Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1999-11-24
2001-10-16
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S320100, C435S325000, C536S023200
Reexamination Certificate
active
06303365
ABSTRACT:
The invention relates to DNA which encodes for Arabidopsis 1-deoxy-D-xylulose-5 phosphate reductoisomerase, and to a method of identifying modulators of an enzyme with 1-deoxy-D-xylulose-5-phosphate reductoisomerase activity and of an enzyme with 1-deoxy-D-xylulose-5-phosphate synthase activity.
Undesired vegetation can be prevented by using herbicides. The demands made of herbicides have risen constantly with regard to activity, costs and ecofriendliness. There is therefore a need for new substances which can be developed into new potent herbicides. In general, it is normal to search for such new guide structures in greenhouse tests. However, such tests are laborious and expensive. The number of substances which can be tested in the greenhouse is, accordingly, limited.
The search is underway for plant-specific biosynthetic routes which constitute advantageous sites of action for herbicides and do not occur in animal organisms.
Recently, it has been found that plants synthesize essential plastid isoprenoids not by exploiting the mevalonate biosynthetic pathway which is found in the animal organism, but the microbial 1-deoxy-xylulose-5-phosphate biosynthetic pathway (Lichtenthaler, K. (1998), Fett/Lipid 100, 128-138; Eisenreich et al). (1998), Chemistry & Biology 5, R221-R233).
This biosynthetic pathway finally leads to the synthesis of, inter alia, carotenoids: and the side chains of plastoquinone and chlorophyll. These products are essential for the photosynthetic growth of plants, Inhibition of one step in this biosynthetic pathway entails the end of plant growth.
This is why the 1-deoxy-xylulose-5-phosphate biosynthetic pathway is of particular interest in the search for new herbicidally active compounds. In particular the two enzymes 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) and 1-deoxy-D xylulose-5-phosphate reductoisomerase (DXPR) are of central importance. It has already been demonstrated that DXPS (CLA I) is essential for the development of a normal plant (Mandel et al. (1996), Plant J. 9, 649-658). This discovery supports the expectation that a herbicidal compound which affects DXPS activity has a herbicidal action. Also, it has been demonstrated that bacterial DXFR is inhibited by the herbicidal compound Fosmidomycin, which is already known (Zeidler et al. (1998), Z. Naturforsch. 53, 980-986; Kuzuyama et al. (1998), Tetrahedron Lett. 39, 7913-7916). However, there are no commercially useable herbicides, which affect DXPS or DXPR activity. In the search for new, improved herbicides, both enzymes are therefore of high importance as sites of action. The 1-deoxy-xylulose-5-phosphate biosynthetic pathway also has importance in microorganisms, especially in parasitic microorganisms as, for example, bacteria or plasmodia. The treatment of infectious diseases, in particular the treatment of malaria, may be based on the inhibition of this metabolic pathway (Jomaa et al. (1999), Science 285, 1573-1576).
So far it has not been possible to determine the enzymatic activity of the enzyme 1-deoxy-xylulose-5-phosphate synthase in a simple test system since the reaction neither entails a measurable change in absorption nor can be coupled in a simple manner with a colour change or fluorescence change (FIG.
1
). The enzymatic activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase entails a measurable change in the optical absorption of the cosubstrate NADPH. However the substrate 1-deoxy-D-xylulose-5-phosphate can only be synthesized with difficulty (FIG.
1
). While 1-deoxy-D-xylulose-5-phosphate can be prepared via a chemical or biochemical route, both methods are expensive and are not well suited for use in test systems with high throughput (Taylor et al. (1998), J. Org. Chem. 63, 2375-2377; Blagg and Poulter (1999), J. Org. Chem. 64, 1508-1511).
The present invention solves these problems by combining the two enzymes in one test system. The
Arabidopsis thaliana
1-deoxy-D-xylulose-5-phosphate synthase gene is already known under the name CLAI (Mandel et al. (1996), Plant J.5. 649-658). The 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene is, as yet, known only from
Mentha piperita
(Lange et al. (1998), Proc. Nati. Acad. Sci. U.S.A. 5, 2100-2104) and from various microorganisms. The
A. thaliana
1-deoxy-D-xylulose-5-phosphate reductoisomerase has also been described and a fragment of the amino acid sequence has been published (Lange und Croteau (1999). Archives of Biochem. and Biophys. 365, 170-174). The combined reaction of pyruvate and glyceraldehyde-3-phosphate to give 2-C-methyl-D-erythrol-4-phosphate is monitored by visually detecting the NADPH consumption (FIG.
1
). The test system is suitable for the search for modulators of both enzymes, that is to say substances which inhibit or else stimulate the activity of the enzymes, and can be used for test series with high throughput (high-throughput screening, HTS). After detection of modulators by one of the two enzymes in an HTS system, the modulators of the two enzymes can be distinguished from each other by using the existing methods for measuring the activity of the two enzymes (Sprenger et al. (1997), Proc. Natl. Acad. Sci. U.S.A. 94, 12857-12862; Kuzuyama et al. (1998), Tetrahedron Lett. 39, 4509-4512; DE 197 52 700-A1).
The present invention relates to the DNA which encodes Arabidopsis 1-deoxy-D xylulose-5-phosphate reductoisomerase, in particular the
Arabidopsis thaliana
1-deoxy-D-xylulose-5-phosphate reductoisomerase, and to fragments of this DNA, which encode functional subunits of 1-deoxy-D-xylulose-5-phosphate reductoisomerase.
The invention furthermore relates to DNA which encodes Arabidopsis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, with an amino acid sequence as shown in SEQ ID NO 2or SEQ ID NO 6.
The invention furthermore relates to DNA as described under SEQ ID NO 1 or SEQ ID NO 5 which encodes the
Arabidopsis thaliana
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
The invention furthermore relates to DNA which shows 80%, preferably 90%, homology to the DNA described under SEQ ID NO 1 or SEQ ID NO 5 and which encodes plant 1-deoxy-D-xylulose-5-phosphate reductoisomerase.
The invention furthermore relates to DNA which is complementary to the DNA which encodes the
Arabidopsis thaliana
1-deoxy-D-xylulose-5-phosphate reductoisomerase, and to RNA which is complementary to the DNA which encodes the
Arabidopsis thaliana
1-deoxy-D-xylulose-5-phosphate reductoisomerase.
The invention furthermore relates to an expression construct which encompasses DNA which encodes the
Arabidopsis thaliana
1-deoxy-D-xylulose-5-phosphate reductoisomerase and is described under SEQ ID NO 1 or SEQ ID NO 5, and to a sequence which is functionally linked herewith and which allows the 1-deoxy-D-xylulose-5-phosphate reductoisomerase to be expressed.
The invention furthermore relates to a vector which comprises DNA which encodes the
Arabidopsis thaliana
1-deoxy-D-xylulose-5-phosphate reductoisomerase and/or is described under SEQ ID NO 1 or SEQ ID NO 5 and which allows the 1-deoxy-D-xylulose-5-phosphate reductoisomerase to be expressed in a host cell.
The invention furthermore relates to a host cell which comprises the abovementioned DNA, an expression construct as mentioned above, or a vector which allows the 1-deoxy-D-xylulose-5-phosphate reductoisomerase to be expressed.
The invention furthermore relates to the use of DXPR and/or DXPS modulators as herbicides , antibiotic agents or anti-malarial agents.
The invention also relates to the use of DXPR and/or DXPS modulators as lead structures for the chemical optimization and the development of improved modulators.
The invention also relates to a method of determining the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase and 1-deoxy-D-xylulose-5-phosphate synthase, which is based on combining the conversion of pyruvate and glyceraldehyde-3-phosphate to give I -deoxy-D-xylulose-5-phosphate by 1-deoxy-D-xylulose-5-phosphate synthase with the conversion of the resulting 1-deoxy-D-xylulose-5-phosphate to give 2-C-methyl-D-erythrol-4-phosphate by 1-deo
Busch Marco
Hain Ruediger
Kloti Andreas S.
Martin William Frank
Tietjen Klaus-Guenther
Biswas Sorojini J.
Majka Joseph T.
Monshipouri Maryam
Paradigm Genetics, Inc. R.T.P
Prouty Rebecca E.
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