Minisymposium 3: Secondary metabolism

Abs # 13005: Biosynthesis of antifungal hydroxycinnamoylagmatine

Presenter:	Rasmussen, Soren K., soren.rasmussen@risoe.dk
Authors	Rasmussen, Soren K. (A)   Divéki, Zoltan  (A)   Poulsen, Tina T. (A)   Kristensen, Brian K. (A)   Burhenne, Kim  (A)
Affiliations:	(A): Risoe National Laboratory
Web Site:	http://www.risoe.dk

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/ mg 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 E. coli had a specific activity of 189 nanokatal/ mg protein, thus confirming the identity of the purified native protein. Wheat has a very similar gene, whereas the rice gene is more distinctly related. 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. Transgenic Brachypodium and barley is being produced, the ACT gene(s) being mapped and the expression of ACT in barley leaves infected with Bgr is being monitored by RT-PCR and by westernblotting.