In vivo and in vitro approaches to studying the effect of osmolytes on enzymes of the folate pathway
Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Elizabeth Fozo, Daniel Roberts, Pratul Agarwal
The cell is a heterogenous, complex environment, while macromolecules are usually studied in dilute solutions. Our lab, as well as many others, has identified several types of weak, solvating,“quinary” interactions that change the behavior of enzymes. One group of molecules that participate in weak interactions are osmolytes. Osmolytes are small molecules involved in osmoregulation in the cell. Previous in vitro studies have identified that osmolytes, including trehalose, preferentially interact with folate, thereby reducing the association of the ligand to E. coli chromosomal and R67 plasmid dihydrofolate reductases. We have employed vapor pressure osmometry to study the weak interactions of trehalose with different atom types on folate. The study indicated trehalose preferentially interacts with phosphate and amide oxygens, but is preferentially excluded from the other atom types. We were able to calculate the predicted preferential interaction coefficient of trehalose with folate and these results concurred with our experimental results. The effect of different osmolytes on the affinity of PtPP and pABA for dihydropteroate synthase was measured byisothermal titration calorimetry. Trehalose and sucrose weakened binding of PtPP to DHPS, and tightened pABA binding. The rest of the osmolytes had opposite effects on ligand binding. The data suggested this pattern was due to the preferential interaction of trehalose with PtPP and exclusion from pABA. For the other osmolytes, there was preferential exclusion from pABA,but preferential interaction with PtPP. Weak osmolyte interactions with ligands weakened their binding to DHPS.We employed an osmotic stress approach to understand the role and relevance of these weak interactions between osmolytes and ligands in vivo. The activities of the enzymes R67 dihydrofolate reductase, methylenetetrahydrofolate reductase, serine hydroxymethyl transferase and chorismate mutase could be titrated by increasing osmotic stress. One reason for titration of enzyme activity was that the osmolytes produced in the cell participated in weak interactions with reduced folates, thereby preventing their association to their respective enzyme. Lesser product formation led to decreased cell growth in these auxotrophic cells. The titration of enzyme activity the cell with osmotic stress indicates that the weak interactions between osmolytes in vitro may also occur in vivo.
Nambiar, Deepika, "In vivo and in vitro approaches to studying the effect of osmolytes on enzymes of the folate pathway. " PhD diss., University of Tennessee, 2019.