(L5398) Insights into the catalytic mechanism of M. tuberculosis indole-3-glycerol phosphate synthase

Nina Goodey (Montclair State University)| Katherine Leon H. (Montclair State University)| Oshane Thomas (Montclair State University, Montclair State University)| Sarah Cho (Montclair State University)| Huma Booter (Montclair State University)| Hanniah Riley (Montclair State University)| Thomas Candela (Montclair State University)| Paige Fadden (Montclair State University)| Joseph LaCap (Montclair State University)| David Konas (Montclair State University)

Tuberculosis is a lung disease, caused by the pathogen M. tuberculosis that affects millions of people world-wide. Several tuberculosis drugs have lost efficacy due to emerging drug resistance and there is a need to discover new inhibitors and targets. Recent research suggests that indole-3-glycerol phosphate synthase (IGPS) in M. tuberculosis (MtIGPS) could be targeted to treat tuberculosis. This enzyme catalyzes the fourth step in the tryptophan biosynthetic pathway. IGPS is an α/β barrel enzyme that catalyzes 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 of 6.7 ± 0.14 and 8.7 ± 0.12, supporting the involvement of both a general acid and a general base in MtIGPS catalysis.  We introduced mutations to three residues that have been proposed to serve as the catalytic base, E57, E158 and E219. The E57Q variant was found to have a >2000-fold reduced catalytic activity compared to the wild type while the catalytic activity of E57D was reduced by only a factor of ~10. The rate-pH profile for the E57D was observed to have shifted to the left, consistent with E57 serving as the catalytic base in the MtIGPS mechanism. The catalytic activity of the E219Q enzyme was reduced by only a factor of 5 compared to the wild type but the E219Q KM was 8 times compared with wild type. Combined with docking predictions, these data support the role of E219 in substrate binding. These data together provide an increased understanding of the structure-function relationship in the MtIGPS active site and insights into catalysis in the IGPS enzyme family.