Pyruvate, condensation with thiamin

First a pyruvate molecule condenses with thiamin pyrophosphate at the thiazolium ring carbon with subsequent loss of carbon dioxide. Then a second pyruvate (or cx-ketobutyrate) condenses followed by loss of acetolactate and regeneration of the thiazolium ring. 

Diacetyl can be produced by either homolactic or heterolactic pathways of sugar metabolism (via free pyruvate) or by utilization of citric acid (see Figs. 1-1 lA and 1-1 IB). In this case, citric acid is first converted to oxaloacetic and acetic acids. The former is then decarboxylated to pyruvate which undergoes a second decarboxylation and condensation with thiamine pyrophosphate (TPP) to yield active acetaldhyde, which reacts with another pyruvate to yield a-acetolactate which undergoes oxidative decarboxylation to yield diacetyl and its equilibrium products see Fig. 1-11 A. In the case of other LAB, the precursor, a-acetolactate is not produced. Here active acetaldehyde, produced as described above, reacts with acetyl CoA to yield diacetyl see Fig. 1-1 IB. 

Diacetyl may be synthesized by either homolactic or heterolactic pathways of sugar metabolism as well as by utilization of citric acid (Fig. 2.9). Citric acid is hrst converted to acetic acid and oxaloacetate the latter is then decarboxylated to pyruvate. Although earlier reports indicated that diacetyl synthesis by lactic acid bacteria does not proceed via a-acetolactate (Gottschalk, 1986), more recent evidence suggests that this pathway is active in lactic acid bacteria (Ramos et al., 1995). Here, pyruvate undergoes a second decarboxylation and condensation with thiamine pyrophosphate (TPP) to yield active acetaldehyde. This compound then reacts with another molecule of pyruvate to yield a-acetolactate, which, in... 

Ketols can also be formed enzymatically by cleavage of an aldehyde (step a, Fig. 14-3) followed by condensation with a second aldehyde (step c, in reverse). An enzyme utilizing these steps is transketolase (Eq. 17-15),132b which is essential in the pentose phosphate pathways of metabolism and in photosynthesis. a-Diketones can be cleaved (step d) to a carboxylic acid plus active aldehyde, which can react either via a or c in reverse. These and other combinations of steps are often observed as side reactions of such enzymes as pyruvate decarboxylase. A related thiamin-dependent reaction is that of pyruvate and acetyl-CoA to give the a-diketone, diacetyl, CH3COCOCH3.133 The reaction can be viewed as a displacement of the CoA anion from acetyl-CoA by attack of thiamin-bound active acetaldehyde derived from pyruvate (reverse of step d, Fig. 14-3 with release of CoA). 

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 pathway also operates in some bacteria and apparently is the sole source of isoprenoid compounds for the unicellular alga Scenedesmus.28 The pathway is outlined in Fig. 22-2. Pyruvate is decarboxylated by a thiamin diphosphate-dependent enzyme,29 and the resulting enamine is condensed with D-glyceraldehyde 3-phosphate to form 1-deoxyxylulose 5-phosphate.28, i0 31a The latter undergoes an isomeroreductase rearrange-... 

Decarboxylation of an a-keto acid like pyruvate is a difficult reaction for the same reason as are the ketol condensations (see fig. 12.33) Both kinds of reactions require the participation of an intermediate in which the carbonyl carbon carries a negative charge. In all such reactions that occur in metabolism, the intermediate is stabilized by prior condensation of the carbonyl group with thiamine pyrophosphate. In figure 13.5 thiamine pyrophosphate and its hydroxyethyl derivative are written in the doubly ionized ylid form rather than the neutral form because this is the form that actually participates in the reaction even though it is present in much smaller amounts. 

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... 

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. 

In nature, an enzyme requiring two co-factors, thiamine diphosphate 2 and flavin adenine dinucleotide, accomplishes the oxidation of pyruvate to acetyl phosphate. The thiazole ring in thiamine condenses at the 2-position with pyruvat eliminating carbon dioxide to give an activated species that is oxidised by the flavin. An enzymatic oxidation process then reactivates the reduced flavin. The redox... 

