2.3- dihydroxyisovaleric

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. 

A promising approach for anaerobic fermentation is the nonnaturally occurring four pathways that process via 2,3-dihydroxyisovalerate (DHIV), a precursor for L-valin biosynthesis which has the correct carbon skeleton of isoprene and requires only two steps from Pyr, with high theoretical yield (0.324 g isoprene/g glucose) [102]. All the proposed pathways are redox-balanced and require the same number of steps as the original routes. In addition, one of them is... 

Dihydroxyacid dehydratase is involved in the biosynthesis of valine and isoleucine. L-a,/3-dihydroxyisovaleric acid and L-aj3-dihydroxy-/3-methylvaleric acid are dehydrated to form the a-keto acid precursors of valine and isoleucine. 

The reaction mixture contained in a total volume of 1 mL 50 mM Tris-HC1 buffer (pH 8.0), 100 tnM MgG2, 3.8 mAf L-a,/3-dihydroxyisovaleric add (sodium salt), and crude extract containing 0.5 mg of protein. The reaction mixture was incubated for various times at 37°C, and the reaction was terminated by transferring 100 L of the assay mixture to a 200 fiL centrifuge tube containing 20 / L of 50% trichloroacetic acid. Predpitated proteins were removed by centrifugation. Then 50 fiL of the supemate was mixed with... 

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. 

Fio. 7. Scheme of biosynthesis of valine and isoleucine. R tepresents a methyl group in valine and an ethyl group in isoleucine biosynthesis. I is pyruvate or a-keto-butyrate II is a-acetolactate or a-aceto-a-hydroxybutyrate HI is a-keto-/3-hydroxy> isovalerate or a-keto- -hydroxy- S-metbylvalerate IV is a,/ -dihydroxyisovalerate or a-0-dihydroxy- -methylvalerate V is a-ketoisovalerate or a-keto-d-methylvalerate and VI is valine or isoleucine. 

As further evidence that acetolactate is on the pathway of valine formation these investigators demonstrated that it is converted to a,/3-dihydroxyisovalerate by appropriately blocked mutants of E. coli. Also, like other five-carbon precursors of L-valine, it inhibited the growth of another strain of E. coli, but not of valine-resistant mutants. 

Umbarger (167), in a study of the conversion of acetolactate to a,j8-dihydroxyisovalerate, concluded that the properties of the enzyme was compatible with the view that a single enzyme performs both the rearrangement and reduction of the carbonyl group for both the valine and isoleucine precursors. 

Subsequently, the enzymic conversion of a-acetolactate to a,6-dihydroxyisovalerate and a-aceto-a-hydroxybutyrate to a,/3-dihydroxy-jS-methylvalerate was shown by Wagner et al. 166) with extracts of Neurospora and by Umbarger 167) with extracts of appropriately blocked E. coU mutants. These acids were then shown to be utUized for valine and isoleucine formation. 

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