Graduation Semester and Year
Spring 2026
Language
English
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Chemistry
Department
Chemistry and Biochemistry
First Advisor
Kayunta Johnson-Winters
Second Advisor
Subhrangsu Mandal
Third Advisor
Sherri McFarland
Fourth Advisor
Morteza Khaledi
Abstract
F420-dependent glucose-6-phosphate dehydrogenase (FGD) is a key enzyme that catalyzes the oxidation of glucose-6-phosphate (G6P) to 6-phosphogluconolactone while simultaneously reducing cofactor F420 to F420H₂. In Mycobacterium tuberculosis (Mtb), this enzyme (Mtb-FGD) is the primary, and possibly the only, source of reduced F420 within the cell, making it central to F420-mediated redox metabolism. During catalysis, Mtb-FGD facilitates a hydride transfer from the C-1 position of G6P to the C-5 position of the F420 cofactor, enabling efficient electron transfer under low redox potential conditions. This activity is particularly important because the reduced cofactor, F420H₂, is required for the activation of several prodrugs used to treat multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. While the cofactor F420 was originally identified in Nocardia and is also found across a range of bacteria and archaea, its biological significance in Mycobacteria is largely defined by the function of FGD, which links central carbon metabolism to clinically relevant drug activation pathways. Chapter 1 of this dissertation provides background about the F420 cofactor and previous work conducted on FGD by various research groups including our lab where we used kinetic characterization methods to determine mechanistic information about the enzyme.
Building on the information obtained in Chapter 1, the Johnson-Winters group conducted a detailed biochemical and biophysical investigation that elucidated the Mtb-FGD mechanism. More specifically, the work presented in Chapter 2 utilizes thermodynamic and kinetic studies to understand the role of the catalytic residue His40 within Mtb-FGD active site. We conducted temperature-dependent pH profiles and affinity-labeling experiments using diethylpyrocarbonate (DEPC) to determine the role of H40 during catalysis. The temperature dependent experiments revealed that a cationic histidine is needed for the reaction to take place, while the affinity labeling experiments determined that the H40 was covalently modified by DEPC which only reacts with a deprotonated histidine. The data obtained suggest that histidine exists in both a protonated and deprotonated state. The protonated His40 first donates a proton to the neighboring Glu13, thereby becoming deprotonated. The deprotonated His40 then abstracts a proton from G6P facilitating the hydride transfer to the F420 cofactor. His40 and Glu13 work together as a dyad during turnover. The work was supplemented with pre steady-state single turnover experiments which revealed the accumulation of intermediate resembling the enzyme-product complex. Additionally, global analysis revealed that Mtb-FGD follows a fast chemistry and slow product release with cofactor association being rate-limiting in catalysis.
Based on recent phylogenetic profiling and kinetic studies, FGD from Nocardioidaceae brasiliensis andCryptosporangium arvum share high sequence identity with Mtb-FGD. However, the FGDs from these new sources are active toward a wider range to sugar substrates such as fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P) in addition to G6P. The focus of Chapter 3 is on the FGD from Cryptosporangium arvum (Cryar-FGD). We used a combination of kinetic and NMR studies to determine the level of substrate promiscuity of. The data suggests that Cryar-FGD possesses a bifunctional role where it has both isomerase and dehydrogenase activity.
In Chapter 4, we report the characterization of FGD from Nocardioidaceae brasiliensis (Noca-FGD), which like Cryar-FGD utilizes multiple sugar phosphates as substrates. However, the enzyme follows a different mechanism. We used a combination of steady-state and pre steady-state kinetics along with NMR spectroscopy to examine the enzyme activity with G6P, F6P, M6P, and glucose-1-phosphate (G1P). These data suggest that Noca-FGD is most efficient with G6P, followed by F6P and M6P, with activity trends mirroring this order. The pre steady-state data reveal a rapid chemistry step followed by slower product, with no detectable accumulation of intermediates. The NMR analysis indicates that Noca-FGD exhibits both dehydrogenase and isomerase activity. However, there is no evidence of the enediol formation as seen with Cryar-FGD. The data supports a mechanism in which the lactone products are released directly, with subsequent hydrolysis occurring nonenzymatically. These results establish that Noca-FGD is mechanistically distinct from both Mtb-FGD and Cryar-FGD, revealing unexpected catalytic diversity amongst the FGD family of enzymes and highlighting the functional flexibility of F420-dependent enzymes.
Keywords
Binding, Steady-state kinetics, pre steady-state kinetics, Mechanism, Enzyme, F420-dependent enzymes, F420 cofactor
Disciplines
Biochemistry
License
This work is licensed under a Creative Commons Attribution-No Derivative Works 4.0 International License.
Recommended Citation
Aziz, Alaa, "Mechanistic Characterization of F420-Dependent Glucose-6-Phosphate Dehydrogenases: Identification of a Catalytic Dyad in FGD from Mycobacterium tuberculosis and Isomerase Activity in FGD from Cryptosporangium arvum" (2026). Chemistry & Biochemistry Dissertations. 10.
https://mavmatrix.uta.edu/chemistry_dissertations2/10