PURIFICATION AND CDNA CLONING OF ANTHRANILATE SYNTHASE FROM RUTA-GRAVEOLENS - MODES OF EXPRESSION AND PROPERTIES OF NATIVE AND RECOMBINANT ENZYMES

Ruta graveolens utilizes anthranilate synthase (AS) for the synthesis both of tryptophan in primary metabolism and acridone alkaloids in sec ondary metabolism. AS has been purified from plants and cell cultures of R. graveolens 670- and 1700-fold, respectively. Glutamine- and ammo nia-dependent AS activities were strictly co-purified in all steps. Th rough cDNA cloning and complementation of Escherichia coli deletion mu tants defective for AS, it is shown that young Ruta plants express two genes for functional AS alpha subunits, AS alpha 1 and AS alpha 2. Th e data indicate that AS alpha from Ruta requires an AS beta subunit wi th a native molecular weight of 60-65 kDa for the glutamine-dependent reaction. Protein synthesized in vitro from cloned cDNA is processed u pon import into isolated chloroplasts, indicating that mature AS alpha subunits are active in plastids in vivo. AS alpha 1 and AS alpha 2 ar e constitutively expressed in Ruta cell cultures, but AS alpha 1 stead y-state mRNA levels are increased 100-fold 6 h subsequent to elicitati on whereas AS alpha 2 expression remains constitutive. Increased AS al pha 1 transcription corresponds to elicitor-induced alkaloid accumulat ion. The data indicate that Ruta regulates anthranilate flux into prim ary and secondary metabolism through differential regulation of AS gen es specific to these pathways.