Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes

Abstract

Ruta graveolens utilizes anthranilate synthase (AS) for the synthesis both of tryptophan in primary metabolism and acridone alkaloids in secondary metabolism. AS has been purified from plants and cell cultures of R. graveolens 670- and 1700-fold, respectively. Glutamine- and ammonia-dependent AS activities were strictly co-purified in all steps. Through cDNA cloning and complementation of Escherichia coli deletion mutants 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. The data indicate that AS alpha from Ruta requires an AS beta subunit with a native molecular weight of 60-65 kDa for the glutamine-dependent reaction. Protein synthesized in vitro from cloned cDNA is processed upon import into isolated chloroplasts, indicating that mature AS alpha subunits are active in plastids in vivo. AS alpha 1 and AS alpha 2 are constitutively expressed in Ruta cell cultures, but AS alpha 1 steady-state mRNA levels are increased 100-fold 6 h subsequent to elicitation whereas AS alpha 2 expression remains constitutive. Increased AS alpha 1 transcription corresponds to elicitor-induced alkaloid accumulation. The data indicate that Ruta regulates anthranilate flux into primary and secondary metabolism through differential regulation of AS genes specific to these pathways.