Thiamin auxotrophy in yeast through altered cofactor dependence of the enzyme acetohydroxyacid synthase

The THI1 gene of Saccharomyces cerevisiae has been identified and found to be allelic with the previously characterized gene ILV2 that encodes acetohy droxyacid synthase (AHAS). This enzyme catalyses the first step in the para llel biosyntheses of the branched-chain amino acids isoleucine and valine, using thiamin pyrophosphate (TPP) as a cofactor. The ilv2-thi1 allele encod es a functional AHAS enzyme with an altered dependence for the cofactor TPP resulting in the thiamin auxotrophic phenotype. Nucleotide sequence analys is and site-directed mutagenesis revealed that the thi1 mutation is a singl e base substitution which causes the conserved amino acid substitution D176 E in the AHAS protein. This study therefore implicates aspartate 176 as ano ther amino acid residue important either for the efficient binding of TPP b y AHAS or for the functional stability of the holoenzyme.