Expression and purification of imidazole glycerol phosphate synthase from Saccharomyces cerevisiae

Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase that catalyzes the formation of IGP and 5-aminoimidazole-4-carboxamide rib onucleotide (AICAR) from N-1-[(5'-phosphoribulosyl)formimino]-5-aminoimidaz ole-4-carboxamide ribonucleotide (PRFAR). This enzyme represents a junction between histidine biosynthesis and de novo purine biosynthesis, The recent characterization of the HIS7 gene in the yeast Saccharomyces cerevisiae IG P synthase established that this protein is bifunctional, representing a fu sion between the N-terminal HisH domain and a C-terminal HisF domain, Catal ytically active yeast HIS7 was expressed in a bacterial system under the co ntrol of T7 polymerase promoter. The recombinant enzyme was purified to hom ogeneity and the native molecular weight and steady-state kinetic constants were determined. The yeast enzyme is distinguished from the Escherichia co li IGP synthase in its utilization of ammonia as a substrate. HIS7 displays a higher K-m for glutamine and a lower turnover in the ammonia dependent I GP synthase activity. As observed with the E. coli IGP synthase, HIS7 shows a low basal level glutaminase activity that can be enhanced 1000-fold in t he presence of a nucleotide substrate or analog. The purification and chara cterization of the S. cerevisiae enzyme will enable a more detailed investi gation of the biochemical mechanisms that mediate the ammonia-transfer proc ess. The fused structural feature of the HIS7 protein and the development o f a high-level production system for the active enzyme elevate the potentia l for determination of its three-dimensional structure through X-ray crysta llography. (C) 2000 Academic Press.