Transient and long-term differential modulations of branched-chain α-keto acid decarboxylase activity in hypophysectomized rats

Hypophysectomy caused a marked but transient increase in branched-chain α-keto acid decarboxylase activities in rat liver mitochondria, peaking at about nine days post-surgery. The magnitude of increase is different for each of the three branched-chain α-keto acids. The activities then fall to a new steady state in three weeks with α-ketoisovalerate decarboxylase activity within the normal range, α-keto-β-methylvalerate decarboxylase activity at twice normal, and α-ketoisocaproate decarboxylase activity decreased to a level too low for accurate measurements.     

The effect of diabetes, nutritional factors, and sex on rat liver and kidney mevalonate kinase, mevalonate-5-phosphate kinase, and mevalonate-5-pyrophosphate decarboxylase

Isopycnic sucrose gradient separation of rat liver organelles revealed the presence of two distinct branched-chain α-keto acid decarboxylase activities; a mitochondrial activity, which decarboxylates the three branched-chain α-keto acids and requires CoA and NAD⁺ and a cytosolic activity, which decarboxylates α-ketoisocaproate, but not α-ketoisovalerate, or α-keto-β-methylvalerate. The latter enzyme does not require added CoA or NAD⁺. Assay conditions for the cytosolic α-ketoisocaproate decarboxylase activity were optimized and this activity was partially characterized. In rat liver cytosol preparations this activity has a pH optimum of 6.5 and is activated by 1.5 m ammonium sulfate. The decarboxylase activity has an apparent K_m of 0.03 mm for α-ketoisocaproate when optimized assay conditions are employed. Phenylpyruvate is a very potent inhibitor. α-Ketoisovalerate, α-keto-β-methylvalerate, α-ketobutyrate, and α-ketononanoate also inhibit the α-ketoisocaproate decarboxylase activity. The data indicate that the soluble α-ketoisocaproate decarboxylase is an oxidase. Rat liver cytosol preparations consumed oxygen when either α-ketoisocaproate or α-keto-γ-methiolbutyrate were added. None of the other α-keto acids tested stimulated oxygen consumption. 1-¹⁴C-Labeled α-keto-γ-methiolbutyrate is also decarboxylated by cytosol preparations. The α-ketoisocaproate oxidase was purified 20-fold from a 70,000g supernatant fraction of a rat liver homogenate. In these preparations the activity was increased 4-fold by the addition of dithiothreitol, ferrous iron, and ascorbate. The major product of this enzyme activity is β-hydroxyisovalerate. Isovalerate is not a free intermediate in the reaction. The data indicate an alternative pathway for metabolism of α-ketoisocaproate which produces β-hydroxyisovalerate.     

Inactivation of the 2-ketoglutarate and pyruvate dehydrogenase complexes of beef heart by branched chain keto acids

Incubation of 2-ketoglutarate dehydrogenase complex with 2-ketoisovalerate, 2-keto-4-methylvalerate, or 2-keto-3-methylvalerate leads to the appearance of a lag phase and of a progressive loss of activity in subsequent measurements of the initial rate of oxidation of 2-ketoglutarate. In the case of 2-ketoisovalerate these effects are shown to be due to the formation of an isobutyryllipoate derivative of the enzyme, as a result of the very slow oxidation of 2-ketoisovalerate by the enzyme complex (Vmax congruent to 0.15% of that for 2-ketoglutarate). Incubation of the enzyme complex with 2-keto[14C]isovalerate or 2-keto[14C]glutarate results in comparable incorporation of radioactivity, amounting to 3.5 to 5.3 nmol of isobutyryl or succinyl residues per mg of protein in the complex. Isobutyryl residues are also incorporated in the enzyme during the simultaneous oxidation of both of these substrates. During the early phase of incubation of the complex with 2-ketoisovalerate the incorporation of isobutyryl residues is much faster than the loss of enzyme activity. This observation seems to support the suggestion that each 2-ketoglutarate decarboxylase subunit of the complex may catalyze the succinylation of more than one lipoate succinyltransferase subunit. Results are also presented showing the inactivation of pyruvate dehydrogenase complex on preincubation with 2-ketoisovalerate and of 2-ketoglutarate dehydrogenase complex with methylenecyclopropylpyruvate, the keto acid corresponding to the toxic amino acid hypoglycin. The relevance of covalent modifications of the two keto acid dehydrogenase complexes to the pathological manifestations of maple syrup urine disease are discussed.     

Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida.

We purified branched-chain keto acid dehydrogenase to a specific activity of 10 mumol/min per mg of protein from Pseudomonas putida grown on valine. The purified enzyme was active with 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate in a ratio of 1.0:0.8:0.7 but showed no activity with either pyruvate or 2-ketoglutarate. There were four polypeptides in the purified enzyme (molecular weights, 49,000, 46,000, 39,000, and 37,000). The purified enzyme was deficient in the specific lipoamide dehydrogenase produced during growth on valine (molecular weight, 49,000). Branched-chain keto acid dehydrogenase required L-valine, oxidized nicotinamide adenine dinucleotide, coenzyme A, thiamine pyrophosphate, and magnesium chloride. A partially purified preparation catalyzed the oxidation of 2-keto-[1-14C]isovalerate to [14C]carbon dioxide, isobutyryl-coenzyme A, and reduced nicotinamide adenine dinucleotide in equimolar amounts. Both the Km and the Vmax for 2-ketoisovalerate were affected by the addition of L-valine to the assay mixture. However, only the Vmax values for oxidized nicotinamide adenine dinucleotide and coenzyme A were affected when L-valine was present. This suggested that valine acted by affecting the binding of branched-chain keto acids to subunit E1 of the complex.     

Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production

2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter(-1)) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter(-1)) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.     

3-Ketoglutarate generation in pancreatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-keto acids

The various neutral amino acids and aliphatic 2-keto acids exhibit differential effects on insulin secretion. The common denominator for all these effects is the 2-ketoglutarate generation in the pancreatic B-cell mitochondria. The neutral amino acidsl-leucine andl-norvaline and the aliphatic ketomonocarboxylic acids 2-ketoisocaproate, 2-ketocaproate, 2-ketovalerate, and 2-keto-3-methylvalerate all stimulate insulin secretion and increase 2-ketoglutarate generation in pancreatic B-cell mitochondria through activation of glutamate dehydrogenase and transamination withl-glutamate andl-glutamine, respectively. The neutral amino acidsl-valine,l-norleucine, andl-alanine and the aliphatic 2-keto acids 2-ketoisovalerate and pyruvate do not stimulate insulin secretion and do not increase 2-ketoglutarate generation in pancreatic B-cell mitochondria. Inhibition of 2-keto acid induced insulin secretion byl-valine andl-isoleucine is accompanied by reduced 2-ketoglutarate generation in pancreatic B-cell mitochondria. Thus intramitochondrial 2-ketoglutarate generation in pancreatic B-cells may regulate the insulin secretory potency of amino acids and 2-keto acids.     

Desilicification from the High Silica Containing Kraft Black Liquor by the Improved Carbon Dioxide Method

Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO₂ production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system.

The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.     

Isolation of a branched chain α-keto acid dehydrogenase from rabbit liver

An enzyme catalysing a series of reactions resulting in the oxidative decarboxylation of branched chain α-keto acids and production of NADH, was extracted from rabbit liver mitochondria with the aid of NaClO₄. Purification yielded a product which appeared homogeneous on electrophoresis. The enzyme is active on three substrates α-ketoisocaproate, α-keto-β-methyl valerate, and α-ketoisovalerate.     

Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum

The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicum l-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67 g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37 g/l 2-methyl-1-butanol and 2.76 g/l 3-methyl-1-butanol in defined medium within 48 h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.     

Common Enzymes of Branched-Chain Amino Acid Catabolism in Pseudomonas putida

Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branched-chain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.     

