Impact of S220A and E168A mutations on M. tuberculosis Indole‐3‐Glycerol Phosphate Synthase (IGPS) catalysis

Abstract

The enzyme indole‐3‐glycerol phosphate synthase (IGPS) catalyzes the conversion of 1‐(o‐carboxyphenylamino)‐1‐deoxyribulose‐5‐phosphate (CdRP) to indole‐3‐glycerol (IGP) in the tryptophan biosynthetic pathway in bacterial organisms (Scheme 1). The tryptophan biosynthetic pathway is necessary for bacterial life, therefore, this enzyme represents a potential target for antibacterial drug development against pathogenic organisms. Because IGPS is absent in humans, no concerns for homolog selectivity need to be considered. Our goal is to investigate substrate/ligand binding and the catalytic mechanism of M. tuberculosis IGPS to facilitate inhibitor discovery. To study how this enzyme interacts with ligands and catalyzes the reaction, mutated plasmids that code for M. tuberculosis IGPS wildtype, as well as mutants S220A and E168A, were expressed in E. coli and purified via a His‐tag. The wildtype and the active site mutants S220A and E168A were studied via kinetic assays. Based on previously reported results, we suspected that Ser220 may be involved in ligand and substrate binding in M. tuberculosis IGPS. We hypothesized that Glu168 plays an important role in either catalysis or binding. We measured wildtype activity following the rate at which the product IGP is produced monitoring fluorescence with an excitation wavelength of 280 nm and an emission wavelength of 340 nm. We have found that the mutant E168A has no detectable activity, indicating that residue E168 plays a critical role in catalysis. We have also found that the catalytic activity of S220A is greatly reduced compared to the wildtype enzyme, indicating that S220 plays an important role as well. Understanding the roles that the active site residues play in M. tuberculosis IGPS ligand binding and catalysis will contribute to future drug discovery efforts.Support or Funding InformationNIH grant 1R15GM126467‐01 titled “Investigation of Substrate Specificity, Mechanism, and Inhibition of IGPS”Scheme 1. Reaction Catalyzed by MtIGPS and a Proposed MechanismFigure 1