Enzyme acetohydroxyacid synthase

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... 

Branched-chain amino acids valine, leucine, and isoleucine are biosynthesized in a pathway catalyzed by the enzyme acetohydroxyacid synthase (AHAS), sometimes referred to as acetolactate synthase (ALS). There are... 

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). 

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. 

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]. 

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). 

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. 

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" ). 

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. 

The subsequent conversion of 2-oxobutyrate to isoleucine involves four enzymes. The same enzymes are considered to participate in the biosynthesis of valine (Fig. 4). Thus, 2-oxobutyrate and its three carbon analogue, pyruvate, would be alternate substrates of acetohydroxyacid synthase. This parallel reaction sequence (Fig. 4) is initiated by the addition of a two-carbon fragment to the 2-carbon of the 2-oxobutyrate or pyruvate. The resultant acetohydroxyacids are reduced with concomitant isomerization to form dihydroxy acids. Dehydration yields oxoacids which are then transaminated to synthesize isoleucine and valine. Both 2-oxoisovalerate and 2-oxo-3-methyl-valerate have been identified as components of plant extracts (Kretovich and Gejko, 1964). 

Although pyruvate and 2-oxobutyrate are substrates of acetohydroxyacid synthase, measurements of the activity of this enzyme have been almost exclusively based on the production of acetolactate from pyruvate. This reaction product is readily decarboxylated under acidic conditions and the acetoin produced can be measured spectrophotometrically. However, ace-toin can be formed during reactions which need not be related to amino acid biosynthesis. Therefore it is unclear whether the enzyme activity characterized by Saytanarayana and Radhakrishnan (1963) can be completely ascribed to acetohydroxyacid synthase. Only a portion of the acetolactate forming activity measured in pea extracts was considered to represent the activity of this enzyme (Davies, 1964). However, the enzyme(s) isolated from barley was shown to facilitate formation of acetohydroxy derivatives of 2-oxobutyrate and pyruvate (Miflin, 1971). Mg or Mn " " as well as the substrate, hydroxyethylthiamine-pyrophosphate, was required for maximum enzyme activity. The fact that the acetolactate forming activity of the barley... 

Regulation of the synthesis of the branched-chain amino acids, like that of the aspartate family, can be viewed in a temporal framework (Fig. 8). However, the nature of the controls associated with the pathway enzymes do not necessarily suggest an obligatory sequence of regulatory interactions. The sequence illustrated in Fig. 8 assumes that each of the end-products would initially be synthesized from its respective precursors. As isoleucine biosynthesis is reduced by inhibition of threonine dehydratase, the competition between pyruvate and 2-oxobutyrate for the active site of acetohydroxyacid synthase would be diminished. This could result in an increased rate of synthesis of leucine and valine (Fig. 8, 2). Leucine would eventually inhibit isopropylmalate synthase and, to a lesser extent, acetohydroxyacid synthase (Fig. 8, 3). The reduced flow of carbon through the pathway would be utilized for the synthesis of valine. As the concentration of valine increased, the activity of acetohydroxyacid synthase would be sharply curtailed due to... 

In the last decade, some new enzymes were added to this set of tools, like acetohydroxyacid synthase or yersinose A. °... 

Three enzymes are involved in the synthesis of 2,3-BD a-acetolactate synthase (EC 4.1.3.18), a-acetolactate decarboxylase (EC 4.1.1.5), and butanediol dehydrogenase (also known as diacetyl [acetoin] reductase Larsen and Stormer 1973 Johansen et al. 1975 Stormer 1975). Two different enzymes form acetolactate from pyruvate. The first, termed catabolic a-acetolactate synthase, has a pH optimum of 5.8 in acetate and is part of the butanediol pathway. The other enzyme, termed anabolic a-acetolactate synthase or acetohydroxyacid synthetase, has been well studied and characterized and will not be discussed here. This enzyme is part of the biosynthetic pathway for isoleucine, leucine, and valine and is coded for by the ilvBN, ilvGM, and ilvH genes in E. colt and Salmonella typhimurium (Bryn and Stormer 1976). 

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