CHARACTERIZATION AND HETEROLOGOUS EXPRESSION OF HYDROXYCINNAMOYL BENZOYL-COA-ANTHRANILATE N-HYDROXYCINNAMOYL/BENZOYLTRANSFERASE FROM ELICITED CELL-CULTURES OF CARNATION, DIANTHUS-CARYOPHYLLUS L/

Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first comm itted reaction of phytoalexin biosynthesis in carnation (Dianthus cary ophyllus L.), and the product N-benzoylanthranilate is the precursor o f several sets of dianthramides. The transferase activity is constitut ively expressed in suspension-cultured carnation cells and can be rapi dly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of th ese sequences differed in a few amino acid residues only suggesting th e presence of isoenzymes. A specific 0.8 kb cDNA probe was generated b y RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)(+) RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1-3, were sequenced and s hown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. T he additional two clones harbored insert sequences mostly homologous w ith pchcbt1 but differing in the 3'-flanking regions due to variable u sage of poly(A) addition sites. The identity of the clones was confirm ed by matching the translated polypeptides with the tryptic enzyme seq uences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of an thranilate N-benzoyltransferase genes. In vitro, the benzoyltransferas es exhibited narrow substrate specificity for anthranilate but accepte d a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNA s described represent also the first hydroxycinnamoyl-transferases clo ned from plants, which classifies the enzymes as hydroxycinnamoyl/benz oyltransferases.