EFFECTS OF EXPRESSING ESCHERICHIA-COLI THREONINE SYNTHASE IN TOBACCO (NICOTIANA-TABACUM-L) SUSPENSION-CULTURE CELLS ON FREE AMINO-ACID LEVELS, ASPARTATE PATHWAY ENZYME-ACTIVITIES AND UPTAKE OF ASPARTATE INTO THE CELLS
Mj. Muhitch, EFFECTS OF EXPRESSING ESCHERICHIA-COLI THREONINE SYNTHASE IN TOBACCO (NICOTIANA-TABACUM-L) SUSPENSION-CULTURE CELLS ON FREE AMINO-ACID LEVELS, ASPARTATE PATHWAY ENZYME-ACTIVITIES AND UPTAKE OF ASPARTATE INTO THE CELLS, Journal of plant physiology, 150(1-2), 1997, pp. 16-22
The amino acids threonine and methionine are derived from aspartate in
a multibranched biosynthetic pathway. In higher planes, the branchpoi
nt enzymes, threonine synthase (TS;EC 4.2.99.2) and cystathionine gamm
a-synthase (C gamma S; EC 4.2.99.9), which lead to threonine/isoleucin
e and methionine synthesis, respectively, compete against each other f
or pathway intermediates. In order to better understand the regulation
and interplay between these competing pathways, the feedback-insensit
ive E. roll TS was constitutively expressed in tobacco (Nicotiana taba
cum L.) suspension cultured cells via Agrobacterium-mediated transform
ation. 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 threonin
e levels. C gamma S activity increased 3.5 fold, apparently to compens
ate for the heightened competition for the common pathway intermediate
homoserine phosphate. Homoserine dehydrogenase and threonine-sensitiv
e aspartate kinase activities were increased by almost 2 fold. Free as
partate was decreased by 55 % in the transformed cells, while free lys
ine and isoleucine levels were not significantly changed, indicating t
hat threonine does not regulate its own synthesis by inhibiting an enz
yme early in the pathway. Transformed cells had a markedly reduced abi
lity to take up (14)[C] aspartate from the medium, suggesting that thr
eonine may regulate its synthesis in vivo in part by limiting aspartat
e availability after Met, Lys, and Ile pools are filled.