The role of osmolytes and crowders in differential binding of ligands to dihydropteroate synthase: A glimpse into how these molecules can affect the function of an enzyme
Authors: Nambiar Deepika K , Sharma Ojaswini , Duff Michael , Howell Elizabeth 
Published: June 2021

It is intriguing to think about how an enzyme functions inside a crowded, complex cell and what parameters influence its activity. In our quest to understand complicated solutions, we found that small molecule osmolytes and crowders affect the enzyme activity in vitro as well as in vivo. Our previous in vitro studies have shown that osmolytes can weakly interact with the ligand, dihydrofolate, shifting the equilibrium towards the free enzyme, dihydrofolate reductase. We have also seen that when E. coli were challenged by osmotic stress leading to production of osmoprotectants and increased crowding in vivo, there is a reduction in the enzyme activity.

The interaction of several of these osmolytes with small molecules containing different functional groups has been studied and atomistic preferential interaction coefficients have been derived. A closer look into these coefficients has shown that certain functional groups have contrasting effects with different osmolytes. For example, trehalose prefers to interact with phosphate groups whereas betaine prefers to exclude them. Why is it important to study these weak differential interactions? Under osmotic stress, E. coli prefers glycine betaine as the major osmolyte. However, trehalose is the major osmolyte under nutrient limiting conditions. Thus, the osmolyte composition changes in the cell depending on its extracellular environment. The resulting weak interactions between osmolyte and folate derived molecules are predicted to influence the behavior of enzymes in the folate pathway.

To understand the effect an osmolyte might have on a folate pathway ligand with phosphate groups, we have calculated the preferential interaction coefficients for the ligands of dihydropteroate synthase (DHPS). DHPS catalyzes the condensation of 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphate (H2PtPP) and p‐aminobenzoic acid (pABA) to form dihydropteroate. The prediction is that trehalose prefers to interact with pteridine pyrophosphate (PtPP) whereas betaine prefers to exclude it. We used ITC to study binding of PtPP and pABA to DHPS. We find weaker binding (0.5 fold) of PtPP to DHPS in the presence of trehalose whereas tighter PtPP binding (6 fold) occurs in the presence of betaine. Conversely, for the case of pABA, a 0.3 fold decrease in Kd occurred upon addition of trehalose whereas a 1.3 fold increase in Kd occurred in upon addition of betaine.

Addition of osmolytes or crowders will also influence the viscosity of the buffer. The change in binding due to the presence of osmolytes/crowders was found not to be dependent on the viscosity of the buffer. Our studies thus shed light on how weak, “quinary” interactions between molecules can affect ligand binding and affect enzyme function. It is of great significance to understand how macromolecules work within a crowded cellular environment. This furthermore may help us gain insights into how effectively drugs will work inside a cell.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.