Recombinant Expression, Purification And Kinetic Characterization Of F₄₂₀-cofactor Dependent Glucose-6-phosphate Dehydrogenase From Mycobacterium tuberculosis

Abstract

The FGD (F₄₂₀-dependent Glucose-6-phosphate dehydrogenase) enzyme is an F₄₂₀ Cofactor (7,8-didemethyl-8-hydroxy-5-deazariboflavin) dependent enzyme found in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). TB is still a prominent cause of illness and death worldwide. Because FGD is not found in humans, makes it a good target for drug development. By understanding the hydride transfer mechanism of the FGD in detail, we can aid in the improvement of drug targeting and development for the treatment of TB. FGD catalyzes the conversion of glucose-6-phosphate to 6-phosophogluconolactone. This project focuses on the purification and kinetic characterization of recombinant FGD using steady-state and pre-steady state kinetic methods. A concurrent goal is to probe the functionality of conserved active site residues that are involved in the hydride transfer reaction. Based upon crystallographic data, it is believed that Histidine 40 acts as an active site base, abstracting a proton from the substrate, glucose-6-phosphate, facilitating the hydride transfer from the substrate to the F₄₂₀ Cofactor. A separate active site amino acid, Tryptophan 44 is believed to stabilize an active site intermediate during turnover. We have mutated these conserved residues, making the following FGD variants, H40A, W44F and W44A. Here, we present purification methods and steady-state kinetic analyses of both wild type FGD and the H40A variant.