Purification and binding features of a pea fructose-1,6-bisphosphatase domain involved in the interaction with thioredoxin f

We previously demonstrated that a cluster in the available (150)Asn-(170)Gl u region of pea chloroplast fructose-1,6-bisphosphatase (FBPase) could be i nvolved in its interaction with the physiological modulator thioredoxin (Tr x), Using as template a cDNA coding for pea chloroplast FBPase, a DNA inser t coding for a 19 amino acid fragment ((149)Pro-(167)Gly) was amplified by PCR, After insertion in the pGEX-4T vector-1, it was expressed in Escherich ia coli as a fusion protein (GST-19) with the vector-coded glutathione tran sferase (GST), This protein appears in the supernatant of cell lysates, and was purified to homogeneity. After thrombin digestion, the 19 amino acid i nsert was isolated as a polypeptide which displayed a positive reaction aga inst pea chloroplast FBPase antibodies, GST-19 linked to glutathione-Sephar ose beads, but not the GST, strongly interacts with pea Trx f, suggesting t hat this binding depends on the 19 amino acid insert. ELISA and Western blo t experiments also demonstrate the existence of a GST-19-Trx f interaction, as well as a negligible quantity of Trx f bound by the vector-coded CST, P utative competitive inhibition assays of FBPase activity carried out in the presence of increasing concentrations of the 19 amino acid insert do not d emonstrate any enzyme inhibition. On the contrary, this protein fragment en hances the enzyme activity proportionally to its concentration in the assay mixture. This indicates that the FBPase-Trx f binding promotes some type o f structural modification of the Trx molecule, or of the FBPase-Trx docking site, thus facilitating the reductive modulation of FBPase.