ANTHRANILATE SYNTHASE FROM RUTA-GRAVEOLENS - DUPLICATED AS-ALPHA-GENES ENCODE TRYPTOPHAN-SENSITIVE AND TRYPTOPHAN-INSENSITIVE ISOENZYMES SPECIFIC TO AMINO-ACID AND ALKALOID BIOSYNTHESIS

Anthranilate synthase (AS, EC 4.1.3.27) catalyzes the conversion of ch orismate into anthranilate, the biosynthetic precursor of both tryptop han and numerous secondary metabolites, including inducible plant defe nse compounds. The higher plant Rota graveolens produces tryptophan an d elicitor-inducible, anthranilate-derived alkaloids by means of two d ifferentially expressed nuclear genes for chloroplast-localized AS alp ha subunits, AS alpha 1 and AS alpha 2. Mechanisms that partition chor ismate between tryptophan and inducible alkaloids thus do not entail c hloroplast/cytosol separation of AS isoenzymes and yet might involve d ifferential feedback regulation of pathway-specific AS alpha subunits. The two AS alpha isoenzymes of R. graveolens were expressed as glutat hione S-transferase fusion proteins in Escherichia coli deletion mutan ts defective in AS activity and were purified to homogeneity. Differen tial sensitivity of the transformed E. coli strains toward 5-methyltry ptophan, a false-feedback inhibitor of AS, was demonstrated. Character ization of affinity-purified AS alpha isoenzymes revealed that the non inducible AS alpha 2 of R. graveolens is strongly feedback inhibited b y 10 mu M tryptophan. In contrast, the elicitor-inducible AS alpha 1 i soenzyme is only slightly affected even by tryptophan concentrations 1 0-fold higher than those observed in planta. These results are consist ent with the hypothesis that chorismate flux into biosynthesis of tryp tophan and defense-related alkaloid biosynthesis in R. graveolens is r egulated at the site of AS alpha isoenzymes at both genetic and enzyma tic levels.