Sulfonylurea synthase inhibition

Lolium biotypes exist which have resistance to the sulfonylurea herbicides chlorsulfuron and metsulfuron methyl (4). The biotype used in the studies presented here is resistant to both these sulfonylurea herbicides. Sulfonylurea herbicides inhibit the chloroplastic enzyme acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS) (16). Inhibition of this enzyme results in disruption of the synthesis of the branched-chain amino acids valine and isoleucine (161. The imidazolinone herbicides also inhibit ALS Q2). In some species auxins can protect against chlorsulfuron inhibition (S. Frear, USDA North Dakota, personal communication) the mechanistic basis for this protection is not known. We have measured the ALS activity in the resistant and susceptible Lolium and have also checked for any induction of ALS activity following treatment with the sulfonylurea herbicide chlorsulfuron. 

Acetolactate synthase inhibition by imidazolinones and triazolopyrimidines, 460 sensitivity to sulfonylurea herbicides, 460 Acetolactate synthase gene activity and inheritance of resistance in tobacco, 461... 

This strategy was used to engineer resistance against glyphosate, and imidazoli-nones and sulfonylureas that inhibit acetolactate synthase (ALS), a key enzyme in the biosynthesis of branched chain amino acids. ALS resistant crops have primarily been generated through selection for an herbicide insensitive ALS allele from natural or mutagenized cell or plant populations [3]. 

Acetolactate synthase inhibition by herbicides, 36,38 inhibition by sulfonylurea herbicides, 33,36,37/... 

Herbicides also inhibit 5- (9/-pymvylshikiniate synthase, a susceptible en2yme in the pathway to the aromatic amino acids, phenylalanine, tyrosine and tryptophan, and to the phenylpropanes. Acetolactate synthase, or acetohydroxy acid synthase, a key en2yme in the synthesis of the branched-chain amino acids isoleucine and valine, is also sensitive to some herbicides. Glyphosate (26), the sulfonylureas (136), and the imida2oles (137) all inhibit specific en2ymes in amino acid synthesis pathways. 

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

There are several commercially available sulfonylurea herbicides that contain a 2-pyrimidine group <2006H(68)561>. These compounds, which function by inhibition of acetolactate synthase (ALS), an enzyme involved in the early stage of branched-chain amino acid synthesis, include sulfometuron-methyl 1095, primisulfuron-methyl 1096, chlorimuron-ethyl 1097, bensulfuron-methyl 1098, ethoxysulfuron 1099, nicosulfuron 1100, and pyrazosulfuron-ethyl 1101. Related nonsulfonylureas include the sulfide pyrftalid 1102 and the ether pyriminobac-methyl 1103. 

Inhibition of acetolactate synthase (ALS) [Acetohydroxy acid synthase (AHAS)] Sulfonylureas Imidazolinones T riazolopyrimidines Pyrimidinylthiobenzoates 2... 

Figure 1. Three chemical families known to exhibit herbicidal activity through the inhibition of acetolactate synthase. A. sulfonylurea (sulfometuron) B. imidazolinone (imazapyr) and C. A representative triazolopyrimidine.

The selective toxicity of sulfonylureas to certain weeds without damage to the cereal crop arises from their rapid metabolism in the crop plant to inactive compounds, whereas in sensitive weeds the metabolism is much slower. The very high herbicidal activity suggests a specific biochemical mode of action, which is concluded to be the inhibition of plant cell division. Sulfonylureas block the enzyme acetolacetate synthase (ALS), which catalyses the biosynthesis of the essential branched chain amino acids valine, leucine and isoleucine. 

In the 1950s and 1960s, Geigy and other firms developed a series of herbicides based on alkyl and dialkyl triazines, such as the root-herbicide atrazine (83)64. They are made by stepwise introduction of suitable nucleophiles into precursors. Other members are ametryn, the non-selective simazine (84) and the cyanuric chloride-derived methylthio triazine desmetryne (85) (Scheme 19). Chlorotoluron is 3-(3-chloro-p-toly 1 )-1,1-dimethylurea (86), a phenylurea herbicide. These products act on the photosynthetic pathway. Both 83 and 84 are used for weed control in maize65. Sulfonylureas such as the triazine chlorosulfone (87) inhibit the enzyme acetolactate synthase. They offer the major advantage of requiring no more than 10 to 20 g per hectare. 

