Structure-Function of Dual NAD-Kinase/NADP-Phosphatase Enzymes from Extremeophiles
Haneen Alkabbani
Department of Chemistry and Biochemistry
Faculty Supervisor: Eric Greene
Organisms maintain distinct pools of reducing equivalent molecules in the cell where NADH is associated with anabolic reactions (energy harvesting) and NADPH is associated with catabolic reactions (energy utilizing). Often, these pools need to be altered for adaptation to new cellular conditions or when engineering hosts to produce (energy utilizing) high level of high value compounds. This project focuses on the expression and characterization of a dual function NAD kinase/NADP phosphatase enzyme that can directly alter levels of NAD(H) and NADP(H) in the cell. To achieve this, we designed and cloned expression plasmids bearing synthesized genes. We expressed the bifunctional enzymes in E. coli and purified each to homogeneity and kinetic characterization is underway. Future directions include mutation studies and cryoEM structural determination to further elucidate enzyme function and adaptation mechanisms. The structure-function relationships forged herein will represent the first for this unique class of enzymes that are the only known to directly rebalance NAD(P)(H) pools in the cell and offer a promising avenue to manipulating reducing equivalents towards bioengineering efforts.