Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Alaa Aziz - University of Texas at Arlington
Co-Author(s): Lindsay Davis, University of Texas at Arlington, Arlington TexasAna Alvarez, University of Texas at Arlington, Arlington TexasGhader Bashiri, The University of Auckland, Auckland 1010, New ZealandEdward N. Baker, The University of Auckland, Auckland 1010, New ZealandKayunta Johnson-Winters, University of Texas at Arlington, Arlington Texas
F420-dependent glucose-6-phosphate dehydrogenase (FGD), although initially discovered in Nocardia, has been extensively studied in Mycobacteria due to health relevance for extreme drug resistant forms of tuberculosis disease (TB). FGD catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, similar to the first step in the pentose phosphate pathway. Our research has focused on elucidating the catalytic mechanism of this enzyme. Previous pH profiles revealed that Glu109 served as the active site acid, while the functionality of His40, previously proposed to be the active site base, still remains unknown1,2. Here, we have continued our investigation of His40, while studying the role of the active site residue, Glu13 in search of which amino acid serves as the active site base. Using site-directed mutagenesis, we created several FGD variants of the beforementioned residues including Glu13Ala, Glu13Gln, His40Ala, and His40Gln. These variants were kinetically characterized using binding experiments, steady-state and pre steady-state kinetic methods, in addition to PROPKA calculations and pH dependence studies. The binding studies suggest that these conserved amino acids are important for the binding of F420 but are not involved in G6P binding. The pH profiles along with the PROPKA calculations suggest that Glu 13 and His40 function as a catalytic dyad, with His40 donating a proton to Glu 13. His 40 can then act as a base, abstracting a proton from G6P facilitation the reduction of the F420 cofactor. The pre steady-state kinetics studies suggest that hydride transfer is not rate-limiting in catalysis, while the global fitting of wtFGD reveal that the reaction follows a fast kinetic/slow product release mechanism with no observable intermediates. Future work will focus on fine-tuning the global analysis of wtFGD along with the FGD variants using Kintek Explorer to fully understand the role of each amino acid during catalysis3.
Funder Acknowledgement(s): I would like to thank the Department of Chemistry and Biochemistry at the University of Texas at Arlington for their support in this project. Dr. Kayunta Johnson-Winters for her guidance and advising. I also thank all current and previous lab members for their help. This work was supported by NIH Grant 1R15GM113223-01A1 (to KJW)
Faculty Advisor: Kayunta Johnson-Winters, firstname.lastname@example.org
Role: I performed the pre steady-steady experiments along with full global analysis of wild type and variants. In addition, I completed binding experiments using the stopped-flow spectrophotometer on wild type and variants.