Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
2001-07-12
2004-01-20
Prouty, Rebecca E. (Department: 1652)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C435S026000, C435S069100, C435S325000, C435S419000, C435S252300, C435S320100, C435S190000, C435S469000, C435S470000, C435S410000, C435S468000, C536S023200
Reexamination Certificate
active
06680428
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 metabolism 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, 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. Tetrahydrofolate (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 convalently 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 purines ring of ATP is involved in histidine biosynthesis.
There are several enzymes involved in tetrahydrofolate metabolism five of which are, methylenetetrahydrofolate dehydrogenase (NADP+), 5,10-methylenetetrahydrofolate reductase, 3-methyl-2-oxobutanoate hydroxymethyltransferase, glutamate formyltransferase, or formyltetrahydrofolate deformnylase. Methylenetetrahydrofolate dehydrogenase (NADP+) is an oxidoreductase which acts on the CH—NH group of donors with NAD+ or NADP+ as acceptor. In eucaryotes it occurs as a trifunctional enzyme also having methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) and formyltetrahydrofolate synthase (EC 6.3.4.3) activity. In some prokaryotes it occurs as a bifunctional enzyme also having methenyltetrahydrofolate cyclohydrolase activity (EC 3.5.4.9). This trifunctional enzyme consists of two major domains: an aminoterminal part, containing the methylene-THF dehydrogenase and methenyl-THF cyclohydrolase activities and a larger formyl-THF synthetase domain.
5,10-Methylenetetrahydrofolate reductase (EC 1.7.99.5) (MTHFR) plays a role in the synthesis of methionine (West et al, (1993)
J. Biol. Chem.
268:153-160 and D'Ari et al. (1991)
J Biol. Chem.
266:23953-23958). S-adenosylmethionine (SAM) an important methyl group donor for many biosynthetic methylation reactions in plants. SAM is formed from methionine by SAM synthetase. Transfer of the methyl group from SAM to an acceptor molecule results in the formation of S-adenosylhomocysteine, which is then hydrolyzed to homocysteine. Methionine is regenerated from homocysteine by methyl group transfer from 5-methyltetrahydrofolate. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. The heavy demand in plant cells for methyl groups derived from SAM necessitate a rapid recycling of S-adenosylhomocysteine, and thus a heavy demand for 5-methyltetrahydrofolate produced by MTHFR.
3-Methyl-2-oxobutanoate hydroxymethyltransferase (EC 2.1.2.11) is the first enzyme in the pantothenate biosynthetic pathway. This enzyme catalyses the conversion of 5,10-methylenetetrahydrofolate and 3-methyl-2-oxobutanoate to tetrahydrofolate and 2-dehydropantoate. Pantothenate is a vitamin required in the diet of animals. It is used in the synthesis of coenzyme A, which in turn, is used in many important enzyme reactions in many pathways, e.g., fatty acid biosynthesis. The production of high levels fatty acids, which require coenzyme A for their synthesis, might be stimulated by production of higher levels of coenzyme A, which in turn would require increased production of pantothenate. Another use might be for the increased production of pantothenate in plants in order to purify this vitamin for sale.
Glutamate formyltransferase (EC 2.1.2.5) catalyses the transfer of a formyl group from 5-formyltetrahydrofolate to L-glutamate. This enzyme serves to channel one-carbon units from formiminoglutamate to the folate pool.
Lastly, formyltetrahydrofolate deformylase (EC 3.5.1.10) catalyses the formation of formate and tetrahydrofolate from 10-formyltetrahydrofolate and water. 10-Formyltetrahydrofolate is required in de novo purine biosynthesis and histidine biosynthesis.
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. Thus 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, 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 tetrahydrofolate metabolism that may be useful as herbicides.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding tetrahydrofolate metabolism enzymes. Specifically, this invention concerns an isolated nucleic acid fragment encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a tetrahydrofolate metabolism enzyme selected from the group consisting of 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase and methylenetetrahydrofolate dehydrogenase.
In another embodiment, the instant invention relates to a chimeric gene encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate 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 a 3-methyl-2-oxobutanoate hydroxymethyltransferase, formyltetrahydrofolate deformylase, glutamate formiminotransferase or methylenetetrahydrofolate dehydrogenase, operably linked to suitable r
Falco Saverio Carl
Famodu Layo O.
Rafalski J. Antoni
E. I. du Pont de Nemours and Company
Ramirez Delia
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