Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Reexamination Certificate
2000-12-19
2002-06-04
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C435S004000
Reexamination Certificate
active
06399352
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel enzymes for converting a compound or substrate known as aminolevulinate (ALA) to another compound or product known as prophobilinogen (PBG). The present invention also relates to novel DNA that encodes for such enzymes.
BACKGROUND OF THE INVENTION
Enzymes are important laboratory tools for discovering new agricultural or related compounds such as insecticides, herbicides, fungicides, nematocides, antimicrobials and the like. In plants, there is a key biochemical pathway in which a pyrrole ring-containing compound known as porphobilinogen (PBG) is produced from two acyclic or open-chain molecules known as aminolevulinate (ALA). PBG is a precursor or substrate for both heme and chlorophyll biosynthesis in plants. Plants employ an enzyme that can catalyze or convert ALA to PBG. Unfortunately, this native enzyme cannot be expressed so that it is active in a non-plant host, such as bacteria, since such expressions have been found to yield an insoluble and inactive enzyme. Thus, it became desirable to develop active, plant-based enzymes that could convert aminolevulinate to porophobilinogen and yet still remain soluble under laboratory conditions.
SUMMARY OF THE INVENTION
The present invention advantageously provides an active, plant-based protein or enzymes known as plant thioredoxin-porphobilinogen synthase (T-PPS) and plant porphobilinogen synthase (PPS) that can convert aminolevulinate to porophobilinogen and yet still remain soluble under laboratory conditions. The present invention has the further advantage of also providing DNA in a non-natural host such as bacteria, virus, yeast, etc. that will produce the desired protein or functional fragments thereof, outside of its native plant source.
Accordingly, in one embodiment, the present invention is directed toward DNA or polynucleotide characterized by
a) SEQ ID NOs: 1 or 7;
b) the complementary sequence thereof; or
c) the double stranded sequence of a) and b).
In a second embodiment, the present invention is directed towards DNA or polynucleotide characterized in that its homology to the sequence as shown in SEQ ID NOs: 1 or 7 is at least 80%. Preferably, the DNA or polynucleotide is characterized in that its homology to the sequence as shown in SEQ ID NOs: 1 or 7 is at least 90%.
In a third embodiment, the present invention is directed towards DNA or polynucleotide that encodes the total: or functional fragments of an amino acid sequence as shown in SEQ ID NOs: 2 or 8.
In a fourth embodiment, the present invention is directed towards RNA characterized in that it is complementary to the DNA of SEQ ID NOs: 1 or 7.
In a fifth embodiment, the present invention is directed towards an expression construct, characterized in that it encompasses DNA or polynucleotide described in the first, second or third embodiments and a sequence that is functionally linked thereto that allows the DNA or polynucleotide to be expressed.
In a sixth embodiment, the present invention is directed towards a plasmid characterized in that it contains DNA or polynucleotide described in the first, second or third embodiments.
In a seventh embodiment, the present invention is directed towards a protein or polypeptide represented by SEQ ID NO: 2, known herein as “Plant Thioredoxin-Porphobilinogen Synthase” (T-PPS) or a protein represented by SEQ ID NO: 8, known as “Plant Porphobilinogen Synthase” (PPS). Preferably, the protein is in a buffered composition.
In an eight embodiment, the present invention is directed towards a method of determining the enzymatic activity of the protein or polypeptide of SEQ ID NO: 2 (i.e. plant thioredoxin-porphobilinogen synthase) or SEQ ID NO: 8 (i.e. plant porphobilinogen synthase), characterized by contacting or converting &dgr;-aminolevulinic acid (a substrate) with said protein and measuring the amount of porphobilinogen (a product) formed therefrom. Preferably, the protein is in a buffered composition.
In a ninth embodiment, the present invention is directed toward a method of identifying a compound which can modify the activity of the protein or polypeptide of SEQ ID NOs: 2, 8 or a functional fragment thereof comprising contacting or converting &dgr;-aminolevulinic acid with said protein or polypeptide or a functional fragment thereof in the presence of a test compound and measuring the amount of porphobilinogen formed therefrom. Preferably, the protein or polypeptide is in a buffered composition. Also preferred is that said identified compound inhibits said protein or polypeptide or functional fragment thereof.
In a tenth embodiment, the present invention is directed towards a method of inhibiting plant growth comprising applying to a plant a compound which inhibits the enzymatic activity of plant thioredoxin-porphobilinogen synthase or plant porphobilinogen synthase. Preferably, the protein or polypeptide is in a buffered composition.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described more fully hereinafter with reference to the accompanying figures or drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The invention also relates to the use of substances which are found with the aid of the above-described method for use as herbicides.
“Buffer” or “buffered composition” refers to a solution in which a buffering agent has been added and which tends to prevent or resist rapid changes in pH upon the addition of small quantities of acid or base.
“Chemically synthesized,” as related to a sequence of DNA, means that the component nucleotides are assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established techniques in the art.
“Complementary” relates to the capability of purine and pyrimidine nucleotides to form base pairs with each other via hydrogen bonds. Complementary base pairs are, inter alia, guanine and cytosine, adenine and thymine, and adenine and uracil.
“DNA” or “polynucleotide” refers to deoxyribonucleic acid.
“Expression” or “expressing” refers to the transcription and/or in the case of a protein gene product, translation, of a heterologous or homologous gene to yield the gene product encoded by the structural portion of the gene or DNA fragment.
“Expression construct” refers to the union of a functional fragment in a plasmid, resulting in a vector that is capable of expressing the functional fragment.
“Functional fragments” describes those DNA fragments which encode for plant porphobilinogen synthase or the fusion protein thereof, or a polypeptide portion therof that still maintains a substantial amount of the activity or function of the plant porphobilinogen synthase or the fusion protein thereof.
“Fusion protein” refers to a chimeric protein or polypeptide in which plant porphobilinogen synthase or functional fragment thereof, is joined to a second protein or polypeptide such as thioredoxin, maltose binding protein, or other proteins. The second protein or polypeptide serves the function of helping or enhancing the solubility and/or the post-translational modification of the plant porphobilinogen synthase or functional fragment thereof.
“Gene” refers to a unit composed of a promoter region, a structural gene region and a transcription termination region.
“Gene product” refers to a specific protein or RNA product derived from the structural portion of the gene.
“Heterologous” is used to indicate that a nucleic acid sequence (e.g., a gene) or a protean has a different natural origin or source with respect to its current host. Heterologous is also used to indicate that one or more of the domains present in a protein differ in their natural origin with respect to other domains present. In cases where a portion of a heterologous gene originates from a different organism
Crawford, Jr. John Milton
Rice John
Sevala Veeresh
Stewart Sandy
Hofmeyer Timothy G.
Kiefer Laura L.
Paradigm Genetics, Inc.
Slobodyansky Elizabeth
Spencer Deborah H.
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