The terpenes, carotenoids, steroids, and many other compounds arise in a direct way from the prenyl group of isopentenyl diphosphate (Fig. 22-1).16a Biosynthesis of this five-carbon branched unit from mevalonate has been discussed previously (Chapter 17, Fig. 17-19) and is briefly recapitulated in Fig. 22-1. Distinct isoenzymes of 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) in the liver produce HMG-CoA destined for formation of ketone bodies (Eq. 17-5) or mevalonate.7 8 A similar cytosolic enzyme is active in plants which, collectively, make more than 30,000 different isoprenoid compounds.910 However, many of these are formed by an alternative pathway that does not utilize mevalonate but starts with a thiamin diphosphate-dependent condensation of glyceraldehyde 3-phosphate with pyruvate (Figs. 22-1,22-2). 

The identity of the enzyme(s) involved in the latter reaction has been debated (13). However, the formation of the above hydro-xyketone, in analogy with acetoin, has been conceptualized as the consequence of the condensation of the "active" form of acetaldehyde, that is formed by decarboxylative addition of pyruvate to thiamine pyrophospate, with benzaldehyde.The role of pyruvate, in fact has been established. The same mechanism can be invoked for the reaction of cinnamaldehyde.lt is known that the pyruvate decarboxylase (E.C. 4.1.1.1) accepts as substrates a-oxoacids... 

Biomimetic Synthesis of Solerone. We applied pyruvate decarboxylase [EC 4.1.1.1] (PDC) as key enzyme for the biomimetic synthesis elucidating the formation of solerone 1 figure 1). The thiamine diphosphate depending enzyme from Saccharomyces cerevisiae is responsible for the decarboxylation of pyruvate in the course of alcoholic fermentation. After loss of carbon dioxide from 2-oxoacids the resulting aldehyde is released. Alternatively, the cofactor-bound decarboxylation product can react with a further aldehyde. By the latter acyloin condensation a new carbon-carbon bond will be formed, thus opening a biosynthetic way to a-hydroxy carbonyl compounds 11J2). 

Pyruvate decarboxylase catalyzes the nonoxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. When an aldehyde is present with pyruvate, the enzyme promotes an acyloin condensation reaction. The mechanistic reason for this fortuitous reaction is well understood and involves the aldehyde outcompeting a proton for bond formation with a reactive thiamine pyrophosphate-bound intermediate (90,91). When acetaldehyde is present, the product formed is acetoin. Benzalde-hyde results in the production of phenylacetylcarbinol (Fig. 26). Both of these condensations are enantioselective, forming the R enantiomer preferentially in both cases. 

Studies on thiamine (vitamin Bi) catalyzed formation of acyloins from aliphatic aldehydes and on thiamine or thiamine diphosphate catalyzed decarboxylation of pyruvate have established the mechanism for the catalytic activity of 1,3-thiazolium salts in carbonyl condensation reactions. In the presence of bases, quaternary thiazolium salts are transformed into the ylide structure (2), the ylide being able to exert a cat ytic effect resembling that of the cyanide ion in the benzoin condensation (Scheme 2). Like cyanide, the zwitterion (2), formed by the reaction of thiazolium salts with base, is nucleophilic and reacts at the carbonyl group of aldehy s. The resultant intermediate can undergo base-catalyzed proton... 

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... 

Yeasts also make use of pyruvic acid to form acetoin, diacetyl and 2,3-butanediol (Figure 2.17). This process begins with the condensation of a pyruvate molecule and active acetaldehyde bound to thiamine pyrophosphate, leading to the formation of cr-acetolactic acid. The oxidative decarboxylation of a-acetolactic acid produces diacetyl. Acetoin is produced by either the non-oxidative decarboxylation of a-acetolactic acid or the reduction of diacetyl. The reduction of acetoin leads to the formation of 2,3-butanediol this last reaction is reversible. 

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