Inhibition of acetolactate synthase

Since the 1980s five new herbicidal classes have emerged that all share the same site of action. These have proved to be potent, selective, broad- spectrum inhibitors of plant growth at field rates measured in grams rather than kilograms per hectare. The sulphony-lureas (SUs), imidazolinones (IMs), triazolopyrimidines (TPs), sulfonylaminocarbonyl-triazolines (often classed as SUs) and pyrimidinyl-oxy-benzoates (pyrimidinyl-carboxy herbicides; PCs) are chemically different (Table 9.1), yet all share the same site of action, namely acetolactate synthase (ALS, E.C. 4.1.3.18, also known as acetohydroxyacid syn-thase, AHAS), a key enzyme in the biosynthesis of the branched-chain amino acids leucine, isoleucine and valine. In each case, growth inhibition may be overcome by the addition of these amino acids. The efficacy and potency of the ALS inhibitors has ensured the continued success of these herbicides, which have rapidly challenged, and in some instances replaced, traditional products, especially in cereals and soybeans. SUs and TPs are active at field rates of 10-100 g ha-1 whereas IMs are required at 100-1000 g ha-1 to give a similar degree of weed control. Currently ALS inhibitors represent the second biggest class of herbicidal active ingredients. A major research effort has progressed to understand their mode(s) of action and to develop new products to inhibit the synthesis of branched-chain amino acids. Further information on this class of herbicide can be found in reviews by Babczinski and Zelinski (1991) and Duggleby and Pang (2000).

The highest activity obtained with the SUs is when the aryl group has an ortho substituent. Thiophene, furan, pyrimidine and naphthalene groups are also active herbicides when replacing the aryl group, but the ortho substitution is still essential with respect to the sulphonylurea bridge. The heterocycle configurations for optimal activity appear to be a symmetrical pyridine or a symmetrical triazine with low alkyl or alkoxy sub-stituents (Beyer et al., 1987). The sulfonylaminocarbonyl moiety also results in the herbicidally active SUs procarbazone and flucarbazone (Amann et al., 2000; Müller et al., 1992- . In the case of the imidazolinones, the highest biological activity has been observed when an imidazolinone ring, ideally sustituted with methyl and isopropyl groups, is attached to an aromatic ring containing a carboxyl group in an ortho position (Los,

Table 9.1 Structures of a selection of amino acid biosynthesis inhibitors.

(a) Sulphonylureas

S CO2CH3 -CO2CH3

R2 R3 R4 Common name (crop/dosage, g ha 1)

-CH3 N -OCH3 Chlorsulfuron (cereals/4-26)

-CH3 N -OCH3 Metsulfuron-methyl (cereals/2-8)

-CH3 N -OCH3 Triasulfuron (cereals/10-40)

-OCH3 CH -OCH3 Bensulfuron-methyl (rice/20-75)

-Cl CH -OCH3 Chlorimuron-ethyl (soybean/8-13)

-CH3 CH -CH3 Sulfometuron-methyl (non-crop/70-840)

CH3 N OCH3

Thifensulfuron (cereals, soybean/17-35)

CH3 N OCH3 Tribenuron (cereals/5-30)

OHCF2 CH OCHF2 Primisulfuron (maize/20-40)

Table 9.1 {Continued)

Table 9.1 {Continued)

Table 9.1 (Continued)
Table 9.1 {Continued)

1986). The more recently discovered PCs demonstrate activity at similar field rates to SUs and TPs. However, the inhibitory mechanism and cross-resistance patterns appear to be a hybrid between the SUs and the IMs. Elegant studies by Shimizu et al. (2002) based upon the synthesis of novel analogues based upon phenoxyphenoxypyrimidine suggest that PCs require both esteric bonding and an appropriate substituted pyrimidine ring in order to demonstrate ALS-inhibiting activity. Highest inhibition was observed when a COOMe group was at the ortho position to the pyrimidinyloxy group. A pyri-midine ring imparted greater inhibiting activity than structures containing other N-heterocyclics. Interestingly, the replacement of the O-bridge with an S-bridge reduced ALS-inhibiting activity but did increase crop tolerance in some cases. The presence of an S-bridge also increased herbicide mobility, both via root uptake and by translocation. The base S-containing compound used in these synthesis studies was pyrithiobac-sodium. Studies of this type not only aid in elucidation of structure-function relationships, but also in the discovery of new ALS-inhibiting herbicides.

All five classes of ALS inhibitors possess remarkable herbicidal properties. They are able to control a very wide spectrum of troublesome annual and perennial grass and broad-leaf weeds at very low doses. Furthermore, formulations have proved to be both foliar- and soil-active with very low mammalian toxicity. Chlorsulfuron, metsulfuron-methyl, and imazamethabenz give selective weed control in cereals, chlorimuron-ethyl and imazaquin are selective in soybean, imazethapyr is selective in other legumes as well as soybean, bensulfuron-methyl is effective in rice, and sulfometuron-methyl and imazapyr have found industrial and non-crop uses for total vegetation control. Imazapyr is effective in forest management by controlling deciduous trees in conifers, and a coformulation of imazapyr and imazethapyr is being developed as a growth retardant in grassland and turf areas, with an additional control of broadleaf weeds.

