Purification and cDNA cloning of isochorismate synthase from elicited cellcultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by t he enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS t o homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The K-m values for chorismate were 558 and 319 mu M for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and r equired Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA libra ry from elicited C. roseus cells with a degenerated primer based on the seq uence of an internal peptide from isoform II resulted in an amplification p roduct that was used to screen the cDNA library. This led to the first isol ation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino a cid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only on e ICS gene in C. roseus.