Methods for the identification of inhibitors of serine...

Details Methods for the identification of inhibitors of serine... Methods for the identification of inhibitors of serine...

2003-03-28

2004-08-03

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

C435S069200, C435S193000

Reexamination Certificate

active

06770452

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to plant molecular biology. In particular, the invention relates to methods for the identification of herbicides.
BACKGROUND OF THE INVENTION
The traditional approach to herbicide development may be characterized as “spray and pray”. Chemicals produced in milligram or greater quantity are sprayed on plants and then plant growth is monitored. While this strategy has resulted in the identification of commercially important herbicides, cost, efficacy and safety challenge the future productivity of the “spray and pray” method. Accordingly, there is a need to identify herbicide targets so that compound libraries can be screened for herbicidal activity in higher through-put in vitro or cell-based assays. Inhibitors of these targets can then be selected and confirmed as having herbicidal activity in conventional herbicide assays.
Cysteine biosynthesis in plants plays a key role in the sulfur cycle in nature. Cysteine is incorporated into proteins and glutathione or serves as the sulfur donor of methionine and sulfur-containing secondary products in plants. Serine acetyltransferase (SAT) plays a regulatory role in the biosynthesis of cysteine by its property of feedback inhibition by cysteine in bacteria and certain plants. Serine acetyltransferase (EC 2.3.1.30) catalyzes the formation of O-acetyl-L-serine (OAS) from acetyl-CoA and L-serine. The final step of cysteine biosynthesis is the formation of L-cysteine from the substrates O-acetyl-L-serine and sulfide by cysteine synthase (CS).
Three cDNA clones encoding SAT isoforms (SAT-c, SAT-p and SAT-m) have been isolated from
Arabidopsis thaliana
. SAT-c is localized in the cytosol while SAT-p and SAT-m are localized in the chloroplasts and mitochondria respectively. In the case of SAT-c, its activity has been shown to be feedback-inhibited by a low concentration of cysteine (Noji et al. (1998)
J. Biol. Chem
. 273:32739-32745). SAT has two different protein-protein interaction domains. An SAT-SAT domain for homomerization, and also a SAT-CS domain located on the C-terminal portion of the protein for heteromerization. When associated in a complex with CS, SAT has been shown to be activated resulting in higher V
max
and substrate affinities. The bound CS is also inactivated by this interaction. The resulting product, OAS, then must diffuse out of the complex to be acted upon by free CS and sulfide to form cysteine. The complex itself is stabilized by sulfide (inactivating CS), but OAS helps dissociate the complex (activating CS). Hence, not only is SAT regulated by the formation of cysteine, it is also regulated by protein interactions and additional substrate-product interactions (Wirtz et al. (2001)
Eur. J. Biochem
. 268:686-693).
The present invention discloses SAT as a target for the evaluation of plant growth regulators, especially herbicide compounds, in plants.
SUMMARY OF THE INVENTION
The present inventors have discovered that antisense expression of a SAT cDNA in Arabidopsis causes reduced growth and altered pigmentation. Thus, the present inventors have discovered that SAT is essential for normal plant development and growth, and is useful as a target for the identification of herbicides. Accordingly, in one embodiment the present invention provides methods for the identification of compounds that inhibit SAT expression or activity, comprising: contacting a candidate compound with a SAT and detecting the presence or absence of binding between the compound and the SAT, wherein binding between the compound and the SAT indicates the compound as a herbicide target. In another embodiment of the invention, methods are provided for the identification of compounds that inhibit SAT enzyme activity, comprising: contacting a SAT polypeptide with acetyl-CoA and L-serine in the presence and absence of a compound or contacting a SAT polypeptide with O-acetyl-L-serine and CoA in the presence and absence of a compound; and determining a change in concentration for at least one of acetyl-CoA, L-serine, O-acetyl-L-serine and/or CoA in the presence and absence of the compound, wherein a change in the concentration for any of acetyl-CoA, L-serine, O-acetyl-L-serine and/or CoA indicates that the compound is a candidate herbicide.


REFERENCES:
patent: WO 02/10210 (2002-02-01), None
Noji, Masaaki et al.,Serine Acetyltransferase Involved in Cysteine Biosynthesis from Spinach: Molecular Cloning, Characterization and Expression Analysis of cDNA Encoding a Plastidic Isoform.Plant Cell Physiol, 42(6): 627-634 (2001).
Mino, Koshiki et al.,Increase in the Stability of Serine Acetyltransferase from Escherichia coli Against Cold Inactivation and Proteolysis by Forming a Biendzyme Complex,Biosci. Biotechnol. Biochem., 65(4), 865-874, 2001.
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Mino, Koshiki et al.,Effects of Bienzyme Complex Formation of Cysteine Synthetase from Escherichia coli on Some Properties and Kinetics,Biosci. Biotechnol. Biochem., 64(8), 1628-1640, 2000.
Droux, Michel et al.,Interactions Between Serine Acetyltransferase and O-Acetylserine(Thiol)Lyase in Higher Plants—Structural and Kinetic Properties of the Free and Bound Enzymes,Eur. J. Biochem., 255, 235-245 (1998).
Noji, Masaaki et al.,Isoform-dependent Differences in Feedback Regulation And Subcellular Localization of Serine Acetyltransferase Involved in Cysteine Biosysynthesis from Arabidopsis thaliana,The Journal of Biological Chemistry, vol. 273, pp. 32739-32745, Issue of Dec. 4, 1998.
Hindson, VJ et al.,Serine Acetyltransferase from Escherichia coli is a Dimer of Trimers,The Journal of Biological Chemistry, vol. 275, No. 1, Issue of Jan., pp 461-466, 2000.
Wirtz, Markus et al.,The Cysteine Synthase Complex From Plants,Eur. J. Biochem, 268, 686-693, 2001.
Harms, Karsten et al.,Expression of a Bacterial Serine Acetyltransferase in Transgenic Potato Plants Leads to Increased Levels of Cysteine and Glutathione,The Plant Journal, 22(4), 335-343, 2000.

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