A New Class of N-Hydroxycinnamoyltransferases: PURIFICATION, CLONING, AND EXPRESSION OF A BARLEY AGMATINE COUMAROYLTRANSFERASE (EC 2.3.1.64)

Abstract

Agmatine coumaroyltransferase (ACT), which catalyzes the first step in the biosynthesis of antifungal hydroxycinnamoylagmatine derivatives, was purified to apparent homogeneity from 3-day-old etiolated barley (Hordeum vulgare L.) seedlings. The enzyme was highly specific for agmatine as acyl acceptor and had the highest specificity for p-coumaroyl-CoA among various acyl donors with a specific activity of 29.7 nanokatal x mg(-1) protein. Barley ACT was found to be a single polypeptide chain of 48 kDa with a pI of 5.20 as determined by isoelectric focusing. The 15 N-terminal amino acid residues were identified by micro-sequencing of the native protein and were used to clone a full-length barley ACT cDNA that predicted a protein of 439 amino acid residues. The sequence was devoid of N-terminal signal peptide, suggesting a cytosolic localization of barley ACT. Recombinant ACT produced and affinity-purified from Escherichia coli had a specific activity of 189 nanokatal x mg(-1) protein, thus confirming the identity of the purified native protein. A partial cDNA sequence for ACT was obtained from wheat that predicted a protein of 353 amino acid residues and had 95% sequence identity to barley ACT. Two motifs in the amino acid sequence reveal that barley ACT represents a new class of N-hydroxycinnamoyltransferases belonging to the transferase superfamily. The barley ACT is unique in producing the precursor of hordatine, a proven antifungal factor that may be directed toward Blumeria graminis.