Inhibition of glycine synthase by branched-chain α-keto acids: A possible mechanism for abnormal glycine metabolism in ketotic hyperglycinemia

The effect of various amino acid metabolites on glycine oxidation by rat liver homogenate was investigated. Three compounds, α-ketoisovaleric acid, α-ketoisocaproic acid, and α-keto-β-methylvaleric acid, were found to inhibit glycine oxidation by 40–60%. In addition, these compounds also inhibited the glycine-CO₂ exchange reaction, a partial reaction of glycine synthase. The reverse reaction, glycine synthesis, was stimulated 4-fold by these α-keto acids. Pyruvate and α-ketoglutarate had no effect on any of these reactions. The parent amino acids, valine, isoleucine, and leucine, also had no effect on the reactions nor did any of their other metabolites with the exception of the branched-chain α-keto acids. The concentration dependence of the inhibition of glycine oxidation and stimulation of glycine synthesis by these branched-chain α-keto acids suggested that the inhibition of glycine oxidation by these compounds was the result of their further oxidation by branched-chain α-keto acid dehydrogenase. However, the products of the branched-chain α-keto acid dehydrogenase, isobutyryl CoA, isovaleryl CoA, or α-methylbutyryl CoA had no effect on glycine oxidation. Thus, it appeared that either the branched-chain α-keto acids altered glycine oxidation by direct binding to glycine synthase or that electrons derived from the oxidation of branched-chain α-keto acids were transferred to the glycine synthase system. It is proposed that glycine synthase and branched-chain α-keto acid dehydrogenase either share a common subunit, possibly lipoamide dehydrogenase, or are so arranged on the mitochondrial membrane that electron transfer between these two enzymes occurs.     

CONTROL OF PYRUVATE AND β-HYDROXYBUTYRATE UTILIZATION IN RAT BRAIN MITOCHONDRIA AND ITS RELEVANCE TO PHENYLKETONURIA AND MAPLE SYRUP URINE DISEASE

—The effects of the amino acids (phenylalanine, valine, leucine and isoleucine) which accumulate in phenylketonuria (PKU) and maple syrup urine disease (MSUD), and their analogue α-keto acids (phenylpyruvate, α-keto isovalerate, α-keto isocaproate, α-keto-β-Me valerate) have been studied on rat brain mitochondrial respiration. Both phenylpyruvate and α-keto isocaproate specifically inhibited the oxidation of pyruvate plus malate and β-hydroxybutyrate plus malate by rat brain mitochondria in the presence of ADP. However, no inhibitory effects of similar concentrations of phenylpyruvate or α-keto isocaproate were observed on the isolated semipurified pyruvate or β-hydroxybutyrate dehydrogenases from rat brain mitochondria. The transport of pyruvate and β-hydroxybutyrate across the brain mitochondrial membrane was studied by both uptake and exchange of radioactively labelled substrates. Both these processes were inhibited by phenylpyruvate and α-ketoisocaproate. The results are interpreted as providing evidence for both pyruvate and β-hydroxybutyrate translocases across the brain mitochondrial membrane, and that the inhibition of these systems by phenylpyruvate and α-keto isocaproate may be important lesions in phenylketonuria and maple syrup urine disease respectively.     

Oxidation of branched-chain amino acids in skeletal muscle and liver of rat Effects of octanoate and energy state

