This dissertation details the investigation of an alternate pathway to isoprenoids that occurs in plants and microorganisms, the non-mevalonate pathway. This exploration of the pathway focuses on the second step, the conversion of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methylerythritol-4-phosphate (MEP) by the enzyme DXP isomeroreductase (DXR). These studies led to an appreciation of the stereochemical course of the enzymatic reduction step and to a better understanding of the structural requirements for inhibitors to have optimal interactions at the active site of the enzyme, DXR. The investigation of the reduction step mediated by DXR revealed that the C1 pro-S hydrogen in 2-C-methylerythritol-4-phosphate derives from C3 of DXP for the DXR from Synechocystis sp PCC6803. The pro-R hydrogen originates from NADPH. The pro-S hydride of NADPH is transferred to the re face of the proposed aldehyde intermediate, which designates DXR as a class B dehydrogenase. Based on the structural features of fosmidomycin, a known inhibitor of DXR, several analogs were synthesized and evaluated for their inhibitory activity against DXR. It was discovered that a polar head group with two ionizable groups, a suitable length of intervening carbons, and an N-acyl N-hydroxy moiety are important factors to demonstrate significant inhibition activity. These studies also provided information that is complementary to structural data obtained from recent X-ray crystal structures of DXR. / Graduation date: 2003

No description available.

Dissertation (Ph.D.)--Auburn University, 2007. / Abstract. Includes bibliographic references (p.147-156).

Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2007. / "A dissertation in molecular biology and biochemistry and cell biology and biophysics." Advisor: Henry M. Miziorko. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Sept. 12, 2008. Includes bibliographical references (leaves 109-126). Online version of the print edition.

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

Graduation date: 2004

Thesis (M.S.)--University of Akron, Dept. of Polymer Science, 2006. / "December, 2006." Title from electronic thesis title page (viewed 12/31/2008) Advisor, Judit E. Puskas; Faculty Reader, Coleen Pugh; Department Chair, Mark D. Foster; Dean of the College, Frank N. Kelley; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

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

A systematic study of highly branched isoprenoids (HBI) was carried out in suspended particulate material (SPM) and Washington coastal sediments to determine their origin and fate. SPM collected at 10 m depth was filtered through Nitex membranes. C₂₅ HBI were found only in the 1.2-40 μm range over the shelf. The particle size fractionation of SPM shows different enrichment for HEH, a common hydrocarbon in phytoplankton, and the sum of C₂₅ HBI in the finer fractions suggesting these hydrocarbons do not share a common source. The distribution of C₂₅ and C₃₀ HBI correlates with the chlorophyll maxima suggesting an upper-water microbial source associated with phytoplankton biomass. It has been hypothesized that sulfur addition into specific biomarkers occurs during the early stages of diagenesis. Incorporation of the HBI into a refractory geomacromolecule via a sulfur linkage or formation of HBI-thiophenes are not evident in the sedimentary lipids. HBI show a rapid decrease in concentration with depth in both midshelf and slope sediments suggesting that biodegradation is the major pathway for their disappearance in Washington coastal sediments. Sediment cores from a midshelf and slope locations show the existence of suboxic/anaerobic conditions within the first 5 cm in the sediments. Elemental sulfur distribution in the midshelf appears to be controlled by bioturbation. On the slope, its profile indicates a quasi steady state regime. Phytane and phytenes (∑Phy) are the major products of Raney nickel desulfurization in both midshelf and slope sediments. Spinach and a strain of Emiliana huxleyi treated with Raney nickel showed strikingly similar patterns to the desulfurization products of sedimentary lipids. The amount of ∑Phy in the slope decreases abruptly by a factor of 6 in the top 2 cm and gradually increases with depth. These results are interpreted as phytyl coming from two sources: (1) chlorophyll-a and (2) S-bound to geomacromolecules. Partial released of phytyl moieties from chlorophyll-a warrants a reevaluation of Raney nickel as a selective desulfurizing agent before its application for paleoenvironmental reconstruction. Reducing micro-environments appear to exist within the bioturbated zone in shelf sediments. However, there is no clear evidence for phytyl moieties S-linked to macromolecules within the mixed layer. Results obtained during this study indicate that sulfur incorporation to biomarkers, although present, does not represent a significant mechanism for the preservation of organic carbon in normal marine sediments. / Graduation date: 1994

Simvastatin, a commonly prescribed statin, has exhibited several unexpected non-cholesterol lowering benefits. For example, patients taking statins have a decreased mortality rate due to bactereamia, a systemic bacterial infection commonly caused by Staphylococcus aureus (S. aureus). To investigate statin protection during bactereamia, human umbilical vein endothelial cells (HUVEC) were pre-treated with simvastatin followed by infection with S. aureus, and infection was significantly decreased. Simvastatin inhibits the cholesterol biosynthesis pathway. Therefore, the protective effect of simvastatin may be due to isoprenoid inhibition, specifically, farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (GGpp). Fpp and GGpp prenylate small G-proteins that function in cytoskeletal rearrangement and endocytosis. When Fpp and GGpp were replenished, infection was not significantly reduced. Furthermore, when farnesyl transferase and geranylgeranyl transferase, enzymes essential to transfer isoprenoid group during prenylation, were inhibited a significant decrease in infection was observed. The data indicates that Fpp and GGpp are essential for S. aureus infection. / Department of Biology