Separation of putrescine and spermidine hydroxycinnamoyl transferases extracted from tobacco callus

Putrescine and spermidine hydroxycinnamoyl transferase activities, which can both be detected in extracts of tobacco callus grown from leaf discs, were separated by ion exchange chromatography on DEAE Trisacryl. Spermidine hydroxycinnamoyl transferase (SHT) is ca 12-fold less active than putrescine hydroxycinnamoyl transferase (PHT). The two enzymes exhibit remarkable differences in specificity towards cinnamoyl-CoA derivatives, putrescine being preferentially conjugated to caffeic acid while spermidine is mainly conjugated to p-coumaric acid. After ion exchange chromatography the PHT fraction is not specific for putrescine and uses other diamines as substrates (mainly cadaverine and diaminopropane), contrary to the SHT fraction which was found to act only on spermidine. Agmatine is conjugated in neither of the two fractions but undergoes rapid hydrolysis to putrescine in the crude enzymic extract. Only N1-feruloylspermidine can be detected when the amide synthesized in vitro by SHT from feruloyl-CoA and spermidine is analysed by mass spectrometry.