Graduate Research Assistant University of Texas at Arlington Fort Worth, Texas, United States
Lindsay Davis (University of Texas at Arlington)| Mercy Oyugi (University of Texas at Arlington)| Ghader Bashiri (University of Auckland)| Ted Baker (University of Auckland)| Kayunta Johnson-Winters (University of Texas at Arlington)
F420-dependent Glucose-6-Phosphate Dehydrogenase (FGD) is vital to the existence of Mycobacteria tuberculosis, the causative agent of Tuberculosis disease (TB). FGD catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone using the F420 cofactor, which becomes reduced during catalysis. The cofactor is necessary for Mycobacteria to exist under stressful conditions and proves to be mechanistically important for activating pro-drugs, like PA-824, to treat TB. A previous crystal structure of wild-type FGD has led to a proposed mechanism, suggesting that the conserved His40 residue serves as the active site base, while conserved Glu109 serves as the acid. Our previous work using pH dependence profiling has suggested that Glu109 is the active site acid, while His40 does not serve as the active site base. Preliminary data from global analysis from pre-steady state kinetic experiments using KinTek Explorer software has suggested accumulation of an F420 cofactor-based intermediate within the active site of wtFGD and FGD variants during turnover. While this intermediate is present within some FGD variants, they are not observed within all. Therefore, we have developed global fit models for both scenarios that will reveal further FGD mechanistic information. Here, we present reaction spectra for wtFGD and several FGD variants along with time-dependent fluorescence binding experiments to aid in identifying the intermediate.