Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2000-07-27
2001-12-04
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S189000
Reexamination Certificate
active
06326164
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to assays for measuring deoxyxylulose 5-phosphate synthase (DXPS) activity. The assays can be used to identify compounds that inhibit or enhance DXPS activity. Such compounds have use in modulating plant and microbial growth and development.
BACKGROUND OF THE INVENTION
Deoxy-D-xylulose 5-phosphate (DXP) is a common precursor of thiamin (vitamin B1), pyridoxyl (vitamin B6) and isoprenoids. Isoprenoids encompass a large family of biomolecules, including vitamins A, D, E and K, cholesterol, plant pigments such as carotenoids and the phytol chain of chlorophyll, natural rubber, many essential oils, plant hormones (gibererellins, abscisic acid), insect juvenile hormone, dolichols, quinone electron carriers in mitochondria and chloroplasts, such as ubiquinone and plastoquinone, structural components of membranes (phytosterols) and Ras protein. In higher plants and bacteria, the first step in the formation of isopentenyl diphosphate, the common precursor of all isoprenoids, by the mevalonate-independent pathway is the formation of DXP from the precursors pyruvate and glyceraldehyde 3-phosphate (FIG.
1
). The reaction is catalyzed by the enzyme deoxyxylulose 5-phosphate synthase (DXPS) (Lange et al. (1998)
Proc Natl Acad Sci
95:2100-2104; Lois et. al. (1998)
Proc Natl Acad Sci
95:2105-2110; Sprenger et al. (1997)
Proc Natl Acad Sci
94:12857-12862).
The DXPS genes or cDNAs from
E. coli
(GenBank AF035440),
Hemophilus influenzae
(Swiss-Prot P54205),
Rhodobacter capsulatus
(Swiss-Prot P26242), Synechocystus sp. PCC6803 (GenBank D90903),
Bacillus subtilis
(Swiss-Prot P54523),
Helicobacter pylori
(GenBank AE000552),
Mycoplasma tuberculosis
(GenBank Z96072),
Glycine max
(GenBank AW278762),
Lycopersicon esculentum
(GenBank AF143812),
Catharanthus roseus
(GenBank AJ011840),
Mentha x peperita
(GenBank AF019383) and
Arabidopsis thaliana
(GenBank AF010383 and 5281015) have been cloned. Also, ESTs encoding fragments of DXPS have been identified in
Oryza sativa, Ricinus communis,
and
Pinus taeda.
However, no homologues of the DXPS genes have been identified in animals.
Disruption of the DXPS gene in Arabidopsis results in an albino phenotype due to a lack of chlorophyll and carotenoid pigments. These results indicate that DXPS is essential for chloroplast function (Lange et al.) and that inhibitors of DXPS activity may have use as herbicides. Accordingly, it would be useful to have a DXPS assay that is amenable to high throughput screening of herbicide candidates.
Several assays for DXPS activity have been reported in the literature. These assays are based on detection of the product, DXP. In these assays, the conversion of [2-
14
C]-pyruvate to [
14
C]-DXP in the presence of glyceraldehyde 3-phosphate and DXPS was measured by detecting [
14
C]-DXP using either reverse phase HPLC (Lange, et. al.) or thin layer chromatography (Lois et al.). However, neither format is suitable for high throughput screening.
SUMMARY OF THE INVENTION
The invention is directed to methods and compositions for the determination of deoxyxylulose 5-phosphate synthase (DXPS) activity. The methods and compositions of the invention are amenable to high throughput screening assays for the identification of inhibitors and enhancers of DXPS activity. Such compounds have use in the modulation of plant growth and development.
The compositions of the invention are DXPS fragments and chimeric polypeptides that have increased solubility in cell extracts as compared to the wild type DXPS polypeptide. These DXPS fragments and chimeric polypeptides can be recombinantly expressed and purified in quantities suitable for high throughput screening assays.
The assays of the invention are based on the detection of substrates of DXPS that remain after a DXPS reaction. Specifically, the invention provides a method for determining deoxyxylulose 5-phosphate synthase activity, comprising:
a) contacting pyruvate and optionally, glyceraldehyde 3-phosphate, with a deoxyxylulose 5-phosphate synthase; and
b) determining the concentration of pyruvate and/or glyceraldehyde 3-phosphate remaining after the contact in step (a).
The assays of the invention are useful for the identification of modulators of deoxyxylulose 5-phosphate synthase activity. Thus, in another aspect, the invention provides a method for identifying modulators of deoxyxylulose 5-phosphate synthase activity, comprising:
a) contacting pyruvate and optionally, glyceraldehyde 3-phosphate, with a deoxyxylulose 5-phosphate synthase, in the presence and the absence of at least one candidate modulator; and
b) comparing the concentration of pyruvate and/or glyceraldehyde 3-phosphate remaining after said contact in the absence of said candidate modulator to said concentration in the presence of said candidate modulator.
REFERENCES:
patent: 98/33936 (1998-02-01), None
patent: 00/08169 (2000-02-01), None
Moran et al. “A rapid beta-NADH-linked fluorescence assay for lactate dehydrogenase in cellular death” (1996) J Pharmacol Toxicol Methods, 36:41-44.*
Singh et al. “A high performance liquid chromatography assay for threonine/serine dehydratase” (1992) Anal Biochem, 208:260-263.*
Eisenreich, W. et al., “The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms,” Chemistry & Biology, Current Biology Publications (Germany), p. R221-R223, (Sep., 1998).
Lange, B. M. et al., “A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway,” Proc. Natl. Sci. USA, The National Academy of Sciences (US), pp. 2100-2104, (Mar. 1998).
Lois, L. M. et al., “Cloning and characterization of a gene fromEscherichia coliencoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis,” Proc. Natl. Acad. Sci. USA, The National Acadamy of Sciences (US), pp. 2105-2110, (Mar. 1998).
Sprenger, G. et al., “Identification of a thiamin-dependent synthase inEscherichia colirequired for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol,” Proc. Natl. Acad. Sci. USA, The National Acadamy of Sciences (USA), pp. 12857-12862, (Nov., 1997).
Crawford, Jr. John M.
Kloti Andreas S.
Lanning Beth
Rice John W.
Stewart Sandy J.
Majka Joseph T.
Nowak Henry
Paradigm Genetics, Inc.
Prouty Rebecca E.
Sale Elaine T.
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