2-Oxoisovaleric acid

Biosynthesis In microorganisms and plants from pyruvic acid 2 pyruvate- 2-acetolactic acid (acetolactate synthase, EC 4.1.3.18 coenzyme thiamin(e) diphosphate)- 2,3-dihydroxyisovaleric acid (2-acetolactate mutase, EC 5.4.99.3)- 2-oxoisovaleric acid (dihydroxy acid dehydratase, EC 4.2.1.9). This is finally am-inated by branched chain amino acid aminotransferase (EC 2.6.1.42). 2-Oxoisovaleric acid is also a precursor of Leu. 

The production of oxoisovaleric acid and oxoisocaproic acids, intermediates in the biosynthesis of valine and leucine respectively, is shown in Fig. 17.16. The decarboxylation and reduction (NAD+ dependent) of these oxo-acids yields the fusel alcohols isobutanol and isoamyl alcohol (Fig. 17.16). Presumably, some oxo-acids in beer result from excretion from the pool and some aldehydes by excretion prior to reduction to the corresponding alcohol. Table 17.7 shows the chemical relationships between alcohols, aldehydes, oxo-acids and corresponding amino acids. 

Pantothenic acid is synthesized in plants and some microorganisms from pantoic acid and p-alanine. Pantoic acid is formed from 2-oxopantoic acid (4-hydroxy-3,3-dimethyl-2-oxobutyric acid) and 2-oxoisovaleric acid (3-methyl-2-oxobu-tyric acid), a precursor of valine. P-Alanine is formed by decarboxylation of l-aspartic acid. Enzymes involved include pantothenate synthetase (EC 6.3.2.1), oxopantoate reductase (EC 1.1.1.169), oxopantoate hydroxymethyltransferase (EC 4.1.2.12), and aspartate 1-decarboxylase (EC 4.1.1.12). 

Pantothenic acid is an essential nutritional factor for a range of yeasts, lactic acid and propionic acid bacteria and other microorganisms. Some bacteria and plants synthesise this acid de novo from pantoic acid and P-alanine. The biosynthesis of pantoic acid uses 3-methyl-2-oxobutanoic (2-oxoisovaleric) acid, which is a precursor of valine, the donor of the hydroxymethyl group is 5,10-methylenetetrahydrofoUc acid, and decarboxylation of aspartic acid yields the P-alanine. 

Fig. 5.18 shows the spectrum of 2-oxoisovaleric acid as its TMS-oxime derivative. The TMS groups give rise to the silyl ions at mjz 73, 75, 147, as already described, which together with M, M —15, M—43 and M-117 are also present in the spectrum of derivatized phenylpyruvic acid (Fig. 5.18). The aromatic ring helps to stabilize the molecular ion, while at the same time directing fragmentation of the benzyl bond to give mjz 91 (CeHgCH. The ion at mjz 189 is formed by the sequential loss of the ester and a methyl radical from the oxime silyl group (-CH3-CO2TMS).

Fig. 10.3 Chromatogram of organic acids extracted from the urine of an untreated patient with branched-chain keto aciduria (maple syrup urine disease), extracted and separated as described in the legend to Fig. 10.2. The chromatogram illustrates the overlapping peaks in the regions occupied by 3-hydroxybutyric, 2-hydroxyisovaleric and 2-oxoisovaleric acids (peak 1) and 2-oxo-3-methyl-valeric, 2-hydroxyisocaprioic and 2-oxoisocaproic acids (peak 2) and phosphate (peak 3). Other peaks of interest are (4) citric, (5) 4-hydroxyphenyl-lactic, (6) 4-hydroxyphenylpyruvic, (7) n-tetracosane (standard) and (8) -hexacosane (standard). (Compare with Fig. 10.4.)...

This enzyme complex [EC 1.2.4.4], also known as 3-methyl-2-oxobutanoate dehydrogenase (lipoamide) and 2-oxoisovalerate dehydrogenase, catalyzes the reaction of 3-methyl-2-oxobutanoate with lipoamide to produce S-(2-methylpropanoyl)dihydrolipoamide and carbon dioxide. Thiamin pyrophosphate is a required cofactor. The complex also can utilize (5)-3-methyl-2-oxopenta-noate and 4-methyl-2-oxopentanoate as substrates. The complex contains branched-cham a-keto acid decarboxylase, dihydrolipoyl acyltransferase, and dihydrolipoa-mide dehydrogenase [EC 1.8.1.4]. 

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. 

A. iEvarsson, K. Seger, S. Turley, J.R. Sokatch, and W.M.J. Hoi. 1999. Crystal structure of 2-oxoisovalerate and dehydrogenase and the architecture of 2-oxo acid dehydrogenase multiple enzyme complexes Nat. Struct. Biol. 6 785-792. (PubMed)... 

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). 

