Acetohydroxyacid synthase

Benzoylformate decarboxylase (BFD EC 4.1.1.7) belongs to the class of thiamine diphosphate (ThDP)-dependent enzymes. ThDP is the cofactor for a large number of enzymes, including pyruvate decarboxylase (PDC), benzaldehyde lyase (BAL), cyclohexane-1,2-dione hydrolase (CDH), acetohydroxyacid synthase (AHAS), and (lR,6] )-2-succinyl-6-hydroxy-2,4-cyclohexadiene-l-carboxylate synthase (SHCHC), which all catalyze the cleavage and formation of C-C bonds [1]. The underlying catalytic mechanism is summarized elsewhere [2] (see also Chapter 2.2.3). 

As indicated in Fig. 24-17, pyruvate is the starting material for the formation of both l- and D-alanine and also the branched chain amino acids valine, leucine, and isoleucine.339,340 The chemistry of the reactions has been discussed in the sections indicated in the figure. The first step is catalyzed by the thiamin diphosphate-dependent acetohydroxyacid synthase (acetolactate synthase), which joins two molecules of pyruvate or one of pyruvate and one of 2-oxobutyrate (Fig. 24-17 Fig. 14-3).340a b In E. coli there are two isoenzymes encoded by genes ilv B and ilv HI. Both are regulated by feedback inhibition by valine, probably... 

Herbicides control weeds and are the most widely used class of pesticides. The latest US EPA data show that some 578 million pounds of herbicides were used in the United States in 1997 and accounts for some 47% of pesticides used. This class of pesticide can be applied to crops using many strategies to eliminate or reduce weed populations. These include preplant incorporation, pre- and postemergent applications. New families of herbicides continue to be developed, and are applied at low doses, are relatively nonphytotoxic to beneficial plants and are environmentally friendly. Some of the newer families such as the imidazolinones inhibit the action of acetohydroxyacid synthase that produces branched-chain amino acids in plants. Because this enzyme is produced only in plants, these herbicides have low toxicities to mammals, fish, insects, and birds. 

Shaner, D.L. and R.G. Lym (1991). Mechanism of resistance to acetolactate synthase/acetohydroxyacid synthase inhibitors. Proc. West. Soc. Weed Sci., 44 122-125. 

Besides optimizing a known enzyme with respect to the desired application, screening for new enzymes catalyzing the wanted transformation might give access to altered reaction parameters. Barak, Chipman and coworkers identified acetohydroxyacid synthase (AHAS) from E. coli as an efficient catalyst for the formation of (1 )-PAC starting from pyruvate and benzaldehyde (Scheme 4.2 B) [20]. 

Figure 3. Relative potencies of N-phthalyl-L-valine anilide, L-valine, the imidazolinone herbicide, Scepter, and the sulfonylurea herbicide, chlorsulfuron, as inhibitors of acetohydroxyacid synthase from Zea mays. (Reproduced with permission from Ref. 53. Copyright 1985 Verlag der Zeitschrift fur Naturforschung.)...

Acetohydroxyacid synthase, inhibition in plants, 66 Acetolactate synthase... 

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. 

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. 

Identification of the mode of action of the imidazolinones occurred while resistant cell lines were being isolated. Imidazolinones inhibit acetohydroxyacid synthase (AHAS EC 4.1.3.18), the first enzyme in the pathway of branched chain amino acid synthesis (8). Imidazolinone-resistant cell lines provide proof that inhibition of AHAS is the site of action of the imidazolinones AHAS activities in extracts from resistant corn cell lines are highly resistant to inhibition by imidazolinone herbicides (7). 

Figure 2. Inhibition of acetohydroxyacid synthase from sensitive corn (B73 — ) and three imidazolinone-resistance mutants (XA17 A—A, XI12B— , and QJ22 Y—Y) by imazaquin.

In these reactions, the C2-atom of ThDP must be deprotonated to allo v this atom to attack the carbonyl carbon of the different substrates. In all ThDP-dependent enzymes this nucleophilic attack of the deprotonated C2-atom of the coenzyme on the substrates results in the formation of a covalent adduct at the C2-atom of the thiazolium ring of the cofactor (Ila and Ilb in Scheme 16.1). This reaction requires protonation of the carbonyl oxygen of the substrate and sterical orientation of the substituents. In the next step during catalysis either CO2, as in the case of decarboxylating enzymes, or an aldo sugar, as in the case of transketo-lase, is eliminated, accompanied by the formation of an a-carbanion/enamine intermediate (Ilia and Illb in Scheme 16.1). Dependent on the enzyme this intermediate reacts either by elimination of an aldehyde, such as in pyruvate decarboxylase, or with a second substrate, such as in transketolase and acetohydroxyacid synthase. In these reaction steps proton transfer reactions are involved. Furthermore, the a-carbanion/enamine intermediate (Ilia in Scheme 16.1) can be oxidized in enzymes containing a second cofactor, such as in the a-ketoacid dehydrogenases and pyruvate oxidases. In principal, this oxidation reaction corresponds to a hydride transfer reaction. 

Acetohydroxyacid synthase (AHAS), formerly referred to as acetolactate synthase, is involved in the biosynthesis of branched-chain amino acids in plants and many microorganisms. " AHAS catalyzes the condensation of two molecules of pyruvate to form acetolactate and CO2, or the condensation of one molecule of pyruvate with one molecule of cr-ketobutyrate to form cr-aceto-cr-hydroxybutyrate and CO2 (Equation (24)). 

Recent work on pyruvate-ferredoxin oxidoreductase also suggested that the enzyme proceeds via a free-radical mechanism. While it is not formally a redox enzyme, the class of enzymes named acetohydroxyacid synthases and acetolactate synthases also have FAD in addition to the ThDP, although the function of FAD is still somewhat uncertain since the carholigase reactions carried out hy these enzymes have no immediately obvious need for an oxidizing coenzyme (there is a detailed discussion of these issues in Kluger and Pike" ). 

Special prize Study of acetohydroxyacid synthase through mutation and QSAR by Z. Xi et al., Nankai Univ., China. 

The first committed step in the biosynthetic pathway of the branched chain amino acids is catalyzed by the enzyme acetohydroxyacid synthase (AHAS, EC 2.2.1.6), which is also referred to as acetolactate synthase (ALS). As depicted in Fig. 2.1.1, the pathway leading to valine and leucine begins with the condensation of two molecules of pyruvate accompanied by loss of carbon dioxide to give (S)-2-acetolactate. A parallel reaction leading to isoleucine involves the condensation of pyruvate with 2-ketobutyrate to afford (S)-2-aceto-2-hydroxybutyrate after loss of carbon dioxide. Both reactions are catalyzed by AHAS, which requires the cofactors thiamin diphosphate (ThDP) and flavin adenine dinudeotide (FAD). A divalent metal ion, most commonly is also required. Several excellent reviews... 

J. Biochem. Mol. Biol., 2000, 33, 1-36. Duggleby, R. G., Guddat, L W., Pang, S. S., Structure and Properties of Acetohydroxyacid Synthase in Thiamine Catalytic Mechanisms in Normal and Disease States, Vol. 11,... 

I midazolinone-Acetohydroxyacid Synthase Interactions in The Imidazolinone Herbicides, Shaner,... 

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. 

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