Quinolinate metabolism enzymes

Details Quinolinate metabolism enzymes Quinolinate metabolism enzymes

1999-08-09

2001-08-07

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Plant cell or cell line, per se ; composition thereof;...

C435S189000, C435S252330, C435S320100, C536S023200

Reexamination Certificate

active

06271031

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 enzymes involved in quinolinate metabolism in plants and seeds.
BACKGROUND OF THE INVENTION
The nicotinamide moiety of the nicotinamide coenzymes (NAD
+
and NADP
+
) is derived, in humans, from dietary nicotinamide, nicotinic acid, or the essential amino acids tryptophan or aspartate. Quinolinate is an intermediary in this pathway. L-aspartate oxidase is one of the two components of the quinolinate synthase complex, which is responsible for the conversion of L-asparate and dihydroxyacetone phosphate into quinolinate. L-aspartate oxidase (EC 1.4.3.16) catalyzes the FAD-dependent oxidation of L-aspartate to iminoaspartate, the intermediate that leads to the formation of quinolinate.
Quinolinic acid is a neurotoxin and has been shown to be present at high levels in the central nervous system of patients with certain diseases, such as AIDS, epilepsy, and meningitis. The enzyme quinolinic acid phosphoribosyltransferase provides the only route for quinolinic acid metabolism and is also an essential step in de novo NAD biosynthesis. Quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) is referred to as nicotinate-nucleotide phosphorylase (carboxylating), or quinolinate phosphoribosyltransferase (decarboxylating). This enzyme catalyzes the synthesis of nicotinic acid mononucleotide from quinolinic acid and 5-phosphoribosyl-1-pyrophosphate. The seven-stranded alpha/beta-barrel domain of quinolinic acid phosphoribosyltransferase is very similar in structure to the eight-stranded alpha/beta-barrel enzymes. The structure shows a phosphate-binding site that appears to be conserved among many alpha/beta-barrel enzymes including indole-3-glycerol phosphate synthase and flavocytochrome b2 (Eads, J. C. et al. (1997)
Structure
5:47-58). Nicotinate-nucleotide phosphorylase from human liver and brain has been cloned, sequenced and analyzed (Okuno, E. et al. (1988)
J. Biochem
. (Tokyo) 103:1054-1059.).
Nicotinate-nucleotide phosphorylase and L-aspartate oxidase have been widely studied in prokaryotes and in mammals but they have not yet been described in plants.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding quinolinate metabolic enzymes. Specifically, this invention concerns an isolated nucleic acid fragment encoding an L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding an L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding L-aspartate oxidase or nicotinate-nucleotide pyrophosphorylase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a quinolinate metabolic enzyme selected from the group consisting of L-aspartate oxidase and nicotinate-nucleotide pyrophosphorylase.
In another embodiment, the instant invention relates to a chimeric gene encoding an L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding an L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase, 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 L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase, 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 L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase; 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 L-aspartate oxidase or nicotinate-nucleotide pyrophosphorylase 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 L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase.
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 L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an L-aspartate oxidase or a nicotinate-nucleotide pyrophosphorylase, 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 L-aspartate oxidase or nicotinate-nucleotide pyrophosphorylase in the transformed host cell; (c) optionally purifying the L-aspartate oxidase or the nicotinate-nucleotide pyrophosphorylase expressed by the transformed host cell; (d) treating the L-aspartate oxidase or the nicotinate-nucleotide pyrophosphorylase with a compound to be tested; and (e) comparing the activity of the L-aspartate oxidase or the nicotinate-nucleotide pyrophosphorylase that has been treated with a test compound to the activity of an untreated L-aspartate oxidase or nicotinate-nucleotide pyrophosphorylase, thereby selecting compounds with potential for inhibitory activity.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.
Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
TABLE 1
Quinolinate Metabolic Enzymes
SEQ ID NO:
Protein
Clone Designation
(Nucleotide)
(Amino Acid)
Corn L-Aspartate Oxidase
p0118.chsay63r
1
2
Soybean L-Aspartate Oxidase
sr1.pk0068.c11
3
4
Wheat L-Aspartate Oxidase
wr1.pk0026.e3:fis
5
6
Tobacco Nicotinate-Nucleotide
tdr1c.pk002.j10
7
8
Pyrophosphorylase
Wheat Nicotinate-Nucleotide
w1m96.pk036.o11
9
10 
Pyrophosphorylase
Wheat Nicotinate-Nucleotide
wr1.pk0104.h5
11 
12 
Pyrophosphorylase
The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defmed in conformity with the IUPAC-IUBMB standards described in
Nucleic Acids Research
13:3021-3030 (1985) and in the
Biochemical Journal
219 (No. 2):345-373 (1984) which are herein incorporate

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