R67, The Other Dihydrofolate Reductase

University Of Tennessee Knoxville, Knoxville TN

Investigators

Abstract

Abstract 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%.