Intellectual Merit: Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using the cofactor, NADPH. Because of the pivotal role of THF in one carbon metabolism, chromosomal DHFRs are targeted by many drugs, including trimethoprim. Bacterial resistance to trimethoprim occurs; one mechanism is production of a novel, R-plasmid encoded (R67) DHFR, which is not homologous in sequence or structure with chromosomal DHFRs. R67 DHFR is interesting as it is one of the smallest enzymes known to self-assemble into an active homotetramer. This DHFR variant possesses 222 symmetry and a single active site pore, which binds both substrate and cofactor utilizing a promiscuous surface. Binding and catalysis in R67 DHFR operate under Catch 222 conditions due to the symmetry in the pore, which allows each binding surface to interact with either DHF or NADPH. Also any catalytic group that might help catalysis will appear four times due to the 222 symmetry, also suggesting a non-optimal active site configuration. From these and other considerations, R67 DHFR appears to be a good model of a primitive enzyme. A study of how this enzyme works will be undertaken using steady state kinetics, isothermal titration calorimetry, directed evolution and other techniques.
Water has recently been found to play a critical role in R67 DHFR function as there is a net uptake of water upon DHF binding, as probed by osmotic stress studies. Requiring water to serve as a co-substrate results in the enzyme being sensitive to water activity. This hypothesis is confirmed in vivo by the decreased abilities of mutant R67 clones to confer trimethoprim resistance in the presence of added osmolytes. To probe the mechanism by which water uptake occurs, the effect of neutral osmolytes on free ligand will be examined. Alternatively, to determine if osmolytes operate on bound DHF due to a poor fit between substrate and enzyme, several mutations will be used to decrease the pore size. To study any contribution of water reorganization on the enthalpy change associated with DHF binding, D2O will be used as the solvent and isothermal titration calorimetry used to monitor the thermodynamics of the interaction. Solvent isotope effects on primary NADPH kinetic isotope effects will be monitored to determine whether protonation is concerted or precedes hydride transfer. As hydrostatic pressure typically results in increased hydration, the effect of hydrostatic pressure on R67 catalytic efficiency will be monitored. A specific role for substrate assisted catalysis will be probed by modification of the carboxyl groups in the substrate tail. Finally, using mutant R67 DHFR genes as well as an in-frame tandem array of four R67 DHFR genes, a directed evolution approach will be taken to select for mutants with increased catalytic efficiency and/or binding specificity. The results gained will detail catalytic strategies associated with a primitive enzyme as well as provide information on how enzymes evolve.
Broader Impact: This research project will provide undergraduate, graduate and postdoctoral researchers with the opportunity to gain a broad background in modern biochemistry and chemistry. This will readily allow them to secure future positions in biochemistry, chemistry, structural biology, drug design and other related fields. More specifically, the PI will continue to judge at local scientific venues, including the TN Jr. Science and Humanities symposium and the Southern Appalachian Science and Engineering Fair; these venues promote science among middle to high school students and feature oral or poster presentation of research. At the undergraduate level, the PI will continue to involve students in research and include them as coauthors on publications. The PI has involved minorities in her research program as well as a hearing disabled graduate student. The PI will maintain her reading of science texts for Recording for the Blind and Dyslexic. This proven track record will be maintained in the future as well. Further, UT Knoxville does a good job of minority student retention, with an overall retention rate of 60%, which is well above the national average of 35%.
Our research studies how the enzyme dihydrofolate reductase (DHFR) works. An enzyme catalyzes a reaction and this particular enzyme changes a double bond to a single bond using a cofactor (helper molecule) named NADPH. DHFR is important in folate (vitamin B9) metabolism. When DHFR activity is inhibited, the cell dies, thus DHFR is a target for anticancer drugs such as methotrexate and antibacterial drugs such as trimethoprim. There are two types of DHFR, one encoded by the chromosome, and another carried by a plasmid (extra-chromosomal element). The first enzyme type is the target for the antibacterial drug trimethoprim and the second enzyme type confers resistance to the drug. In this grant, we studied how water affects substrate and cofactor binding in a type II DHFR named R67 DHFR. Water is ubiquitous but its effects are generally ignored in scientific experiments. One way to study the role of water is to vary water concentration by adding small molecules to the solution. The molecules take up space and lower the water concentration. We find that low water concentrations favor cofactor binding to R67 DHFR, while low water concentrations disfavor substrate, dihydrofolate (DHF), binding. (Note: the concentration of pure water is 1.0. Once molecules are added to water such as in coffee, tea or soda, the water concentration decreases.) We also observe when different small molecules (osmolytes) are added to the solution that cofactor binding is affected to the same extent. This behavior suggests a preferential exclusion model where water prefers to interact with the protein and cofactor surface while small osmolytes are excluded. In contrast, binding of dihydrofolate is sensitive to the identity of the added osmolyte; this result is called preferential interaction, where molecule 1 (such as sucrose) interacts well with the protein while molecule 2 (such as dimethylsulfoxide) doesn’t interact as well and produces a smaller effect. Since R67 DHFR uses the same (promiscuous) interaction surface to bind both DHF and cofactor, these are puzzling results. How can the same binding surface provide opposite results? We come to the proposal that the difference must come from DHF, that it is a sticky molecule, allowing osmolyte interaction. We have tested this hypothesis by redoing the binding experiments using the type I DHFR, which possesses an entirely different protein structure. We again find weaker binding of DHF to chromosomal DHFR in the presence of osmolytes and tighter binding of NADPH, consistent with our model. While water is ubiquitous and occurs at high concentration, it is often ignored. Our experiments in the lab (in vitro) typically use high water concentrations. In contrast, our experiments in bacteria (in vivo) take place under conditions where the concentration of water is decreased due to the presence of high concentrations of molecules in the cellular environment. Thus our hypothesis of weak osmolyte interaction with folate/DHF is novel. High osmolyte concentrations can exist in the mammalian kidney, some plants, cartilaginous fish, bacteria, etc), and our model predicts varying osmolyte concentration will impact function. We propose that weak interactions are unavoidable in the cell due to intracellular crowding. In other words, our studies address the basic question of whether the in vitro behavior of folate and its derivatives accurately reflects their behavior in vivo. (An analogy considers how easy it might be to cross Times Square at midnight on New Year’s Eve (crowded condition as inside a cell) vs. walking in the desert on a cold morning in late February (not crowded experiment as in the test tube)? It is important to understand how weak interactions occur as they will impact function in the cell. While specific interactions are productive, it is generally thought that nonspecific (weak) interactions will compete and be nonproductive. For example, if 100 dihydrofolate molecules are present in the cell and 40 are stuck to surfaces, then only 60 are left to do their job. (An analogy might be if someone is at work for 8 hrs/day but spends 3 hrs on email/phone, then there is less work done.) The broader impacts of this research are diverse. Numerous undergraduates have been engaged in the above research. Two students were involved this year, while last year involved 4 students. One honors undergrad won the Chancellor’s award for academic achievement, was a top 10 graduate in the Natural Sciences Division, College of Arts & Sciences, was a first author on one paper and a coauthor on three other papers. Several other undergrads have also been co-authors on publications. All students have continued in scientific careers. Pairing undergrads with graduate students/postdocs provides mentorship to the undergrad as well as facilitates the teaching & organizational skills of the more advanced students. Students receive training in various biochemical disciplines, including basic enzymology, fluorescence, calorimetry, cloning, etc. This training provides a solid grounding for future positions.
