Indole-3-glycerol Phosphate Synthase Substrate and Ligand Binding Interactions

Indole-3-glycerol Phosphate Synthase (IGPS) catalyzes the irreversible ring closure of 1- (o-carboxyphenylamino)-1-deoxyribulose 5- phosphate (CdRP) to form indole-3-glycerol phosphate (IGP) during the fourth step of tryptophan biosynthesis. Tryptophan biosynthesis required for the survival of Mycobacterium tuberculosis, which makes it an ideal candidate for anti-tuberculosis treatment. Crystal structures for E. coli IGPS have been reported and previous computational analysis suggested nine amino acid residues that could be important for drug binding. Site directed mutagenesis was used to change the amino acids being tested and five out of nine mutants were generated successfully. The mutant enzymes were expressed in E. coli BL21 (DE3) host cells and purified. The chemical synthesis and purification of the substrate, CdRP, needed for kinetic studies was optimized. Kinetic studies will be conducted using CdRP, and the enzyme turnover rate (kcat) and Michaelis constant (KM) will be determined. Any changes in the kcat and KM for the mutants compared to the wild type IGPS will suggest that the residue in question participates in substrate binding or catalysis.