Graduate Thesis Or Dissertation
 

Studies on 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Synechocystis sp. PCC6803 : characterization of mutants and inhibitors

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  • 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.
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