One of the most effective classes of herbicide are the sulfonylureas. They are applied in quantities of no more than a few grams per hectare. The action of sulfonylureas is based on inhibition of acetolactate synthase (ALS) [9]. The fluorine-containing derivative Primsulfuron methyl has selectivity in the cultivation of maize [10]. 

Inhibits ALS activity by 50%. A good correlation exists between the herbicidal activity of sulfonylureas and their ability to inhibit acetolactate synthase (2). This in vitro assay using the target enzyme along with the three-dimensioFTI structure of the enzyme should aid in the generation of a substantial data base that can be used to design potent inhibitors. 

Second, a key enzyme or receptor in the pathway should be identified as the target. It is best to select enzymes whose products are important for several functions in the species. Cellular response to such a metabolic blockade should also be considered (e.g., cascading effects). Often end-product limitation results in more metabolites entering the pathway. After sufficient substrate accumulation, catalysis may occur even in the presence of an inhibitor (10). However, accumulation of toxic intermediates would prevent tTTTs cellular response and lead to death. Again using sulfonylureas as an example, acetolactate synthase is a common enzyme in the pathway for two essential amino acids rather than just one. Also, inhibition of acetolactate synthase leads to high levels of a-ketobutyrate which is thought to have deleterious effects (11). 

The sulfonylureas, an extremely potent class of herbicides, act by inhibiting acetolactate synthase (ALS), which is the first common enzyme in the biosynthetic pathways leading to the branched chain amino acids. Two other unrelated classes of herbicides also act by interfering with this enzyme. We have cloned and characterized the genes encoding ALS from several higher plants. The ALS genes isolated from herbicide sensitive and herbicide resistant plants have been compared, and several mutations which confer the herbicide resistant phenotype have been identified. 

The sulfonylurea herbicides are a new family of chemical compounds, some of which are selectively toxic to weeds but not to crops. The selectivity of the sulfonylureas results from their metabolism to non-toxic compounds by particular crops, but not by weeds. In addition to efficient weed control, the sulfonylurea herbicides provide environmentally desirable properties such as field use rates as low as two grams/hectare and very low toxicity to mammals. The high specificity of the herbicides for their molecular target contributes to both of these properties. In addition, the low toxicity to mammals results from their lack of the target enzyme for the herbicides. Sulfonylureas inhibit the enzyme acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS), which catalyzes the first common step in the biosynthesis of the branched chain amino acids leucine, isoleucine and valine. In mammals these are three of the essential amino acids which must be obtained through dietary intake because the biosynthetic pathway for the branched chain amino acids is not present. The prototype structure of a sulfonylurea herbicide is shown in Figure 1. 

A great number of herbicides that work through the inhibition of acetolactate synthase (ALS) have been commercialized. They belong to four chemical groups sulfonylureas (23), triazolopyrimidines (2), imidazolinones (5), and pyrimidinyloxybenzoic analogues (3). (The number of active ingredients in parentheses is taken from The Pesticide Manual.) Also in this case, potent herbicides were developed (e.g., chlorsulfuron) before the site of action was found. 

Sulfonylureas are systemic herbicides absorbed by the foliage and roots. They act by inhibiting acetolactate synthase, a key enzyme in the biosynthesis of branched chain aminoacids." This results in stopping cell division and plant growth. The most important degradation pathways of sulfonylureas are chemical hydrolysis and microbial degradation. 

For example, glyphosate inhibits the enzyme, EPSP (5-enolpyruvylshikimate 3-phosphate) synthase, that catalyzes a step in the synthesis of the aromatic amino acids. Similarly, both the imidazolinones and sulfonylureas inhibit acetolactate synthase (ALS), the enzyme that catalyzes the first step in the formation of branched-chain amino acids (11). Triazine herbicides act by binding to a specific protein in the thylakoid membranes of the chloroplasts, preventing the flow of electrons and inhibiting photosynthesis (12). 

