Abstract
Summary The amino acids threonine and methionine are derived from aspartate in a multibranched biosynthetic pathway. In higher plants, the branchpoint enzymes, threonine synthase (TS; EC 4.2.99.2) and cystathionine γ-synthase (CγS; EC 4.2.99.9), which lead to threonine/isoleucine and methionine synthesis, respectively, compete against each other for pathway intermediates. In order to better understand the regulation and interplay between these competing pathways, the feedback-insensitive E. coli TS was constitutively expressed in tobacco ( Nicotiana tabacum L.) suspension cultured cells via Agrobacterium -mediated transformation. Expression of E. coli TS in tobacco cells resulted in a 7-fold increase in total TS activity and a 5.7-fold increase in free threonine levels. CγS activity increased 3.5 fold, apparendy to compensate for the heightened competition for the common pathway intermediate homoserine phosphate. Homoserine dehydrogenase and threonine-sensitive aspartate kinase activities were increased by almost 2 fold. Free aspartate was decreased by 55 % in the transformed cells, while free lysine and isoleucine levels were not significantly changed, indicating that threonine does not regulate its own synthesis by inhibiting an enzyme early in the pathway. Transformed cells had a markedly reduced ability to take up 14 [C] aspartate from the medium, suggesting that threonine may regulate its synthesis in vivo in part by limiting aspartate availability after Met, Lys, and lie pools are filled.
Published Version
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