ALS, like EPSP synthase, is a nuclear-encoded, chloroplast-localised enzyme in higher plants, and also occupies a strategic location in the biosynthetic pathway of essential amino acids. This pathway has been well studied in microorganisms and is becoming increasingly understood in higher plants. Essentially, synthesis occurs in the stroma from threonine and pyruvate in a common series of reactions (Figure 9.7; Ray, 1989). In isoleucine synthesis, threonine is first deaminated to 2-oxobutyrate by threonine dehydratase, which is controlled by feedback regulation by valine and isoleucine. ALS catalyses the first common step of branched-chain amino acid biosynthesis to yield acetohydroxy acids which undergo oxidation and isomerisation to yield derivatives of valeric acid. Dehydration and transamination then produces isoleucine and valine. 2-Oxoisovalerate reacts with acetyl-CoA to form a--sopropylmaleate which is then isomerised, reduced and transaminated to yield leucine. ALS demonstrates feedback inhibition to leucine, valine and isoleucine.

ALS has been extensively studied in microorganisms and is the subject of increasing scrutiny in plants, although plant ALS is labile and constitutes less than 0.01% of total plant protein. As many as six ALS isozymes have been reported in bacteria to allow carbon flux through this pathway at varying concentrations of pyruvate. However, isozymes are not required for ALS in the chloroplast stroma where more reliable concentrations of substrates are assumed. Study of ALS extracted from pea seedlings identified a 320-kDa ALS that dissociated to a 120-kDa ALS in the absence of flavin adenine dinucleotide (FAD). The larger ALS demonstrated feedback inhibition from valine, leucine and isoleucine whereas the smaller ALS, although still demonstrating enzyme activity, did not exhibit feedback inhibition. This suggests that there are separate, regulatory, subunits that require FAD in order to remain attached to the catalytic subunits of ALS. Current understanding is that the ALS enzyme in pea seedlings consists of at least 4 catalytic and 2 regulatory subunits (Shimizi et al., 2002). Regulation of ALS activity is carried out by leucine, valine and isoleucine (feedback inhibition). This inhibition is also observed for leucine/isoleucine and leucine/valine mixtures but for valine/isoleucine mixtures an antagonism of feedback was observed. This suggests that there are two regulatory sites on the enzyme, one for leucine and one for valine/isoleucine. Two regulatory sites are also suggested by regulatory promoter studies (Hershey et al., 1999). Analysis of the ALS gene from a number of plant species has revealed that it is highly conserved, with very little difference between the rice, maize and barley ALS genes. The isozyme II from S. typh-imurium requires FAD, thiamine pyrophosphate (TPP), and Mg2+ for complete activation, and the reaction proceeds in a biphasic manner. First, a pyruvate molecule binds to TPP at the active site and is decarboxylated to yield an enzyme-substrate complex plus CO2. A second pyruvate then reacts with this complex and acetolactate is released. A number of ALS herbicides bind slowly, but tightly, to the enzyme-substrate complex to prevent the addition of the second pyruvate molecule (LaRossa and Schloss, 1984). However, imidazolinones are uncompetitive inhibitors with respect to pyruvate (Shaner et al., 1984), and in the case of sulfonylureas and triazolopyrimides both competitive and non-competitive inhibition is exhibited (mixed-type inhibition) (Subramanian and Gerwick, 1989; Durner et al., 1991). This indicates that the herbicide-binding site for ALS is distinct from the enzyme's active site. Other studies have shown that the herbicide does not bind at the allosteric site either.

OH NH2

Threonine dehydratase

CH3 C COOH

pyruvate pyruvate i

O CH3

Acetolactate Synthase

CH3 CH2 C COOH 2-ketobutyrate

. pyruvate OH O

2-acetolactate NADPH—s NADP+<-^f CH3 OH

Acetohydroxyacid Reductoisomerase

CH3 C CH COOH

2,3-dihydroxyisovalerate CH3

CH3 CH CH COOH

2-oxoisovalerate glutamate-<—

Dihydroxyacid Dehydratase

Aminotransferase

CH3 CH2 C C CH3

COOH 2-acetohydroxybutyrate NADPH "V~~>NADP+ OH OH

CH3 CH2 C CH COOH

2,3-dihydroxy-3-methylvalerate H2O

CH3 CH2 CH C COOH

2-oxo-3-methylvalerate glutamate 2-oxoglutarate NH2

CH3—CH —CH —COOH CH3— CH2—CH —CH —COOH

NH2 CH3

valine isoleucine

Figure 9.7 Biosynthesis of branched-chain amino acids (from Ray, 1989).

The absolute requirement for FAD where no oxidation or reduction reactions are involved has puzzled many workers. Schloss and colleagues (1988) demonstrated that ALS shows considerable sequence homology with pyruvate oxidase, suggesting that both enzymes may have evolutionary similarities. Indeed, ALS does demonstrate an oxygen-consuming side-reaction when its activity is inhibited (Durner et al., 1994). They have discovered that pyruvate oxidase binds both FAD and a quinone for redox reactions, and that the binding of pyruvate oxidase from Escherichia coli with ubiquinone-40 is tightest in the presence of pyruvate. Since the binding of ALS with herbicides is also tightest in the presence of pyruvate, these authors have concluded that the herbicide binding site is derived from an evolutionary vestige of a quinone-binding cofactor site that is no longer functional in ALS. Hence, the herbicide site is extraneous to, or outside of, the ALS active site. McCourt et al. (2006) have recently reported that SUs and IMs block a channel in ALS through which substrates access the active site of the enzyme. These studies further reported that SUs approach to within 5 A of the catalytic centre, whereas IMs bind at least 7 A from it. This confirms earlier observations of different binding sites for these two classes of herbicide. It seems likely that TPs and PCs will bind to ALS in a similar manner to SUs. Ten amino acids have been identified that are involved in the binding of both SUs and IMs, six that are only involved in SU binding and two that are only involved in IM binding. These observations may lead to the development of novel ALS inhibitors that are unaffected by mutations in ALS genes that currently result in target site-mediated herbicide resistance. Zhou et al. (2007) have recently reviewed current understanding of the mechanisms of action of ALS-inhibiting herbicides.

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