Inhibitors of plant peptide deformylase for use as...

Details Inhibitors of plant peptide deformylase for use as... Inhibitors of plant peptide deformylase for use as...

2000-07-14

2004-05-04

Clardy, S. Mark (Department: 1616)

Plant protecting and regulating compositions

Plant growth regulating compositions

C504S269000, C435S228000

Reexamination Certificate

active

06730634

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to a method of identifying herbicides and to the use of inhibitors of plant peptide deformylase as broad spectrum herbicides.
BACKGROUND
Ribosome-mediated synthesis of proteins begins with a methionine residue. In prokaryotes and eukaryotic organelles (mitochondria and chloroplasts), the methionyl moiety carried by the initiator tRNA is N-formylated prior to its incorporation into a polypeptide (Meinnel and Blanquet, J. Bacteriol. 175:7737-7740 (1993)). N-formylmethionine is therefore always incorporated at the N-terminus of a nascent polypeptide in prokaryotes (Adams, J. M. and Capecchi, M., Proc. Natl. Aca. Sci. U.S.A. 55:147-155 (1966); Webster et al., Proc. Natl. Acad. Sci. U.S.A. 55:155-161 (1966)). However, most mature proteins do not retain the N-formyl group (Marcker, K. and Sanger, F. J. Mol. Biol. 8:835-840 (1964)). Instead, the N-formylmethionine group is removed post- or co-translationally in a process known as deformylation, which is catalyzed by peptide deformylase.
Deformylation is catalyzed by peptide deformylase which cleaves the formyl group from the nascent polypeptide chain (Adams, J., J. Mol. Biol. 33:571-589 (1968); Livingston, D. M. and Leder, P., Biochemistry 8:435-443 (1969); Takeda, M. and Webster, R. E., Proc. Natl. Acad. Sci. U.S.A. 60:1487-1494 (1968)).
Prior to the present invention, research in the area of peptide deformylase activity and enzymology focused on the bacterial enzyme. For example, the structure of the core domain of
E. coli
peptide deformylase was solved by NMR (Meinnel, T. et al., J. Mol. Biol. 262:375-386 (1996)) and the structure of the full-length protein by X-ray crystallography (Chan et al., Biochemistry 36:13904-13909 (1997)). Becker et al., J. Biol. Chemistry 273(19): 11413-11416 (1998) solved the structure of the catalytically active
E. coli
enzyme in the nickel-bound form (PDF-Ni) and in inhibitor-complexed form.
More recently, Ragusa et al., J. Mol. Biol. 289: 1445-1457 (1999), investigated the substrate specificity of
Escherichia coli
peptide deformylase by measuring the efficiency of the enzyme to cleave formylpeptides. Durand et al., Archives Biochemistry and Biophysics, 367(2):297-302 (1999), tested a variety of peptide aldehydes and identified calpeptin as a potent inhibitor of
E. coli
and
B. subtilis
peptide deformylase.
The overall focus in the field has been engineering site-specific inhibitors of peptide deformylase with a goal of developing broad-spectrum antibiotics with low to no mammalian toxicity. Rajagopalan et al., Biochemistry 36:13910-13918 (1997), identified specific and potent inhibitors of the bacterial enzyme which are potentially novel and new antibiotic agents. Meinnel et al., Biochemistry 38:4287-4295 (1999) designed and synthesized substrate analogue inhibitors of bacterial peptide deformylase and studied their capacity to undergo hydrolysis. To aid in the design of both the cobalt and zinc containing
E. coli
peptide deformylase, Hao et al., Biochemistry 38:4712-4719 (1999), studied the structure of the protein-inhibitor complexes of the cobalt and zinc containing
E. coli
enzyme.
Prior to the present invention, it was generally accepted that peptide deformylase genes and proteins were absent from eukaryotic cells. The present invention unexpectedly establishes, for the first time, the existence of a peptide deformylase gene and protein in eukaryotic cells, particularly in plant cells. The inventors have found that the deformylase is a novel and suitable target for identifying new broad spectrum herbicides.
SUMMARY OF THE INVENTION
In a first aspect, the present invention relates to a plant peptide deformylase gene which is expressed in a higher plant. A particularly preferred higher plant is Arabidopsis. More particularly preferred is the strain
Arabidopsis thaliana.
In additional aspects, the present invention relates to a nucleotide and amino acid sequence encoding a plant peptide deformylase.
In another aspect, the present invention relates to a recombinant vector comprising the peptide deformylase nucleotide sequence described above.
In another aspect, the invention relates to a host cell that is transformed or transfected with the recombinant vector of the invention. The host cell may be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a plant cell or a mammalian cell.
In yet another aspect, the present invention relates to an isolated or recombinantly expressed peptide deformylase polypeptide which is encoded by a plant peptide deformylase gene as described above.
In still another aspect, the present invention relates to a method for introducing the plant peptide deformylase gene into a cell which does not express said gene, which method comprises transforming a higher plant seed crop with the recombinant vector (vector containing a peptide deformylase gene) such that the cell expresses the peptide deformylase enzyme encoded by the gene. The cell may be a prokaryotic cell or a eukaryotic cell.
In another aspect, the present invention provides a method of identifying an inhibitor of plant peptide deformylase, said method comprising:
(a) incubating a catalytically effective amount of a plant peptide deformylase, with a suitable substrate, in the presence or absence of a candidate inhibitor compound, and
(b) detecting and quantifying the enzyme product formed.
The method may further comprises comparing the amount of enzyme product formed in the presence and absence of said candidate inhibitor.
In a preferred embodiment, the method of the invention permits identification of inhibitors of peptide deformylase derived from Arabidopsis thaliana.
The invention also provides a method of identifying herbicides which comprises testing a compound in a peptide deformylase inhibition assay, and where a measurable reduction of plant peptide deformylase is observed, subjecting the compound to an in vivo test for herbicidal activity.
In another aspect, the invention relates to herbicides which act by inhibiting plant peptide deformylase and which are identified by the methods of the invention.
In another aspect, the invention provides a method of combating weeds comprising treating said weeds with a herbicide, wherein said herbicide is a compound which is an inhibitor of peptide deformylase. A preferred herbicide is actinonin, which is a potent inhibitor of plant peptide deformylase.
The invention also provides a method of controlling vegetation comprising applying to plant foliage a herbicidally effective amount of an inhibitor of peptide deformylase.
In a further aspect, the invention provides a method of inhibiting the activity of plant peptide deformylase comprising exposing said peptide deformylase to an effective amount of an inhibitor of said peptide deformylase.
Still further, the invention provides an antibody directed against peptide deformylase isolated from
Arabidopsis thaliana
. The antibody may be a polyclonal or a monoclonal antibody.
In an additional aspect, the invention also provides a transgenic plant, wherein said transgenic plant is engineered to be resistant to an inhibitor of plant peptide deformylase.
In a further aspect, the invention provides a herbicidal composition comprising an inhibitor of plant peptide deformylase. In accordance with the present invention, the inhibitor is a herbicide, i.e. a compound having herbicidal activity. The composition of the invention may also contain a herbicidally acceptable surfactant.
In yet another aspect, the present invention provides a method of controlling vegetation comprising applying to plant foliage a herbicidal composition, wherein said herbicidal composition comprises an inhibitor of plant peptide deformylase. Those of skill in the art would recognize that the herbicide actinonin may be included in compositions of the present invention.
Additional aspects of the invention will be described throughout the specification.


REFERENCES:
patent: 5985273 (1999-11-01), Reed et al.
patent: 0911409 (1999-04-01), None
patent: 1033405 (2000-09-01), None
Chen

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