Toward the Construction of a Kinetic Model of Methionine and Thereonine Biosynthesis to Increase Seed Nutritional Value: Characterization of Threonine Synthase.

Improving the nutritional quality of crop species, like Cicer arietinum and Lens culinaris, is of particular interest to agricultural biotechnology. These crops are deficient, from a human dietary perspective, in the essential amino acid L-methionine (L-Met). In plants, the biosynthesis of L-Met is initiated by the formation of L-cystathionine from O-phospho-L-homoserine (OPHS) catalyzed by cystathionine γ-synthase (CGS). OPHS occupies the branch-point between L-Met and L-threonine (L-Thr) biosynthesis, catalyzed by threonine synthase (TS), making these two enzymes targets for metabolic engineering studies to increase L-Met production. The construction of a kinetic model recreating the branch-point can facilitate the metabolic engineering of important crop species by providing a tool for anticipating disturbances in metabolic flux caused by manipulations to the branch-point. The focus of the research described in this thesis is to provide a starting point for the creation of a kinetic model of the OPHS branch-point in the target species. The characterization of plant TS requires a continuous spectrophotometric assay, which was developed using a non-allosteric variant of threonine deaminase from Escherichia coli (eTDL447F) and hydroxyisocaproate dehydrogenase (HO-HxoDH) from Lactobacillus delbrueckii. The assay was verified for suitability for use under the various pH optima of TS across phyla. The functional coding sequences of TS from C. arietinum (CaTS) and L. culinaris (LcTS) share approximately 80% amino acid sequence identity with TS from Glycine max, and the residues of the allosteric site are conserved with those of TS from other plant species. The PLP-binding motif, which is conserved across plant and microbial species, is present in the active site of both enzymes. The kinetic parameters of CaTS, LcTS, and AtTS2 were measured with 100 µM of the allosteric activator S-adenosyl-L-methionine (SAM), with the parameters of AtTS1 measured as a control. SAM increases the kcat/KmOPHS of AtTS2, LcTS, CaTS, and AtTS1 by 10-, 20-, 25-, and 80-fold, respectively. Due to the varying flux control patterns of TS from A. thaliana, C. arietinum, and L. culinaris, the construction of a species-specific kinetic model of the branch-point is needed for future aimed at increasing L-Met biosynthesis to nutritionally significant levels.