A-Acetolactate

INSULIN AND OTFiER ANTIDIABETIC AGENTS] (Vol 14) a-Acetolactate decarboxylase [9025-02-9]... 

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])...

In addition, cofactor engineering has been used to deliberately modify the intracellular NADH/NAD+ ratio that plays a predominant role in controlling the Lactococcus lactis fermentation pattern. The introduction of the nox gene, which codes for a NADH oxidase (NOX) that converts molecular oxygen to water at the expense of NADH, to a strain with an inactivated copy of the aldB gene for a-acetolactate decarboxylase led to the efficient metabolism of the na-... 

Figure 13.8 Pathway for metabolism of citrate by Leuconostoc spp. and S. lactis subsp. diacetylactis. (1) Citrate permease, (2) citrate lyase, (3) oxaloacetic acid decarboxylase, (4) pyruvate decarboxylase, (5) a-acetolactate synthetase, (6) a-acetolactate carboxylase, (7) diacetyl synthetase, (8) diacetyl reductase, and (9) acetoin reductase.

Enterobacter aerogenes, B. subtilis, P. fluorescens, and Serratia marces-cens produce acetoin by decarboxylation of a-acetolactate. However, yeasts and E. coli form acetoin from the acetaldehyde-TPP complex and free acetaldehyde (Rodopulo et al 1976). These organisms do not decarboxylate a-acetolactate, but use it to produce valine and pantothenic acid. In lactic acid bacteria, a-acetolactate is not used for valine or pantothenic acid synthesis, since these substances are required for growth (Law et al. 1976B Reiter and Oram 1962). In those microorganisms which can synthesize valine, this amino acid inhibits a-acetolactate synthesis (Rodopulo et al 1976). 

Formation of a-ketols from a-oxo acids also starts with step b 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 R-a-acetolactate, a reaction catalyzed by acetohydroxy acid synthase (acetolactate synthase).128 Acetolactate is the precursor to valine and leucine. A similar ketol condensation, which is catalyzed by the same synthase, is... 

The third type of carbon-branched unit is 2-oxoisovalerate, from which valine is formed by transamination. The starting units are two molecules of pyruvate which combine in a thiamin diphosphate-dependent a condensation with decarboxylation. The resulting a-acetolactate contains a branched chain but is quite unsuitable for formation of an a amino acid. A rearrangement moves the methyl group to the (3 position (Fig. 24-17), and elimination of water from the diol forms the enol of the desired a-oxo acid (Fig. 17-19). The precursor of isoleucine is formed in an analogous way by condensation, with decarboxylation of one molecule of pyruvate with one of 2-oxobutyrate. 

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. 

Richelieu, M., Hoalberg, U., and Nielsen, J. C. (1997). Determination of a-acetolactic acid and volatile compounds by head-space gas chromatography. ]. Dairy Sci. 80, 1918-1925. 

As a side activity, many decarboxylases catalyze the formation of C-C bonds. In the reaction of two pyruvate molecules, catalyzed by pyruvate decarboxylase (PDC, E.C. 4.1.1.1), a-acetolactate is formed, an important intermediate of valine biosynthesis. In turn, a-acetolactale can be oxidatively decarboxylated by oxygen to diacetyl or enzymatically decarboxylated by acetolactate decarboxylase (ADC, E.C. 4.1.1.5) to (] )-acetoin (Figure 7.29). 

D- or L-lactic acid -- pyruvic acid ------------ - a-acetolactic acid... 

The newest enzyme for use in beer is acetolactate decarboxylase, used to decrease the fermentation time, by avoiding the formation of diacetyl. Externally or internally produced a-acetolactate decarboxylase transforms the a-acetolactate to acetoin (acetylmethylcarbinol) without the enzyme, acetolactate goes to diacetyl, and then a secondary fermentation slowly reduces it to acetoin. Avoiding or reducing the secondary fermentation results in significant reduction in storage capacity and money tied up in inventory Q). Normally acetolactate forms by the thiaminepyrophosphate-catalyzed acyloin condensation of acetaldehyde and pyruvic acid (2) or by the condensation of two pyruvic acid molecules to yield acetolactate and CC. Acetolactate is important in the synthesis of isoleucine and valine by the yeast. The acetolactate left at the end of the primary fermentation is oxidized spontaneously in a nonenzymatic reaction to diacetvl and C0.> (Eqn. 1)... 

Application and Principle This procedure is used to determine a-acetolactatedecarboxylase (ALDC) activity, expressed as ADU/g. The assay is based on the decarboxylation of a-aceto-lactate to acetoin by a-Acetolactatedecarboxylase. Acetoin is reacted with a mixture of naphthol and creatine to form a red color for which absorbance is measured at 522 ran. The assay is based on a 20-min decarboxylation of the a-acetolactate substrate at 30° and at pH 6.0. 

Procedure Preheat the Enzyme Sample solutions, the MES Buffer, and the a-Acetolactate Substrate in a 30° water bath for approximately 10 min. Prepare the following solutions for each enzyme sample in the following order ... 

