Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L.

摘要

Anthranilate N-benzoyltransferase (BT, EC 2.3.1.144) catalyses the first committed reaction of phytoalexin biosynthesis in D. caryophyllus, and the product N-benzoylanthranilate is the precursor of several sets of dianthramides, reportedly essentialfor the expression of disease resistance in many carnation cultivars. BT activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. BT 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 these sequences differed in a few amino acid residues only, suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by reverse transcriptase PCR, employing degenerated oligonucleotide primers complementary to 2 of the tryptic peptides and using poly(A)+ RNA from elicited carnation cells. Five distinct BT clones were isolated from a cDNA library, and 3 cDNAs (pchcbt1 to 3), were sequenced and shown to encode full-size N-benzoyltransferases. Translated peptide sequences revealed more than 95% identity among these 3 clones. The additional 2 clones harboured insert sequences mostly homologous with pchcbt1 but differing in the 3'-flanking regions due to variable usage of poly(A) addition sites. Identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequencesas well as by BT activity expressed in Escherichia coli. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceededthose observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Nucleotide sequence data reported here have been submitted to the EMBL/GenBank/DDBJ database under accession numbers Z84383, Z84384, Z84385, Z84386 andZ84571.