A new class of N-hydroxycinnamoyltransferases. Purification, cloning, and expression of a barley agmatine coumaroyltransferase (EC 2.3.1.64).
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.
Burhenne
Kim
K
Risoe National Laboratory, Plant Research Department, PRD-301, Plant Products, DK-4000 Roskilde, Denmark.
Kristensen
Brian K
BK
Rasmussen
Søren K
SK
D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D020224:Expressed Sequence Tags; D001467:Hordeum / Q000201:enzymology; D007525:Isoelectric Focusing; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000201:enzymology / Q000378:metabolism
D018076:DNA, Complementary; D004798:Enzymes; D010455:Peptides; D011994:Recombinant Proteins; D000217:Acyltransferases; C474024:agmatine coumaroyltransferase