Sample Value HI Pipet 200 pL of the Enzyme Sample solution into a 10-mL test tube in the 30° water bath. Add 200 pL of a-Acetolactate Substrate, mix, and immediately replace the test tube into the water bath. 

Calculation One a-acetolactatedecarboxylase unit (ADU/g) is the amount of enzyme that produces 1 pmol of acetoin per min by decarboxylation of a-acetolactate under the given reaction conditions. 

Diacetyl, and its reduction products, acetoin and 2,3-butanediol, are also derived from acetaldehyde (Fig 8D.7), providing additional NADH oxidation steps. Diacetyl, which is formed by the decarboxylation of a-acetolactate, is regulated by valine and threonine availability (Dufour 1989). When assimilable nitrogen is low, valine synthesis is activated. This leads to the formation of a-acetolactate, which can be then transformed into diacetyl via spontaneous oxidative decarboxylation. Because valine uptake is suppressed by threonine, sufficient nitrogen availability represses the formation of diacetyl. Moreover, the final concentration of diacetyl is determined by its possible stepwise reduction to acetoin and 2,3-butanediol, both steps being dependent on NADH availability. Branched-chain aldehydes are formed via the Ehrlich pathway (Fig 8D.7) from precursors formed by combination of acetaldehyde with pyruvic acid and a-ketobutyrate (Fig 8D.7). 

Unfortunately diacetyl formation is still not well understood. Acetoin formation occurs either by nonspecific interaction of acetaldehyde with the a-hydroxyethyl thiamine pyrophosphate intermediate in pyruvate decarboxylation (209) or by decarboxylation of a-acetolactate (210), which in turn arises either from interaction of pyruvate with a-hydroxyethyl thiamine pyrophosphate (211) or as a specific intermediate in valine biosynthesis (212, 213). Diacetyl does not appear to be formed directly from acetoin (208, 214). It is formed from a-acetolactate, in absence of cells, by O2 oxidation (215), and even under N2 (216), although an oxidation must occur. It is also formed from acetyl CoA (217, 218), probably by interaction with a-hydroxyethyl thiamine pyrophosphate [cf. stimulation by acetyl CoA addition to a solution of pyruvate and pyruvate decarboxylase (2i5)]. It is not known whether this involves a specific enzyme or is a mere side reaction. 

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... 

Production of organic acids is found among various bacterial and fungal species. This is particularly common among all lactic acid bacteria (LAB) (Vesterlund et al., 2004). Heterofermentative LAB are able to ferment various organic acids, predominantly citrate, malate, and pyruvate (Zaunmuller et al., 2006). In various species and strains of LAB, organic acid production may, however, vary. For example, in Lactococcus ladus pyruvate is partially converted to a-acetolactate when electron acceptors (such as citrate) are present, whereas Lactobacillus sanfranciscensis... 

The syntheses of valine, leucine, and isoleucine from pyruvate are illustrated in Figure 14.9. Valine and isoleucine are synthesized in parallel pathways with the same four enzymes. Valine synthesis begins with the condensation of pyruvate with hydroxyethyl-TPP (a decarboxylation product of a pyruvate-thiamine pyrophosphate intermediate) catalyzed by acetohydroxy acid synthase. The a-acetolactate product is then reduced to form a,/3-dihydroxyisovalerate followed by a dehydration to a-ketoisovalerate. Valine is produced in a subsequent transamination reaction. (a-Ketoisovalerate is also a precursor of leucine.) Isoleucine synthesis also involves hydroxyethyl-TPP, which condenses with a-ketobutyrate to form a-aceto-a-hydroxybutyrate. (a-Ketobutyrate is derived from L-threonine in a deamination reaction catalyzed by threonine deaminase.) a,/3-Dihydroxy-/3-methylvalerate, the reduced product of a-aceto-a-hydroxybutyrate, subsequently loses an HzO molecule, thus forming a-keto-/kmethylvalerate. Isoleucine is then produced during a transamination reaction. In the first step of leucine biosynthesis from a-ketoisovalerate, acetyl-CoA donates a two-carbon unit. Leucine is formed after isomerization, reduction, and transamination. 

The product, a-acetolactate, is formally derived from the acyl carbanion generated by the decarboxylation of pyruvate with a second molecule of pyruvate. The formation of lactylthiamin diphosphate on the enzyme generates initially the unprotonated adduct, which contains the enamine equivalent of a carbanion. This can add to the carbonyl group of a second molecule of pyruvate to form acetolactate (Scheme 35). 

In the biosynthesis of valine 179 the first step is a benzoin condensation of 2 mol of pyruvate 95 to yield a-acetolactate 217. A single enzyme, a-acetohydroxyacid reductoisomerase (EC 1.1.1.86), then catalyzes the re-arrangement/reduction steps 217->218- 219 (Scheme 61). The same enzyme catalyzes a similar sequence implied in 222->223 in the biosynthesis of isoleucine 212. The substrates 217 and 222 for these reactions have been shown to have the (2S) configuration (189,190), the product 223 to have the (2R,3R) configuration (191-194), and the product 219 to have (IR) configuration (192,193). 

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