Acetohydroxyacid synthase: A proposed structure for regulatory subunits supported by evidence from mutagenesis

Valine inhibition of acetohydroxyacid synthase (AHAS) plays an important ro le in regulation of biosynthesis of branched-chain amino acids in bacteria. Bacterial AHASs are composed of separate catalytic and regulatory subunits ; while the catalytic subunits appear to be homologous with several other t hiamin diphosphate-dependent enzymes, there has been no model for the struc ture of the small, regulatory subunits (SSUs). AHAS III is one of three iso zymes in Escherichia coli. Its large subunit (encoded by ilvl) by itself ha s 3-5 activity of the holoenzyme and is not sensitive to inhibition by vali ne. The SSU (encoded by ilvH) associates with the large subunit and is requ ired for full catalytic activity and valine sensitivity. The isolated SSU b inds valine. The properties of several mutant SSUs shed light on the relati on between their structure and regulatory function. Three mutant SSUs were obtained from spontaneous Val bacterial mutants and three more were designe d on the basis of an alignment of SSU sequences from valine-sensitive and r esistant isozymes, or consideration of the molecular model developed here. Mutant SSUs N11A, G14D, N29H and A36V, when reconstituted with wild-type la rge subunit, lead to a holoenzyme with drastically reduced valine sensitivi ty, but with a specific activity similar to that of the wild-type. The isol ated G14D and N29H subunits do not bind valine. Mutant Q59L leads to a vali ne-sensitive holoenzyme and isolated Q59L binds valine. T34I has an interme diate valine sensitivity. The effects of mutations on the affinity of the l arge subunits fur SSUs also vary. D. Fischer's hybrid fold prediction metho d suggested a fold similarity between the N terminus of the ilvH product an d the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. On the basis of this prediction, together with the properties of the mutants, a model for the structure of the AHAS SSUs and the location of the valine-b inding sites can be proposed. (C) 2001 Academic Press.