The effect of octanoate on the oxidative decarboxylation of ¹⁴C-labeled amino acids has been studied in perfused hindquarter and liver of rat. Regulation of the branched-chain α-keto acid dehydrogenase has been further studied with α-[¹⁴C-1]ketoisovalerate in isolated rat muscle and liver mitochondria. (1) Octanoate has a stimulatory effect on the oxidation of branched-chain amino acids in perfused hindquarter. The oxidative decarboxylation of other amino acids are inhibited. Octanoate inhibits the oxidative decarboxylation of all amino acids in perfused liver. (2) The oxidation of valine is stimulated by octanoate and hexanoate also in isolated muscle mitochondria. The stimulatory effect is probably related to activation of the fatty acids since acyl-carnitines inhibit the oxidation. (3) The oxidation of α-ketoisovalerate in mitochondria is inhibited by competing substrates (pyruvate, α-ketoglutarate and succinate). This inhibition is counteracted by octanoate and ADP. (4) Low concentrations (1–5 μM) of 2,4-dinitrophenol (DNP) activates wheras higher concentrations inactivates the branched-chain α-keto acid dehydrogenase in intact but not in solubilized muscle mitochondria. The inactivation is counteracted by ATP, but is increased by octanoate. (5) The observations seem to suggest that the activation (like the inactivation) of branched-chain α-keto acid dehydrogenase in skeletal muscle is dependent on the mitochondrial energy state which therefore may regulate both activation and inactivation of the dehydrogenase.     

Transamination of neutral amino acids and 2-keto acids in pancreatic B-cell mitochondria

High aminotransferase activities catalyzing the reactions between L-glutamate and L-glutamine and the aliphatic ketomonocarboxylic acids 2-ketoisocaproate, 2-ketocaproate, and 2-ketoisovalerate were observed in pancreatic B-cell mitochondria. While maximal rates of transamination with L-glutamate were observed in the presence of micromolar concentrations of keto acid, maximal rates of transamination with L-glutamine were recorded only in the presence of millimolar concentrations of keto acid. The insulin secretagogue 2-ketoisocaproate was the most effective transamination partner for L-glutamate, while the insulin secretagogue 2-ketocaproate was the most effective transamination partner for L-glutamine. Since B-cell mitochondria are well supplied with L-glutamate and L-glutamine, 2-ketoglutarate generation in the presence of these two neutral 2-keto acids may be an important prerequisite for their insulin secretory potency. High rates of transamination of 2-ketoglutarate were observed in the pancreatic B-cell mitochondria with the branched-chain amino acids L-leucine and L-valine, but not with L-norleucine. In connection with the ability of L-leucine to activate glutamate dehydrogenase, this high activity of the branched-chain amino acid aminotransferase in pancreatic B-cell mitochondria may provide an explanation for the insulin secretory potency of this amino acid.     

Uptake of alpha-ketoisocaproic acid in lymphoblast line WI-L2.

The uptake of α-ketoisocaproate by the cultured human lymphoblast line WI-L2 appears to be mediated by a transport system which has an apparent Km of 125 μM. The rate of uptake of α-ketoisocaproate decreases with increasing pH values, i.e., pH 6 > 7 > 8 and is stimulated by sodium at all pH values. Closely related branched chain α-ketoacids, α-keto-β-methylvaleric and α-ketoisovaleric exhibited the greatest inhibition of α-ketoisocaproate transport. Straight chain α-keto acids inhibited α-ketoisocaproic acid uptake to a lesser degree as did the α-hydroxy analogs of the branched chain α-keto acids. Inhibitors of the general anion transport system of erythrocytes, 1-anilino-8-napthalene sulfonic acid and 4-acetamido-4-isothiocyanostilbene-2-1′-disulfonic acid did not affect α-ketoisocaproate transport. A reduced sulfhydryl group is critical for α-ketoisocaproate acid uptake; transport is partially or completely inhibited by sulfhydryl reagents such as dithio-bis-nitrobenzoate, iodoacetamide, and p-chloromercuribenzoate. Inhibition by the sulfhydryl reagents is reversed with β-mercaptoethanol or partially with dithiothreitol.     

Comparative characterisation of thiamin diphosphate-dependent decarboxylases

Several 2-keto acid decarboxylases catalyse an acyloin condensation-like carboligase reaction beside their physiological decarboxylase activity. Although many data concerning stability and catalytic potential of these enzymes are available, a standard evaluation under similar reaction conditions is lacking. In this comprehensive survey we assemble already published data combined with new studies of three bacterial pyruvate decarboxylases, yeast pyruvate decarboxylase, benzoylformate decarboxylase from Pseudomonas putida (BFD) and the branched-chain 2-keto acid decarboxylase from Lactococcus lactis (KdcA). The obtained results proof that the optima for activity and stability are rather similar if comparable reaction conditions are used. Although the substrate ranges of the decarboxylase reaction of the various pyruvate decarboxylases are similar as well, they differ remarkably from those of BFD and KdcA. We further show that the range of acceptable donor aldehydes for the carboligase reaction of a respective enzyme can be reliably predicted from the substrate range of decarboxylase reaction.     

