Mutations in two distinct regions of acetolactate synthase regulatory subunit from Streptomyces cinnamonensis result in the lack of sensitivity to end-product inhibition

Acetolactate synthase small subunit encoding ilvN genes from the parental S treptomyces cinnamonensis strain and mutants resistant either to valine ana logues or to 2-ketobutyrate were cloned and sequenced. The wild-type IlvN f rom S. cinnamonensis is composed of 175 amino acid residues and shows a hig h degree of similarity with the small subunits of other valine-sensitive ba cterial acetolactate synthases. Changes in the sequence of ilvN conferring the insensitivity to valine in mutant strains were found in two distinct re gions. Certain point mutations were located in the conserved domain near th e N terminus, while others resulting in the same phenotype shortened the pr otein at V(104) or V(107). To confirm whether the described mutations were responsible for the changed biochemical properties of the native enzyme, th e wildtype large subunit and the wild-type and mutant forms of the small on e were expressed separately in E. coil and combined in vitro to reconstitut e the active enzyme. (C) 1999 Academic Press.