Insights into the catalytic mechanism of M. tuberculosis indole‐3‐glycerol phosphate synthase

Abstract

The pathogen M. tuberculosis causes the lung disease tuberculosis (TB). Emerging TB drug resistance worldwide indicates the need for new inhibitors and targets. Recent research suggests that the tryptophan biosynthetic pathway in M. tuberculosis can be targeted to treat TB. Indole‐3‐glycerol phosphate synthase (IGPS) in M. tuberculosis(MtIGPS) is an α/β barrel enzyme that catalyzes the fourth step in the tryptophan biosynthetic pathway: the conversion of 1‐(o‐carboxyphenylamino)‐1‐deoxyribulose 5’‐phosphate (CdRP) into indole‐glycerol‐phosphate (IGP) via cyclization, decarboxylation and dehydration steps. The pH profile of MtIGPS was observed to have a bell shaped curve with pKa1 and pKa2 values or 6.7 ± 0.14 and 8.7 ± 0.12, supporting the involvement of both a general acid and a general base in MtIGPS catalysis. Mutations were introduced to three residues that had been previously proposed to serve as the catalytic base: E57, E168 and E219. The E57Q variant had a >200‐fold reduced catalytic activity compared to the wildtype and the E168Q variant had a >187‐fold increased KM. The E219 sidechain interacts with K119, which is proposed to be the catalytic acid. The E219D variant had a >233‐fold decreased catalytic activity. Combined with docking predictions, these data were consistent with E57 as the catalytic base, E168 playing a role in substrate binding, and E219 in orienting the catalytic acid K119. These data together provide an increased understanding of the structure‐function relationship in the MtIGPS active site and insights in the IGPS enzyme family.