Purification, characterization, and immunolocalization of hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase from opium poppy

A development-specific and elicitor-inducible acyltransferase [hydroxycinna moyl-CoA: tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110)] tha t catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to hydroxyphen-ethylamines was purified 988-fold to apparent homoge neity from opium poppy (Papaver somniferum L.) cell-suspension cultures. Th e purification procedure, which resulted in a 6.8% yield, involved hydropho bic interaction and anion-exchange chromatography, followed by affinity chr omatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA ) as eluent. Purified THT had an isoelectric point of 5.2, a native molecul ar mass of approximately 50 kDa, and consisted of two apparently identical 25-kDa subunits as determined by two-dimensional polyacrylamide gel electro phoresis. The purified enzyme was able to synthesize a variety of amides du e to a relatively low specificity for cinnamoyl-CoA derivatives and hydroxy phenethylamines. The best substrates were feruloyl-CoA (V K-m(-1) 13.4 mkat g(-1) M-1) and tyramine (V K-m(-1) 6.57 mkat g(-1) M-1). The THT activity increased during development of opium poppy seedlings, occurred at high lev els in roots and stems of mature plants, and was induced in cell-suspension cultures after treatment with a pathogen-derived elicitor. Immunoblot anal ysis using THT mouse polyclonal antibodies did not always show a correlatio n between THT polypeptide and enzyme activity levels. For example, despite low THT activity in leaves, an abundant 25-kDa immunoreactive polypeptide w as detected. Immunohistochemical localization showed that THT polypeptides occur in cortical and xylem parenchyma, immature xylem vessel elements, roo t periderm, anthers, ovules, and the inner layer of the seed coat, but are most abundant in phloem sieve-tube members in roots, stems, leaves, and ant her filaments.