Metabolic acetolactate synthase

Hydroxy-2-butanone (acetoin) is a characteristic constituent of butter flavour used for flavouring margarine and can be obtained as a by-product of molasses-based and lactic acid fermentations [49, 71]. The closely related 2,3-butanedione (diacetyl) has a much lower organoleptic threshold than acetoin and is an important strongly butter-like flavour compound in butter and other dairy products [72] in buttermilk, for instance, the diacetyl concentration is only about 2-4 mg [73]. a-Acetolactate (a-AL) is an intermediate of lactic acid bacteria mainly produced from pyruvate by a-acetolactate synthase. In most lactic acid bacteria, a-AL is decarboxylated to the metabolic end product acetoin by a-AL decarboxylase (ALDB) [71] (Scheme 23.5). 

Scheme 23.5 Metabolic pathways of lactic acid bacteria leading from pyruvate to a-acetolactate and acetoin and chemical diacetyl formation. ALS a-acetolactate synthase, ALDB a-acetolactate decarboxylase, DDH diacetyl dehydrogenase. (Adapted from [72])...

Most known thiamin diphosphate-dependent reactions (Table 14-2) can be derived from the five halfreactions, a through e, shown in Fig. 14-3. Each half-reaction is an a cleavage which leads to a thiamin- bound enamine (center. Fig. 14-3) The decarboxylation of an a-oxo acid to an aldehyde is represented by step h followed by fl in reverse. The most studied enzyme catalyzing a reaction of this type is yeast pyruvate decarboxylase, an enzyme essential to alcoholic fermentation (Fig. 10-3). There are two 250-kDa isoenzyme forms, one an tetramer and one with an (aP)2 quaternary structure. The isolation of a-hydroxyethylthiamin diphosphate from reaction mixtures of this enzyme with pyruvate provided important verification of the mechanisms of Eqs. 14-14,14-15. Other decarboxylases produce aldehydes in specialized metabolic pathways indolepyruvate decarboxylase in the biosynthesis of the plant hormone indole-3-acetate and ben-zoylformate decarboxylase in the mandelate pathway of bacterial metabolism (Chapter 25). Formation of a-ketols from a-oxo acids also starts with step h of Fig. 14-3 but is followed by condensation with another carbonyl compound in step c, in reverse. An example is decarboxylation of pyruvate and condensation of the resulting active acetaldehyde with a second pyruvate molecule to give l -a-acetolactate, a reaction catalyzed by acetohydroxy acid synthase (acetolactate synthase). Acetolactate is the precursor to valine and leucine. A similar ketol condensation, which is catalyzed by the same S5mthase, is... 

Aristidou, A.A. San, K.Y. Bennett, G.N. Modification of central metabolic pathway in Escherichia coli to reduce acetate accumulation by heterologous expression of the Bacillus subtilis acetolactate synthase gene. Biotechnol. Bioeng. 1994, 44 (8), 944-951. 

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 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. 

Acetolactate synthase inhibitors, response patterns from metabolic profiling, 293,294/... 

Acetolactate synthase (ALS) is the enzyme target site of the sulfonyl-ureas. In common with the imidazolinone aryl carboxylates, these herbicides inhibit valine and isoleucine biosynthesis. The imidazolinones are exemplified by Assert (8), which consists of a mixture of m- and p-isomers. The selectivity of ALS inhibitors invariably can be accounted for by differential metabolism or uptake or related phenomena rather than by any significant inherent difference in the properties of the ALS enzymes of crop and weed species. Assert is another example of a herbicide activated in plants by deesterification to the phytotoxic acid, and susceptible species such as Avena fatua (wild oat) are unable to metabolize the molecule further. Facile ring-methyl hydroxylation to the m- and p-benzyl alcohols, however, followed by glycosylation confers tolerance to maize and wheat." ... 

Figure 1.2. Citrate metabolism in Lactococcus, Leuconostoc, and Weissella species. Key for the enzymes CL, citrate lyase OAD, oxaloacetate decarboxylase LDH, lactate dehydrogenase PDC, pyruvate decarboxylase ALS, a-acetolactate synthase ADC, a-acetolactate decarboxylase DAR, diacetyl acetoln reductase BDH, 2,3-butanediol dehydrogenase Tppi, thiamine pyrophosphate.

Platteeuw, C., Hugenholtz, J., Starrenburg, M., et al. (1995) Metabolic engineering of Lactococcus lactis influence of the overproduction of alpha-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions. AppZ Environ Microbiol 61, 3967—3971. 

The first step in valine biosynthesis is a condensation between pyruvate and active acetaldehyde (probably hy-droxyethyl thiamine pyrophosphate) to yield a-acetolactate. The enzyme acetohydroxy acid synthase usually has a requirement for FAD, which, in contrast to most flavopro-teins, is rather loosely bound to the protein. The very same enzyme transfers the acetaldehyde group to a-ketobutyrate to yield a-aceto-a-hydroxybutyrate, an isoleucine precursor. Unlike pyruvate, the a-ketobutyrate is not a key intermediate of the central metabolic routes rather it is produced for a highly specific purpose by the action of a deaminase on L-threonine as shown in figure 21.10. 

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