Application of Global Sequence Similarity to Find an Inhibitor of Acetolactate Synthase. Acetolactate synthase (ALS) Is the site of action of sulfonylurea, Imldazollnone, and trlazolo pyrimidine herbicides (10-14). Their mode of Inhibition and binding sites on ALS were ambiguous, because (1) these herbicides bear no obvious similarity In their chemical structures to those of ALS substrates (pyruvate and acetolactate), cofactors (thiamine pyrophosphate, FAD, and Mg ) and effectors (valine, Isoleuclne, and leucine) and (2) they Inhibit ALS In a mode too complex to be analyzed. 

Inhibitors of Acetolactate Synthase (ALS/AHAS) The enzyme acetolactate synthase (ALS) plays in plants an essential role in branched-chain amino acid biosynthesis. In the pathway leading to valine and leucine, ALS catalyzes the formation of 2-acetolactate from two pyruvate molecules, and in the pathway to isoleucine the formation of 2-acetohydroxybutyrate from 2-ketobutyrate and pyruvate. Due to this double function the enzyme is also called with a more general term aceto-hydroxyacid synthase. ALS is inhibited by several groups of herbicides, mainly the sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), pyrimidinylthiobenzoates(PTBs) and sulfonylaminocarbonyltriazolinone (SCTs) (see Chapter 2.1, M. E. Thompson). 

Herbicidal sulfonylureas have a unique mode of action they interfere with a key enzyme required for plant cell growth - acetohydroxyacid synthase (AHAS, EC 2.2.1.6) [1, 2, 3] (see also Mark E. Thompson in this volume, Chapter 2.1 Biochemistry of the Target and Resistance ). AHAS is the enzyme responsible for the synthesis of the branched-chain amino acids valine, leucine and isoleucine. Inhibition of this enzyme disrupts the plant s ability to manufacture proteins, and this disruption subsequently leads to the cessation of all cell division and eventual death of the plant. 

Triazine herbicides and quinoclamine, having the mode of action in inhibition of PS II (Photosynthesis at photosystem II) had low variability on sensitivities in different algal taxa. On tiie other hand. Amide herbicide such as Pretilachlor and Cafenstrole as well as sulfonylurea herbicides of bensulfiironmethyl and imazosulfuron had great variability on sensitivities in different algal taxa. These herbicide have in other the mode of action in inhibition of cell division or in inhibition of acetolactate synthase rather tiian in inhibition of PS II. Carbamate herbicide showed relatively low toxicity on algae. Daimuron and bentazone exhibit low toxicity on all tire tested species (Figure 3). 

Imazaquin, an example of the imidazolinone class of herbicides, is particularly selective to soybeans. These compounds have a similar mode of action to that of the sulfonylurea herbicides, which will be discussed later. They block branched chain amino acid biosynthesis by inhibition of the enzyme acetolactate synthase (ALS). 

Nicosulfuron ALS-Sulfonylurea Aceto-lactate synthase (ALS) Inhibition of branched chain amino acid synthesis 1991 Post- emergence Maize Grasses, broadleaf weeds, 35... 

Some groups of herbicides depend upon phloem translocation to reach their sites of action. For example, the phenoxyalkanoic acid herbicides, which act on cell growth and development, and glyphosate, which inhibits the biosynthesis of aromatic amino acids, are translocated from leaves to all sites of active growth within the plant. Similarly, the sulfonylureas and imidazolinones, which inhibit acetolactate synthase, are, when applied postemergence, translocated in the phloem to all parts of the plant. 

The sulfonylureas are compounds of intermediate lipophilicity (log estimated in the range 1.5 to 2.5), and all possess the acidic sulfonylurea function (pKa 3.3 to 5.2). They are applied pre- or postemergence and kill plants by slowing cell division due to inhibition of the enzyme acetolac-tate synthase. The compounds move in both the xylem and phloem, " and their phytotoxic action is believed to be due both to direct uptake at the sites of action (e.g., root tips) and to phloem transport to the growing points. 

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