The discovery of the methylerythritol phosphate pathway (the MEP pathway) as an alternate pathway for isoprenoid biosynthesis in some organisms including most bacteria, malarial parasites and plants, but not in animals, has stimulated extensive studies in this area. Research has revealed the potential of finding novel antibacterials, antimalarial drugs, and herbicides from enzyme inhibitors of this pathway. The natural products fosmidomycin and FR900098 appear to be very promising antibacterial and antimalarial compounds. Both compounds have inhibition activities against the second enzyme in the MEP pathway, deoxyxylulose- 5-phosphate reductoisomerase (DXR), which mediates the conversion of deoxyxylulose-5-phosphate (DXP) into methylerythritol-4-phosphate (MEP). This thesis presents one aspect of the MEP pathway studies. Six different analogs of DXP were designed based on the structural features of DXP to understand the requirements of the DXR-substrate binding. Compounds with the trivial names 1-Me-DXP (containing an ethyl ketone moiety), DX-phosphonate (DXP having a phosphonate group rather than a phosphate group), 4-epi-DXP (possessing the opposite stereochemistry at the C��� position compared to DXP), 4-deoxy-DXP (lacking the hydroxyl group at the C��� position), 3-deoxy-DXP (lacking the hydroxyl group at the C��� position), and DXP carboxamide (having a primary amide group rather than the methyl ketone) were synthesized and tested as alternate substrates and enzyme inhibitors against DXR. The compound DX-phosphonate was the only alternate substrate among the synthesized compounds. The remaining analogs of DXP acted as weak competitive inhibitors against DXR. Kinetic studies of these compounds provided an overall picture of how the substrate DXP binds to DXR. Further studies of the compound 1-Me-DXP, using the published X-ray crystal structures of DXR and DXR mutagenesis demonstrated more detail of the DXR active site. The results present useful information for designing better enzyme inhibitors. The mechanism for the rearrangement of DXP to MEP by DXR was also studied. Two possible mechanisms for this rearrangement have been proposed, the ��-ketol rearrangement and the retroaldol/aldol rearrangement. Several approaches including the use of the potential alternate substrates, 4-deoxy-DXP and 3-deoxy-DXP were tried. Unfortunately none of the results obtained can definitively rule out either of the mechanisms. Further studies are needed to completely understand this mechanism and establish additional strategies for inhibition of DXR. Syntheses of an intermediate from the DXR reaction, methylerythrose-4-phosphate, were also attempted in order to better understand the chemistry mediated by DXR. Even though the target compound was not successfully obtained, several synthetic approaches to this compound were useful for the syntheses of the different DXP analogs mentioned above. / Graduation date: 2004

In recent years, the methyl erythritol phosphate (MEP) pathway to isoprenoids has been the subject of intensive research. The interest is because isoprenoids have important roles in many cellular processes essential for the survival of several pathogenic organisms, making the inhibition of this pathway an attractive target for the drug discovery. The second enzyme in the MEP pathway is 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). DXR is a promising target for the development of new antibiotics, antimalarials and herbicides. The overall objective of this research was a better understanding of DXR by using site-directed mutagenesis guided by crystal structure analysis and inhibition studies. One set of mutants was designed to expand the selectivity of DXR. An analog of DXP, 1,2-dideoxy-D-threo-3-hexulose 6-phosphate (1-methyl-DXP or Me-DXP), that differs from DXP by having an ethyl ketone, rather than a methyl ketone, was reported to be a weak competitive inhibitor. Using the x-ray crystal structures of DXR as a guide, a highly conserved tryptophan residue in the flexible loop was identified as a potential steric block to the use of this analog as a substrate. Four mutants of Synechocystis sp. PCC6803 DXR, named W204F, W204L, W204V and W204A, were prepared and characterized. The W204F mutant was found to utilize the analog Me-DXP as a substrate. The roles of amino acids residues shown to be in the DXR active site in the available E. coli crystal structures were also studied. Mutants at the positions Dl52, S153, E154, H155, M206 and E233, were prepared. The kinetic characterization of these mutants showed that the amino acid substitution, conservative or not, in these residues reduced the DXR catalytic activity, confirming that these are key amino acids responsible for the DXR catalytic efficiency. Inhibition studies of the E. coli DXR by fosmidomycin in the presence of Co²⁺, Mg²⁺ and Mn²⁺ showed that this inhibition is not dependent on a specific divalent cation. Inhibition of the Synechocystis sp. PCC6803 DXR by fosmidomycin and its hydroxamate and FR 900098 analogs was conducted showing that these compound are potent inhibitors of this enzyme. Fosmidomycin and FR900098 have inhibition constants in the low nM range. In addition the patterns of the progress curves for fosmidomycin, its hydroxamate analog and FR900098 were shown to be prototypical for slow, tight-binding inhibitors, as was seen for these inhibitors with the E. coli enzyme. / Graduation date: 2005

No description available.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 5 uppsatser.

Thesis (Ph.D.)--Baylor University, 2008. / In abstract the '50' in IC50 is subscript. Includes bibliographical references (p. 349-357).

Typescript (photocopy). / Digitized by Kansas Correctional Industries

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Master / Master of Dental Surgery

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences