Plant isocitrate dehydrogenase homologs

Details Plant isocitrate dehydrogenase homologs Plant isocitrate dehydrogenase homologs

1998-11-19

2001-03-20

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S006120, C435S069100, C435S410000, C435S325000, C435S252300, C435S091200, C435S320100, C435S025000, C530S333000

Reexamination Certificate

active

06204039

ABSTRACT:

FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding AND+ isocitrate dehydrogenase in plants and seeds.
BACKGROUND OF THE INVENTION
AND+ isocitrate dyhdrogenase (AND+ IDH; EC 1.1.1.41) is a key step in the citric acid cycle and catalyzes the oxidative decarboxylation of isocitrate to form &agr;-ketoglutarate, CO
2
, and NADH. Its key regulatory role in the TCA cycle is well documented. Traditionally the enzyme was also considered to be responsible for the production of 2-oxoglutarate which is a precursor in ammonia assimilation and amino acid biosynthesis (Bray (1983) Nitrogen Metabolism in Plants. Longman, London); however, NADP+ IDH (EC 1.1.1.42) has recently been regarded as an alternative pathway when large quantities of 2-oxoglutarate are required (Chen et al. (1990a)
Plant Physiol Biochem
28:141-145; Gàlvez et al. (1995)
Plant Sci
105:1-14).
AND+ IDH is localized exclusively in the mitochondria in association with the TCA cycle. This enzyme has been purified from several nonphotosynthetic eukaryotes such as fungi (Keys et al. (1990)
Bacteriol
172:4280-4287; Alvarez-Villafañe et al. (1996)
Biochemistry
35:4741-4752) and animals (Giorgio et al. (1970)
J. Biol Chem
245:5469-5477), in which it appears to be a 300-kD octamer. AND-IDH cDNAs have been cloned from yeast (Cupp et al. (1991)
J. Biol Chem
266: 22199-22205) and animals (Nichols et al. (1995)
Biochem J
310:917-922). In these organisms, the enzyme is composed of two (yeast) or more (animals) different subunits encoded by different genes.
Accordingly, the availability of nucleic acid sequences encoding all or a portion of an isocitrate dehydrogenase would facilitate studies to better understand carbon and nitrogen metabolic pathways in plant cells and provide genetic tools to enhance or otherwise alter these pathways which in turn could provide mechanisms to modulate the citric acid cycle and possibly ammonia assemilation in plant cells. Additionally, the instant isocitrate dehydrogenase proteins can be used as a targets to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding an enzyme of the citric acid cycle. Specifically, this invention concerns an isolated nucleic acid fragment encoding an isocitrate dehydrogenase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding an isocitrate dehydrogenase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of an isocitrate dehydrogenase. In another embodiment, the instant invention relates to a chimeric gene encoding an isocitrate dehydrogenase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding an isocitrate dehydrogenase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding an isocitrate dehydrogenase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of an isocitrate dehydrogenase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an isocitrate dehydrogenase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of isocitrate dehydrogenase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding an isocitrate dehydrogenase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of an isocitrate dehydrogenase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an isocitrate dehydrogenase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of isocitrate dehydrogenase in the transformed host cell; (c) optionally purifying the isocitrate dehydrogenase expressed by the transformed host cell; (d) treating the isocitrate dehydrogenase with a compound to be tested; and (e) comparing the activity of the isocitrate dehydrogenase that has been treated with a test compound to the activity of an untreated isocitrate dehydrogenase, thereby selecting compounds with potential for inhibitory activity.


REFERENCES:
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Keys et al., (1990), Bacteriol., 172:4280-4287.
Nichols et al., (1995), Biochem J., 310:917-922.
Alvarez-Villafane et al., (1996), Biochemistry, 35:4741-4752.
Galvez et al., (1995), Plant Sci., 105:1-14.
Chen et al., (1990), Plant Physiol. Biochem., 28:141-145.
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Cupp et al., (1991), J. Biol. Chem., 266:22199-22205.*
Martinez-Rivai et al., (1998), Plant Physiol., 118:249-255.

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