Purification of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase from cell-suspension cultures ofSolanum tuberosumL. cv. Datura

摘要

A pathogen-elicitor-inducible acyltransferase tyramine hydroxycinnamoyltransferase (THT); EC 2.3.1, which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to tyramine in the formation ofN-hydroxycinnamoyltyramine, was purified to apparent homogeneity from cell-suspension cultures of potato (Solanum tuberosumL. cv. Datura), with a 1400-fold enrichment, a 5 recovery and a final specific activity of 208 mkat·(kg protein)−1. Affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent was the decisive step in the purification sequence. The purified protein showed a native molecular mass of ca. 49 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and in the absence of a reducing agent (2-mercaptoethanol) indicated that THT is a heterodimer in which the protein subunits (ca. 25 kDa) are non-covalently associated. The enzyme was stimulated fivefold by 10 mM Ca2+. The apparentKmvalue for tyramine was dependent on the nature of the hydroxycinnamoyl-CoA present. Thus, theKmvalue for tyramine was about tenfold greater (174 μM) in the presence of 4-coumaroyl-CoA than in the presence of feruloyl-CoA (20