The sequences of biochemical transformations involved in the synthesis of the aspartate family and branched-chain amino acids in multicellular plants are similar to those that occur in microorganisms. Support for this conclusion has been derived principally from isolation of a number of the requisite enzymes. Information on the kinetic and physical properties of enzymes is best achieved after extensive purification. In contrast, useful predictions of the physiological function of regulatory enzymes depend upon effective enzyme extraction and complete preservation of native properties. Since the latter objective has been emphasized during most investigations of enzymes associated with amino acid biosynthesis in plants, the bulk of our knowledge has been obtained from comparatively crude enzyme preparations. Results of both direct and competitive labeling experiments have added demonstrations of many of the predicted precursor-product relationships and a few metabolic intermediates have been isolated from plants. The nature of a number of intermediate reactions does, however, remain to be clarified notably, the reactions associated with the conversion of dihydropicolinate to lysine and those involved in the synthesis of leucine from 2-oxoisovalerate. 

Fusel alcohol formation is linked to amino acid biosynthesis, and the presence of an amino acid in wort may inhibit the formation of the corresponding fusel alcohol. This usually results from the end product of an anabolic pathway (e.g. valine. Fig. 17.16) inhibiting the operation of the first step (a-acetolactate synthetase) and thus preventing synthesis of the oxo-acid (oxoisovaleric). In defined media, such regulatory effects are... 

Fig. 4.3 Chromatogram of standard oxo acids separated as their trimethysilyl-oxime derivatives on 3 per cent OV-17 using temperature programming from 110°C to 220°C. Peak identifications are 1, pyruvate 2, 2-oxobutyrate 3, 2-oxoisovalerate 4, 2-0X0valerate 5, L-2-oxo-3-methylvalerate 6, 2-oxoisocaproate plus 7, d-2-oxo-3-methylvalerate 8, 2-oxo-4-methylthiobutyrate 9, 2-oxoglutarate 10, phenylpyruvate. (Redrawn with modifications from Sternowsky etal., 1973)...

Oast house urine disease (methionine malabsorption syndrome, 2-hydroxybutyric aciduria) Methionine, 2-hydroxybutyric, phenylpyruvic and the branched-chain keto acids especiaUy 2-oxoisovaleric Methionine malabsorption (amino acid-transport defect) (Chapter 16)... 

Fig. 10.1 Metabolites in the urine of an untreated patient with branched-chain keto aciduria (maple syrup urine disease). Extracted using ethyl acetate and separated as their trimethylsilyl-oxime derivatives on a 25 m SE-30 capillary column, using temperature programming from 80°C to 110°C at 0.5°C min and an injection split ratio 1 12 at a temperature of 250°C. The peaks marked R are due to solvent and reagents. Peak identifications are 1, lactic 2, 2-hydroxyisobutyric 3, 2-hydroxybutyric 4, pyruvic 5, 3-hydroxybutyric 6, 2-hydroxyisovaleric 7, 2-oxobutyric 8, 2-methyl-3-hydroxy-isovaleric 10, a and b, 2-oxoisovaleric 11, acetoacetic 12, 2-hydroxyisocaproic 13, 2-hydroxy-3-methyl- -valeric 14, 2-oxo-3-methyl-/i-valeric (14a L- 14b D-) 15, 2-oxoisocaproic acids. The internal standard was malonic acid. (Redrawn with modifications from Jellum etal., 1976)...

Fig. 10.4 Chromatogram of organic acids extracted from the same urine of the patient illustrated in Fig. 10.3, separated as their O-n-butyloxime and trimethylsilyl derivatives under the conditions described in the legend to Fig. 10.2. Peak identifications are 1, lactate 2, glycollate 3, unidentified 4, 2-hydroxy-n-butyrate plus 3-hydroxy-propionate 5, sulphate 6, 3-hydroxybutyrate and 3-hydroxyisobutyrate 7, 2-hydroxyisovalerate 8, 3-hydroxyisovalerate 10, 2-hydroxyisocaproate 11,2-hydroxy-3-methyl-n-valerate 12, phosphate 13,2-oxoisovalerate (peak 1) 14,2-oxoisovalerate (peak 2) 15, 2-oxo-3-methyl-/i-valerate (peak 1) 16, 2-oxo-3-methyl-n-valerate (peak 2) 17, 2-oxoisocaproate 18, citrate 19, 4-hydroxyphenyl-lactate 20, n-tetracosane (standard) 21, n-hexacosane (standard). The separation of O-n-butyloxime-TMS derivatives of the keto acids from the TMS-hydroxy acids and other components is apparent (compare with Fig. 10.3). Note the apparent loss of 4-hydroxyphenylpyruvate. (The horizontal axis represents the time elapsed in minutes from sample injection.)...

Fig. 10.5 Mass spectra of the ethoxime-trimethylsilyl derivatives of (a) 2-oxoisovaleric, (b) 2-oxo-3-methyl-n-valeric (peak 1), (c) 2-oxo-3-methyl-/i-valeric (peak 2) and (d) 2-oxoisocaproic acids and the trimethylsilyl derivatives of (e) 2-hydroxyisovaleric, (f) 2-hydroxy-3-methyl-rt-valeric and (g) 2-hydroxyisocaproic acids.

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