosphate at the expense of an ATP molecule. Typically, plants have either 2 or 3 AK isoforms that each differ in sensitivity to feedback inhibition by end-products the aspartate pathway. For example, in barley one isoform, AK-I, is inhibited by threonine, and two other isoforms, AK-II and AK-III, are inhibited by lysine, and syntergistically with lysine, by S-adenosylmethionine (SAM) (Galili, 1995, Zhu-Shimoni et al, 1997).
Bifunctional proteins that contain both AK and homoserine dehydrobenase activity were first found in Escherichia coli where two AK bifunctional AK isoforms, AK-I and AK-II, have been observed, both of which are lysine-insensitive. Bifunctional AK and homoserine dehydrogenase (HSD) [EC 1.1.1.3] (e.g. Weisemann and Matthews, 1993) cDNAs similar to the E. coli AK-I/HSD-I and AK-II/HSD-II have subsequently been found in higher plants (Weisemann and Matthews, 1993). E. coli also contains a lysine-sensitive AK isoform, AK-III, that lacks HSD activity (Galili, 1995), and Frankard et al (1997) have isolated a cDNA clone encoding a monofunctional AK from Arabidopsis thaliana having greatest similarity with the Escherichia coli AKIII, the enzyme that is feedback-inhibited by lysine (encoded by lysC) (Frankard et al, 1997). The inferred amino acid sequence of te Arabidopsis clone lacks an 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.
In Arabidopsis AK-III appears to be regulated at the transcriptional level by light and photosynthetic metabolites, e.g. sucrose and phosphate, but apparently not by N availability (Zhu-Shimoni and Galili, 1998). Expression of AK/HSD appears to be coordinated with the expression of asparagine synthetase which is repressed by light and sucrose, and stimulated by dark and nitrogen. The hypothesis is that such reciprocal control of AK-III-like and AK-II-like genes may favor asparagine synthesis at night and the conversion of aspartate to aspartate family amino acids during the day (Zhu-Shimoni and Galili, 1998).
HSD catalyzes the first reaction uniquely associated with threonine, methionine, and isoleucine biosynthesis in the pathway (see above), i.e the conversion of aspartate-4-semialdehyde to homoserine. Higher plants generally contain a threonine-sensitive and a threonine-insensitive isoform of HSD. Interconversion by threonine between a threonine-sensitive trimeric form of HSD and a threonine-insensitive dimeric form has been demonstrated for the enzyme from carrot(Galili, 1995), but it is not clear whether such mechanisms operate in other plants or in vivo.
The formation of O-phospho-L-homoserine is catalyzed by Homoserine kinase [EC 2.7.1.39]. In plants O-phospho-L-homoserine is an important branch point intermediate leading to either methionine and sulfur metabolism or to threonine and branched chain amino acids. An open reading frame (located on the top arm of chromosome II), corresponding cDNA for homoserine kinase has been identified in Arabidopsis (Lee and Luestek, 1999). This HSK gene has homology with homoserine kinase from bacteria and fungi and contains a conserved motif thought to comprise the ATP binding site of ATP-dependent metabolite kinases. The N-terminal 50 amino acids of the HSK protein show features of a transit peptide directing localization to plastids.
Threonine synthase [EC 4.2.99.2] from Arabidopsis thaliana has been identified by using a cDNA to functionally complement 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 activity of the recombinant protein is allosterically activated 85-fold by S-adenosylmethionine, 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; Laber et al, 1999). The Arabidopsis threonine synthase is a dimer, while the E. coli and yeast enzymes are monomers (Laber et al, 1999).
Arabidopsis thaliana threonine synthase has been crystallized, and its structure solved at 2.25 A (Thomazeau et al, 2001). The homodimeric structure reveals four functional domains with a two-stranded beta-sheet arm protruding from one monomer to 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 their substrate, O-phospho-L-homoserine (OPH) (Bartlem et al, 2000). 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 (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).
|Synthesis of threonine from aspartate|Isoleucine and valine biosynthesis|Leucine biosynthesis|Lysine biosynthesis|Cysteine and methionine biosynthesis|Sulfate assimilation|Serine and glycine metabolism|Three-dimensional structure of Arabidopsis thaliana threonine synthase|Inhibition of branched chain amino acid biosynthesis by chlorsulfuron|Feedback inhibition of valine, leucine and isoleucine biosynthesis|Feedback inhibition control by lysine and threonine|Cyanogenic glucosides derived from branched chain amino acids|References|
