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
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/29747 |
| Date | 11 March 2005 |
| Creators | Fernandes, Roberta P. M. |
| Contributors | Proteau, Philip J. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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