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

Abstract

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 Ophospho-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 (eTD L447F ) 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 iii allosteric activator S-adenosyl-L-methionine (SAM), with the parameters of AtTS1 measured as a control. SAM increases the k cat /K m OPHS 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. iv PREFACE This thesis follows the integrated thesis format and so the main chapters represent work that has already been published in a peer-reviewed journal (Chapters 2 and 3) at the time of submission of this thesis, or will soon be submitted for publication (Chapter 4). . Development of a continuous assay and steady-state characterization of Escherichia coli threonine synthase, Analytical Biochemistry. 423 (2012) 78-85. Synthase and Threonine Synthase from Cicer arietinum and Lens culinaris, Biochemistry and Cell Biology. 91 (2013) 95-101.

Interkingdom Comparison of Threonine Metabolism for Stem Cell Maintenance in Plants and Animals

“…Interestingly, THREONIN SYNTHASE 2 (AT1G72810) may compensate TS1 mutation in mto2 mutant. SAM acts an allosteric regulator of enzymatic activity of the TS1 ( Curien et al, 1998 ; Mas-Droux et al, 2006 ) and TS2 ( Morneau, 2014 ). At low SAM levels, TS2 has a two orders of magnitude lower activity compared to that of TS1.…”