Effect of osmotic stress on folate pathway enzyme.

Abstract

How enzymes behave in cells may differ from how they behave in the test tube. Previous in vitro studies of three different dihydrofolate reductases as well as biophysical studies of folate find that osmolytes interact weakly with folate and dihydrofolate thereby replacing water in the hydration shell at certain functional groups. Removal of the osmolyte from the solvation shell of dihydrofolate is more difficult than removal of water, which weakens the Kd value. To determine if this phenomenon occurs in vivo, osmotic stress titrations of several folate pathway enzymes were performed in E. coli. Knockout and rescued strains of folate pathway enzymes were made in E. coli and subjected to osmotic stress. The bacterium synthesizes osmolytes in response to osmotic stress. Comparison of the growth of the knockout and rescued strains of the folate pathway enzymes would give us a measure of the effect of osmotic stress on the particular enzyme activity. We have found that the knockout strain could grow to high osmolalities on supplemented media while the rescued strain stopped growing at lower osmolalities on minimal media. This growth pattern was observed for an R67 dihydrofolate clone rescuing a ΔfolA strain of E. coli, for a methylenetetrahydrofolate reductase clone rescuing a Keio ΔmetF strain and for a serine hydroxymethyl transferase clone rescuing a Keio ΔglyA strain. While many parameters may be involved in these osmotic stress titrations, math modeling of the folate pathway suggests that these results could be an effect of direct titration of the enzyme activity or may be due to domino/cascading effects. We have further explored the effect of osmotic stress on E. coli DH5α strain with the dihydropteroate synthase (DHPS) gene in a tunable plasmid. The ability of the enzyme to function and rescue DH5α cells in the presence of sulfamethoxazole was determined. This was done to study the effect of osmotic stress on the functioning of DHPS with respect to growth of the cells in minimum inhibitory concentrations of the drug. From our previous osmotic stress titrations, the in vivo enzyme activity can be titrated directly by osmotic stress or can be inhibited due to domino effects. We are studying the effects of osmolytes to DHPS by using Isothermal Titration Calorimetry. Thus, by combining in vitro and in vivo studies, we will be able to answer if the activity of DHPS is affected in vivo. This will help to bridge the gap between in vivo and in vitro studies with enzymes. Model of osmolyte interaction with DHF that results in weaker binding to DHFR. For enzyme assays in buffer, DHF binds tightly to DHFR (target enzyme) and water (blue) is released. Added osmolytes (magenta spheres) interact weakly with DHF. For DHF to bind to DHFR, both osmolytes and water need to be released. While these interactions are weak, if the osmolyte-DHF interaction is stronger than the water-DHF interaction, then binding to DHFR is weakened. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.