Assay for screening inhibitors of C4 plant enzymes

Details Assay for screening inhibitors of C4 plant enzymes Assay for screening inhibitors of C4 plant enzymes

2000-12-18

2002-12-31

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

C435S190000, C435S194000, C435S232000

Reexamination Certificate

active

06500632

ABSTRACT:

TECHNICAL FIELD
The invention described below relates to an assay procedure for screening potential inhibitors of plant enzymes. In particular, the invention relates to an assay procedure which can be used for the high-throughput screening of potential inhibitors of enzymes of the C
4
acid cycle in plants.
BACKGROUND ART
The majority of plants can be divided into C
3
and C
4
plants, depending on the mechanism the plant uses to incorporate CO
2
into organic compounds. In C
3
plants, CO
2
is initially added onto a five-carbon compound forming an unstable six carbon compound which dissociates into two stable three carbon compounds, hence the C
3
name. On the other hand, the first stable compound formed in C
4
plants is a four carbon compound. An extra biochemical pathway exists in the leaves of C
4
plants which allows them to fix CO
2
more efficiently and, under certain environmental conditions, to grow more rapidly than their C
3
counterparts. In addition, C
4
plants use water and nitrogen more efficiently than C
3
plants. Together, these properties enable C
4
plants to compete favourably with many of the world's crops, most of which are C
3
plants for example, wheat, rice, barley and oats. It follows then that many of the weeds which have an adverse effect on agricultural production throughout the world are C
4
plants. Typical examples are nutgrass (
Cyperus rotundus
), couch grass (
Cynodon dactylon
), barnyard grass (
Echinochloa
spp.), Johnson grass (
Sorghum halopense
), and goose grass (
Eleusine indicia
). Nutgrass is a particularly serious global problem being present in more than 100 countries and affecting more than 50 crop species. For efficient agricultural production there is an obvious and pressing need for control of C
4
weed species. To date, however, no herbicide specific for C
4
weeds has been provided.
Crucial to the C
4
acid cycle this being the cycle that fixes CO
2
in C
4
plants are the following enzymes: pyruvate orthophosphate dikinase (pyruvate,Pi dikinase); phosphoenolpyruvate carboxylase; and, NADP-malate dehydrogenase. The pathway involving these enzymes includes the step which incorporates atmospheric CO
2
and creates the products which feed into the sugar-producing Calvin cycle. Interruption of this biochemical pathway should, therefore, adversely affect photosynthesis in C
4
plants.
Attempts to develop a C
4
-specific herbicide have involved designing structural analogues of substrates of the C
4
acid cycle enzymes. Those exhibiting inhibitory effects have been further modified to maximise their effect. The only report of a compound which specifically inhibited a C
4
enzyme related to 3,3-dichloro-2(dihydroxy-phosphinoylmethyl)propenoate which acts on phosphoenolpyruvate carboxylase (see C. L. D. Jenkins et al.,
Biochem. Int.
14, 219-226 [1987]; H. G. McFadden et al.,
Aust. J. Chem.
40, 1619-1629 [1987]; C. L. D. Jenkins,
Plant Physiol.
89, 1231-1237 [1989]; and, H. G. McFadden et al.,
Aust. J. Chem.
4, 301-314 [1989]). However, the compound was found to have no effect on the growth of C
4
plants. At present, none of the compounds known to inhibit enzymes of the C
4
acid cycle has herbicidal activity.
Nevertheless, inhibiting the C
4
acid cycle to kill C
4
plants remains a promising herbicidal strategy. It has been shown that C
4
plants transformed by molecular means (antisense technology) to decrease the level of pyruvate, Pi dikinase or phosphoenolpyruvate carboxylase, enzymes specific to the C
4
acid cycle, are incapable of surviving unless grown under high CO
2
conditions (see J. P. Maroco et al.,
Plant Physiol.
116, 823-832 [1998]). Therefore, it follows that a compound that inhibits either pyruvate, Pi dikinase or phosphoenolpyruvate carboxylase might be an efficient and selective herbicide, thus preventing the deleterious effect of C
4
weeds on C
3
crops.
Marine organisms are an abundant source of compounds of benefit to humans. Many pharmaceuticals are isolated from plants or are derivatives of compounds first identified in marine organisms. Compounds of marine origin are also known which are enzyme inhibitors. Thus, it is reasonable to assume that because of the diversity of the compounds produced by marine organisms there are likely to be some that are inhibitors of C
4
acid cycle enzymes.
In the absence of an indication that an organism produces a compound having desired properties, identification of useful compounds in marine organisms usually entails the screening of extracts from thousands of organisms. However, the known assays for the above three C
4
enzymes, which are spectrophotometric assays, are large-volume assays and are not suitable for the screening of large numbers of samples. There is thus a need for an assay which can be used to screen large numbers of samples for potential inhibitors of the C
4
acid cycle enzymes.
SUMMARY OF THE INVENTION
The object of the invention is to provide a high-throughput assay which can be used to screen potential inhibitors of enzymes of the C
4
acid cycle in plants.
According to a first embodiment of the invention, there is provided an assay for inhibitors of C
4
acid cycle enzymes of plants, the assay comprising:
a) testing for inhibition of at least one of pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase or malate dehydrogenase by:
i) including a sample containing the potential inhibitor in a test mixture comprising pyruvate orthophosphate dikinase and substrates thereof, phosphoenolpyruvate carboxylase and the substrate bicarbonate, and malate dehydrogenase and the substrate NADH;
ii) incubating said test mixture under conditions appropriate for the conversion of pyruvate to malate with oxidation of NADH; and
iii) detecting inhibition of at least one of said pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase or malate dehydrogenase by comparing the level of NADH or NAD
+
in said test mixture with the level of NADH or NAD
+
in a control mixture incubated under the same conditions as in (a)(ii);
b) testing for inhibition of phosphoenol pyruvate carboxylase or malate dehydrogenase with any sample which contains an inhibitor of at least one of said pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase or malate dehydrogenase by:
i) including said sample in a test mixture comprising phosphoenolpyruvate carboxylase and substrates thereof, and malate dehydrogenase and the substrate NADH;
ii) incubating said test mixture under conditions appropriate for the conversion of phosphoenolpyruvate to malate with oxidation of NADH; and
iii) detecting inhibition of said phosphoenolpyruvate carboxylase or malate dehydrogenase by comparing the level of NADH or NAD
+
in said test mixture with the level of NADH or NAD
+
in a control mixture incubated under the same conditions as in (b)(ii);
c) testing for inhibition of malate dehydrogenase with any sample which contains an inhibitor of said phosphoenolpyruvate carboxylase or malate dehydrogenase by:
i) including said sample in a test mixture comprising malate dehydrogenase, oxaloacetate and NADH or including oxaloacetate in said test mixture from a(ii) or b(ii);
ii) incubating said test mixture under conditions appropriate for the conversion of oxaloacetate to malate with oxidation of NADH; and
iii) detecting inhibition of said malate dehydrogenase by comparing the level of NADH or NAD
+
in said test mixture with the level of NADH or NAD
+
in a control mixture incubated under the same conditions as in (c)(ii).
According to a second embodiment of the invention, there is provided an assay for inhibitors of C
4
acid cycle enzymes of plants, the assay comprising:
a) testing for inhibition of at least one of pyruvate orthophosphate dikinase, phospho-enolpyruvate carboxylase or malate dehydrogenase by:
i) including a sample containing the potential inhibitor in a test mixture comprising pyruvate orthophosphate dikinase and substrates thereof, phosphoenolpyruvate carboxylase and the

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