Dihydroxyacid dehydratase

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. 

4-dinitrophenylhydrazone derivatives of a-ketoisovaleric acid and a-keto-jS-methylvaleric acid are separated from 2,4-dinitrophenylhydrazine by chromatography at room temperature on a Zorbax Cjg column (4.6 mm x 250 mm). Solvent A was 25% acetonitrile in water containing 0.1% triethyla-mine (v/v) and adjusted to pH 4.5 with acetic acid. Solvent B was acetonitrile. A linear gradient from 20 to 50% B was made within 20 minutes. The effluent was monitored at 254 nm. 

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 

SURVEY OF ENZYMATIC ACTIVITIES ASSAYED BY THE HPLC METHOD 

30 fiL of 0.2% 2,4-dinitrophenylhydrazine in 2 M HQ (w/v) in a 2.5 mL polypropylene centrifuge tube. After 30 minutes at room temperature, samples were diluted with 60% acetonitrile in water to a final volume of 1 mL. After filtration, 20 nL was injected onto an HPLC column. 

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Dihydroxyacid dehydratase of the branched-chain amino acid biosynthetic pathway catalyzes the dehydration and tautomerization of 2,3-dihydroxy-3-methyl-(butyrate and pentanoate) to 2-keto-3-methyl(butyrate and pentanoate). The enzyme isolated from spinach recently has been shown to have not a [4Fe-4S] cluster, but rather a spectroscopically unusual [2Fe-2S] cluster in its active site (68,69). The EPR spectrum of the reduced enzyme is similar to that seen for Rieske Fe-S proteins (71) with a g-average of 1.91. Upon addition of substrate the g-average of the EPR spectrum shifts to 1.96 (opposite the effect of substrate on aconitase), and then reverts back to a g-average of 1.90 when only the product is present The dramatic changes in the EPR spectra upon addition of substrate suggest, in analogy to aconitase, that the Fe-S cluster may be directly involved in catalysis. 

The role of the iron-sulfur clusters in many of the proteins that we have just considered is primarily one of single-electron transfer. The Fe-S cluster is a place for an electron to rest while waiting for a chance to react. There may sometimes be an associated proton pumping action. In a second group of enzymes, exemplified by aconitase (Fig. 13-4), an iron atom of a cluster functions as a Lewis acid in facilitating removal of an -OF group in an a,P dehydration of a carboxylic acid (Chapter 13). A substantial number of other bacterial dehydratases as well as an important plant dihydroxyacid dehydratase also apparently use Fe-S clusters in a catalytic fashion.317 Fumarases A and B from E. coli,317 L-serine dehydratase of a Pepto-streptococcus species,317-319 and the dihydroxyacid... 

Dihydroxyacid dehydratase (Val, lie biosynthesis) 2,3-dihydroxycarboxylic acid/ 2-keto carboxylic acid... 

The substrates of serine dehydratase [45,46] and dihydroxyacid dehydratase [47] differ from citrate in more than just R. The different substrates are compared in Figure 4. They all have in common the central HO —C—CH COO- fragment, indicating that all enzymes should have the following features ... 

As with fumarase, the reactions catalyzed by serine dehydratase and dihydroxyacid dehydratase do not require release and rebinding of intermediate. The reaction mechanisms should be describable as adapted versions of one-half only of Figure 3. 

This section will be concluded with a note on an apparently exceptional system. The dihydroxyacid dehydratase from spinach has been reported to contain a [2Fe-2S] cluster [23], This would then be the very first example of a noncubane iron-sulfur enzyme. The authors list three observations that, together, conclu-... 

Dihydroxyacid dehydratase (E.C. 4.2.1.9) is a ubiquitous enzyme that is involved in the biosynthesis of the branched-chain amino acids (lie, Leu and Val) and of pantothenic acid and coenzyme A. The enzyme catalyzes the elimination of water from 2,3-dihydroxyalkanoic acids (23) to 2-hydroxy-2-alkenoic acids (24), which tautomerize to 2-ketoalkanoic acids (25). The enzyme from spinach has the highest activity towards 2,3-dihydroxy-3-methylbutanoic acid (Val precursor, Scheme 11.5-4) but also accepts other substrates1341. Thus, 2,3-dihydroxybutanoic acid, 3-cyclopropyl-2,3-dihydroxybutanoic add as well as 2,3-dihydroxy-3-methylpentanoic acid are substrates. With the latter substrate a slight preference for (2R,3S)-2,3-dihydroxy-... 

Figure 7 Other dehydratases that may use a [4Fe-4S] cluster in an analogous manner to aconitase. A. Isopropylmalate isomerase. B. Fumarase. C. Dihydroxyacid dehydratase. D. Maleic acid hydratase.

Biosynthesis Leu is formed from pyruvic acid - 2-acetolactic acid [acetolactate synthase (EC 4.1.3.18.)+(l-hydroxyethyl)-TPP] - 2,3-dihydroxy-isovaleric acid [reductase+NAD(P)H] - 2-oxoisova-leric acid [dihydroxyacid dehydratase] - 2-isopropyl-malate [2-isopropylmalate synthase + acetyl-CoA (EC 4.1.3.12)] -> 3-isopropylmalate [isopropylmalate dehydratase (EC 4.2.1.33) -HjO+HiO] 2-oxo-isocaproate [3-isopropylmalate dehydrogenase (EC 1.1.1.85) + NAD ] L. [leucine aminotransferase (EC... 

DHAD dihydroxyacid dehydratase (encoded by ilvD, Ilv3)... 

Acetolactate synthase 2 acetolactate reducto-isomerase 3 dihydroxyacid dehydratase 4 aminotransferase... 

Dihydroxyacid dehydratase activity in microorganisms with diverse fermentation patterns. Proc Soc Exp Biol Med 137 292-298... 

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