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Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Dr. Allen M. Orville, Committee Chair ; Dr. Loren D. Williams, Committee Member ; Dr. Dale E. Edmondson, Committee Member ; Dr. Frank E. Lffler, Committee Member ; Dr. Nicholas V. Hud, Committee Member

Nitrochemicals are currently widely used as solvents, drugs, biocides, fuels and explosives and are consequently widely distributed in the environment. The reductive nitrite elimination from explosive compounds is catalyzed by two FMN-dependent, xenobiotic reductases (XenA or XenB). These genes for these regiospecific enzymes were cloned from Pseudomonas putida and P. fluorescens I-C respectively and isolated from the soil of a contaminated World War II munitions manufacturing plant. These enzymes enable the microbes to fulfill their nitrogen requirements from nitroglycerin by catalyzing the regiospecific, NADPH dependent, reductive denitration of nitroglycerin with differing selectivities. The two enzymes also transform a number of additional nitrocompounds in vitro, e.g. TNT and metronidazole, a leading drug in the treatment of Helicobacter pylori, a causative agent of human ulcers. Single crystals were obtained for XenA and XenB and complete X-ray diffraction datasets have been collected and analyzed to better understand these characteristics. The 1.6 Å resolution structure of XenA reveals a dimer of β/α)₈-TIM barrels, but the 2.3 Å resolution structure for XenB is a monomer. The (β/α)₈-TIM barrel protein fold is the most common fold in the PDB. However, the XenA structure exhibits a unique, C-terminal domain-swapped topology. Thus a portion of each active site is comprised of residues from the neighboring monomer. To probe the reaction cycle, crystal structures of ligand complexes and the reduced enzyme have been refined. For example, our structure of the XenA-metronidazole complex shows that ligands bind parallel to the FMN si-face. Our 1.5 Å resolution structure for reduced XenA reveals an FMN isoalloxazine ring with an angle of ~165° along the N5-N10 axis. We have also generated models of the reduced enzyme-nitroglycerin complexes by molecular dynamics. The results with both XenA and XenB reveal differences in enzyme-ligand hydrogen bonding. These differences correlate remarkably well with the regiospecific differences observed for nitrite elimination from nitroglycerin and reduction of TNT by the two enzymes.

The activity of dihydrofolate reductase, a key enzyme in the de novo biosynthesis of thymidylate, purines, and some amino acids, had previously been found to be increased in a methotrexate-resistant mouse leukemia (L5178Y) cell line as compared to the activity in the parental methotrexate-sensitive cell line. The increased activity was composed of two forms of the enzyme, one of which, form 2, was highly insensitive to inhibition by methotrexate. Both forms were purified to near homogeneity and using the antibodies prepared against them, it could be demonstrated that the two forms are antigenically distinct. The increased dihydrofolate reductase activity present in the methotrexate resistant cells resulted from an overproduction of both forms of the enzyme due to the presence of abundant mRNA coding for these enzymes. An increase in the dihydrofolate reductase gene copy number could be demonstrated in the resistant cells. mRNA coding for form 1 and form 2 enzymes was greatly enriched by polysome immunoprecipitation and complimentary DNA (cDNA) was synthesized in vitro from these enriched mRNA molecules. Evidence was found for the presence of methotrexate-insensitive forms of dihydroflate reductase in the blast cells of three out of eight acute myelogenous leukemia patients, and in two (distinct from the above) of the eight patients the activity was significantly increased. In contrast to the overproduction of dihydrofolate reductase protein in the methotrexate-resistant mouse cell line, increased enzyme activity in a methotrexate-resistant human promyelocytic leukemia (HL-60) cell line could not be correlated with an increase in the enzyme protein. Furthermore, the amounts of dihydrofolate reductase mRNA and gene-dosages were similar in the parental metho-trexate-sensitive and methotrexate-resistant cells. The enzyme from the resistant cells differed significantly in some of its physical and kinetic properties from that present in the parental cells. An increase in dihydrofolate reductase activity resulting from a modification of the enzyme rather than gene amplification has not to date been reported in the literature and may present a novel mechanism of resistance to methotrexate. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate