Masters Theses

Date of Award

12-1993

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Elizabeth E. Howell

Committee Members

Cynthia B. Peterson, Jorge Churchich

Abstract

R67 Dihydrofolate reductase (DHFR) is a plasmid encoded DHFR which confers a high level of resistance upon host bacterial cells to the antimicrobial drug trimethoprim (TMP). R67 DHFR shares no sequence or structural homology with the wildtype (wt.) E. coli chromosomal DHFR yet the two enzymes carry out identical reactions. The R-plasmid encoded enzyme is active as a homo-tetramer which is held together mainly by hydrophobic interactions which occur at the dimer-dimer interface. An interesting feature of R67 DHFR is that all four monomers share in the formation of the active site pore which is found in the center of the protein as an 18 A long pore with openings at both ends. The mechanism of R67 DHFR is not well understood; However it is thought to occur by binding a protonated substrate and the cofactor NADPH in the center of the active site pore.

To begin to determine R67 DHFR origins, and to investigate any functional similarity of R67 DHFR to wt. E. coli DHFR, substrate tolerance and ligand binding studies were performed. These studies show that R67 DHFR has a broader range of substrate tolerance and can bind more types of ligands than its chromosomal counterpart wt. E.coli DHFR. The results from this study also suggest that R67 DHFR could not have originated from any of the chromosomally encoded DHFRS, and possibility that R67 DHFR is a primitive enzyme which was elicited by bacteria to reduce a wide variety of substrates quickly.

This study also describes experiments which involve two genetically engineered R67 DHFRS, an artificially duplicated monomer, R67 DHFRdouble, and H62C R67 DHFR. Both of these enzymes have been engineered with the goal of breaking the unique symmetry of the enzyme and also to increase enzyme stability at lower pH. The results from these studies have shown that, in the case of the R67 DHFRdouble, we have shown that the engineered R67 DHFRdouble protein is as active as native R67 DHFR. The results from the studies of the H62C R67 DHFR protein show that the protein is kinetically and fluorometrically different from the native enzyme. H62C R67 DHFR has also been shown to possess the ability to utilize acetyl-pyridine NADP+ and NADPH to perform a transhydrogenase reaction, which is not present in the native R67 DHFR.

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