Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1999-12-17
2002-08-20
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S193000, C435S320100, C435S419000, C435S235100, C435S468000, C435S091200, C435S252300, C435S325000, C435S254110, C435S440000, C536S023200, C800S295000, C800S288000
Reexamination Certificate
active
06436657
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 tetrahydrofolate metabolic enzymes in plants and seeds.
BACKGROUND OF THE INVENTION
Tetrahydrofolic acid and its derivatives N
5
,N
10
-methylenetetrahydrofolate, N
5
,N
10
-methenyltetrahydrofolate, N
10
-formyltetrahydrofolate, and N
5
-methyltetrahydrofolate are the biologically-active forms of folic acid, a four-electron-oxidized form of tetrahydrofolate (THF). The tetrahydrofolates are coenzymes which are not enzyme-bound and are specialized cosubstrates for a variety of enzymes involved in one-carbon metabolism. THF is a 6-methylpterin derivative linked to p-aminobenzoic acid and glutamic acid residues. Its function is to transfer C1 units in several oxidation states. The C1 units are covalently attached to THF at its N5 and/or N10 positions and enter into the THF pool through the conversion of serine to glycine by serine hydroxymethyl transferase and the cleavage of glycine by glycine synthase. A C1 unit in the THF pool can have several outcomes: it may be used in the conversion of the deoxynucleotide dUMP to dTMP by thymidylate synthase, it may be reduced for the synthesis of methionine, or it may oxidized for the use in the synthesis of purines, since the purine ring from ATP is involved in histidine biosynthesis.
Serine hydroxymethylase, phosphoribosylglycinamide formyltransferase, phosphoribosylaminoimidazolecarboxamide formyltransferase, formate-tetrahydrofolate ligase and aminomethyltransferase are five enzymes involved in tetrahydrofolate metabolism. Serine hydroxymethylase (EC 2.1.2.1) is also called serine aldolase, glycine hydroxymethyltransferase or threonine aldolase. This enzyme catalyzes the conversion of 5,10-methylenetetrahydrofolate and glycine to tetrahydrofolate and L-serine. This enzyme is involved in multiple pathways such as glycine, serine and threonine metabolism, lysine degradation, cyano-amino acid metabolism and one carbon pool by folate and methane metabolism. In pea, two mitochondrial forms and a non-mitochondrial form of the enzyme are found. The mRNA appears to be expressed predominantly in leaves (Turner et al. (1992)
J. Biol. Chem.
267:13528-13534).
Phosphoribosylglycinamide formyltransferase (EC 2.1.2.2), also called GAR transformylase or 5′-phosphoribosylgycinamide transformylase is involved in the purine metabolism pathway and the one carbon pool folate. It is located in the chloroplast and catalyzes the conversion of 10-formyltetrahydrofolate and 5′-phosphoribosylglycinamide into tetrahydrofolate and 5′-phosphoribosyl-N-formylglycinamide. It is the third enzyme in the 10-step de novo purine biosynthetic pathway and its cDNA has been identified in
Arabidopsis thaliana
where it was shown to encode a single monofunctional enzyme (Schnorr et al. (1994)
Plant J.
6:113-121).
Phosphoribosylaminoimidazolecarboxamide formyltransferase (EC 2.1.2.3), also called 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, catalyzes the ninth step of the de novo purine biosynthesis pathway converting 10-formyltetrahydrofolate and 1-(5′-phosphoribosyl)-5-amino-4-imidazolecarboxamide to tetrahydrofolate and 1-(5′-phosphoribosyl)-5-formamido-4-inidazolecarboxamide. Two
Saccharomyces cerevisiae
genes encoding isozymes of AICAR transformylase have been described. Yeast is the only organism where two different isozymes have been identified (Tibbetts and Appling (1997)
Arch. Biochem. Biophys.
340:195-200).
Formate—tetrahydrofolate ligase (EC 6.3.4.3) is also called formyltetrahydrofolate synthetase or 10-formyltetrahydrofolate synthetase. In eukaryotes it occurs as a trifunctional enzyme also having methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) activities. It is involved in the glyoxylate and dicarboxylate metabolism pathways and one carbon pool by folate and folate biosynthesis. The first plant formate-tetrahydrofolate ligase has been purified from spinach leaves where it appears to be monofunctional and where it was found to be a dimer with a subunit molecular weight of 67,000 (Nour and Rabinowitz (1991)
J. Biol. Chem.
266:18363-18369).
Aminomethyltransferase (EC 2.1.2.10), is also called T-Protein of the glycine cleavage system, tetrahydrofolate aminomethyltransferase or S-aminomethyldihydrolipoylprotein (6S)-tetrahydrofolate aminomethyltransferase (ammonia-forming). It catalyzes the conversion of (6S)-tetrahydrofolate and S-aminomethyldihydrolipoylprotein to (6R)-5,10-methylenetetrahydrofolate, ammonia and Dihydrolipoylprotein. Aminomethyltransferase from pea has been purified to homogeneity and its cDNA identified. Using Northern blot analysis, a high steady state level of mRNA was found to accumulate in green leaves compared to etiolated leaves. The mRNA was also found in roots where the protein is detectable by Western blot analysis (Bourguignon et al. (1993)
Eur. J. Biochem.
217:377-386).
Because these enzymes are involved in tetrahydrofolate metabolism, amino acid synthesis, fatty acid biosynthesis and de novo synthesis of purines, inhibition of their activity may be lethal, thus suggesting that they would be attractive herbicide targets. Production of these plant enzymes in bacteria for use in a high throughput screen for chemical inhibitors would be desirable. Alternatively, overproduction of these enzymes in transgenic plants could be used to enhance the production of many secondary metabolites, amino acids, purine nucleic acids and vitamins. Accordingly, the availability of nucleic acid sequences encoding all or a portion of an enzyme involved in tetrahydrofolate metabolism would facilitate studies to better understand tetrahydrofolate metabolism in plants and provide genetic tools to enhance the production of secondary metabolites, amino acids and vitamins. These enzymes may also provide targets to facilitate design and/or identification of inhibitors of tetrahydrofolate metabolism that may be useful as herbicides.
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 100 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a serine hydroxymethylase polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 34, 36, 38, and 40. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 70 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a phosphoribosylglycinamide formyltransferase polypeptide selected from the group consisting of SEQ ID NOs:10, 12, 42, 44, and 46. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 70 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a phosphoribosylamino-imidazolecarboxamide formyltransferase polypeptide selected from the group consisting of SEQ ID NOs:14, 16, 48, 50, 52, and 54. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 70 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a formate—tetrahydrofolate ligase polypeptide selected from the group consisting of SEQ ID NOs:18, 20, 22, 24, 56, 58, 60, and 62. The present invention also relates to an isolated polynucleotide comprising the complement of the nucle
Famodu Omolayo O.
Orozco, Jr. Emil M.
E. I. du Pont de Nemours and Company
Prouty Rebecca E.
Steadman David
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