Two Forms of Herbicide-Sensitive Acetolactate Synthase under Different Control by 2-Oxobutyrate in Rhodospivillum rubrum

Regulation of acetolactate synthase (ALS, EC 4.1.3.18) in the phototrophic prokaryote Rhodospirillum rubrum was studied. In cell free extracts of 5 strains investigated, enzyme activity was very labile (about 80% loss of activity within 12h during storage at 4 °C) but was stabilized to some extent by 10 (μM FAD and 20 vol.% glycerol. By filtration of extracts through Superose 6 HR gels (FPLC technique), ALS activity of all strains was separated in two fractions of 200 and 600 kDa, respectively. The enzyme fractions had about the same affinity to pyruvate (ATm = 1.6 - 1.8 m M) , the same sensitivity to L-valine (50 and 65% inhibition by 0.1 m M valine in the standard test mixture) and the herbicide sulfometuron methyl (90 and 92% inhibition by 1 μM herbicide), but differed greatly in their sensitivity to inhibition by 0.4 m NaCl. In culture media with 2-oxobutyrate (2-OB), growth began only after a lag-phase of several days (5 days with 1 mM of the inhibitor). Cells grown in the presence of 2-OB had a reduced total ALS activity and did not contain the 200 kDa fraction. The inhibition of ALS by valine was noncompetitive in respect to pyruvate (K1= 0.l m M ) . From other branched-chain amino acids tested (L-leucine, L-isoleucine, norvaline, norleucine) only isoleucine was inhibitory (K1; = 3.1 m M )