Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-08-09
2002-06-11
Prouty, Rebecca E. (Department: 1652)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S006120, C435S183000, C435S194000, C435S410000, C435S419000, C435S320100, C435S252100, C435S252300, C435S254200, C536S023100, C536S023200, C536S023600, C800S295000
Reexamination Certificate
active
06403859
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 proteins involved in vitamin B6 metabolism in plants and seeds.
BACKGROUND OF THE INVENTION
Vitamins are organic nutrients required in small quantities for a variety of biochemical functions. Vitamins, generally, can not be synthesized by the body and must therefore be supplied by the diet. Besides being water soluble, vitamins in the B complex have little in common from the chemical point of view. Excess B vitamins are rarely accumulated or stored and thus must be provided regularly.
Vitamin B1 (thiamin) and vitamin B6 (pyridoxine, pyridoxal, and pyridoxamine) are essential for animal nutrition. Vitamin B6 consists of three closely related pyridine derivatives: pyridoxine, pyridoxal, and pyridoxamine. All forms of vitamin B6 are absorbed from the intestine, but some hydrolysis of the phosphate esters occurs during digestion. In the presence of magnesium ions and using ATP as a cofactor, pyridoxal kinase (EC 2.7.1.35) converts pyridoxine to pyridoxine 5′-phosphate. At least two pyridoxal kinases involved in the salvage pathway of vitamin B6 biosynthesis have been identified in
Escherichia coli
(Yang et al. (1998)
J. Bacteriol
. 180:1814-1821). Pyridoxine 5′-phosphate is converted to pyridoxal 5′-phosphate by the action of pyridoxamine 5′-phosphate oxidase (EC 1.4.3.5). Pyridoxal 5′-phosphate is the major form of B6 transported in plasma. Pyridoxal phosphate is used in transamination and is an integral part in the enzymes which breakdown glycogen. Studies in yeast and bacteria show that loss of function perturbs amino acid, fatty acid, and sterol metabolism (Lam, H. M. and Winkler, M. E. (1992)
Bacteriol
. 174:6033-6045; Zhao and Winkler (1995)
J. Bacteriol
. 177:883-891; Loubbardi et al. (1995)
J Bacteriol
. 177:1817-1823).
Thiamin and Vitamin B6 are present in almost all plant and animal tissues commonly used as foods, but the content is usually small. Accordingly, enzymes responsible for their biosynthesis are potential targets for future antibiotics, fungicides, and herbicides. Thus, a detailed understanding of the activation, structure, mechanism, kinetics, and substrate-binding properties of the vitamin B1 and vitamin B6 biosynthetic enzymes from plants (and other organisms) would aid in the rational design of chemical or other kinds of herbicides. Isolation and purification of the enzymes from plants would provide a valuable tool for the in vitro screening of inhibitors of vitamin B1 and vitamin B6 biosynthesis.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding enzymes involved in vitamin B6 metabolism. Specifically, this invention concerns an isolated nucleic acid fragment encoding a pyridoxal kinase or a pyridoxamine-phosphate oxidase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a pyridoxal kinase or a pyridoxamine-phosphate oxidase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding pyridoxal kinase or pyridoxamine-phosphate oxidase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a vitamin B6 metabolic enzyme selected from the group consisting of pyridoxal kinase and pyridoxamine-phosphate oxidase.
In another embodiment, the instant invention relates to a chimeric gene encoding a pyridoxal kinase or a pyridoxamine-phosphate oxidase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a pyridoxal kinase or a pyridoxamine-phosphate oxidase, 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 pyridoxal kinase or a pyridoxamine-phosphate oxidase, 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 a pyridoxal kinase or a pyridoxamine-phosphate oxidase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a pyridoxal kinase or a pyridoxamine-phosphate oxidase; 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 pyridoxal kinase or pyridoxamine-phosphate oxidase 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 a pyridoxal kinase or a pyridoxamine-phosphate oxidase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a pyridoxal kinase or a pyridoxamine-phosphate oxidase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a pyridoxal kinase or a pyridoxamine-phosphate oxidase, 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 pyridoxal kinase or pyridoxamine-phosphate oxidase in the transformed host cell; (c) optionally purifying the pyridoxal kinase or the pyridoxamine-phosphate oxidase expressed by the transformed host cell; (d) treating the pyridoxal kinase or the pyridoxamine-phosphate oxidase with a compound to be tested; and (e) comparing the activity of the pyridoxal kinase or the pyridoxamine-phosphate oxidase that has been treated with a test compound to the activity of an untreated pyridoxal kinase or pyridoxamine-phosphate oxidase, 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
Vitamin B6 Metabolic Enzymes
SEQ ID NO:
(Nucleo-
(Amino
Protein
Clone Designation
tide)
Acid)
Corn Pyridoxal Kinase
Contig of:
1
2
cc1.mn0001.h9
cr1n.pk0161.c12
p0004.cb1hc14r
p0038.crvae17r
Rice Pyridoxal Kinase
rlr6.pk0096.a8
3
4
Soybean Pyridoxal Kinase
Contig of:
5
6
sgc6c.pk001.o1
srm.pk0038.g4
Wheat Pyridoxal Kinase
Contig of:
7
8
wdk4c.pk006.119
wl1n.pk0106.e8
Corn Pyridoxamine-
Contig of:
9
10
Phosphate Oxidase
cbn10.pk0048.g12
cpd1c.pk007.15
cr1n.pk0063.f3
csi1n.pk0050.f8
p0010.cbpcs54r
p0072.comfr39r
p0072.comfu19r
Rice Pyridoxamine-Phosphate
Contig of:
11
12
Oxidase
rlr48.pk
Cahoon Rebecca E.
Falco Saverio Carl
Rafalski J. Antoni
E. I. du Pont de Nemours and Company
Hutson Richard
Prouty Rebecca E.
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