Biosynthesis of branched-chain fatty acids in Bacillus subtilis. A decarboxylase is essential for branched-chain fatty acid synthetase

Branched long-chain fatty acids of the iso and anteiso series are synthesized in many bacteria from the branched-chain alpha-keto acids of valine, leucine, and isoleucine after their decarboxylation followed by chain elongation. Two distinct branched-chain alpha-keto acid (BCKA) and pyruvate decarboxylases, which are considered to be responsible for primer synthesis, were detected in, and purified in homogenous form from Bacillus subtilis 168 strain by procedures including ammonium sulfate fractionation and chromatography on ion exchange, reversed-phase, and gel absorption columns. The chemical and catalytic properties of the two decarboxylases were studied in detail. The removal of BCKA decarboxylase, using chromatographic fractionation, from the fatty acid synthetase significantly reduced its activity. The synthetase activity was completely lost upon immunoprecipitation of the decarboxylase. The removal of pyruvate decarboxylase by the above two methods, however, did not affect any activity of the fatty acid synthetase. Thus, BCKA decarboxylase, but not pyruvate decarboxylase, is essential for the synthesis of branched-chain fatty acids. The very high affinity of BCKA decarboxylase toward branched-chain alpha-keto acids is responsible for its function in fatty acid synthesis.     

Transamination of 3-phenylpyruvate in pancreatic B-cell mitochondria

High aminotransferase activities catalyzing the reaction between L-glutamate and the aromatic ketomonocarboxylic acid, 3-phenylpyruvate, were observed in the mitochondria from pancreatic B-cells. At very low concentrations of 3-phenylpyruvate, L-glutamine was an effective amino group donor. The aminotransferase activities for the aliphatic ketomonocarboxylic acids, pyruvate and 2-ketoisovalerate, were lower in B-cell mitochondria. High rates of transamination of 2-ketoisocaproate with L-glutamine were observed and may be an important prerequisite for the insulin secretory potency of this 2-keto acid. Since B-cell mitochondria are well supplied with L-glutamine and L-glutamate, 3-phenylpyruvate-induced 2-ketoglutarate production may explain the insulin secretory potency of 3-phenylpyruvate which is not a fuel for pancreatic islet cells.     

Regulation of valine catabolism in Pseudomonas putida

The activities of six enzymes which take part in the oxidation of valine by Pseudomonas putida were measured under various conditions of growth. The formation of four of the six enzymes was induced by growth on d- or l-valine: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-hydroxyisobutyrate dehydrogenase, and methylmalonate semialdehyde dehydrogenase. Branched-chain amino acid transaminase and isobutyryl-CoA dehydrogenase were synthesized constitutively. d-Amino acid dehydrogenase and branched-chain keto acid dehydrogenase were induced during growth on valine, leucine, and isoleucine, and these enzymes were assumed to be common to the metabolism of all three branched-chain amino acids. The segment of the pathway required for oxidation of isobutyrate was induced by growth on isobutyrate or 3-hydroxyisobutyrate without formation of the preceding enzymes. d-Amino acid dehydrogenase was induced by growth on l-alanine without formation of other enzymes required for the catabolism of valine. d-Valine was a more effective inducer of d-amino acid dehydrogenase than was l-valine. Therefore, the valine catabolic pathway was induced in three separate segments: (i) d-amino acid dehydrogenase, (ii) branched-chain keto acid dehydrogenase, and (iii) 3-hydroxyisobutyrate dehydrogenase plus methylmalonate semialdehyde dehydrogenase. In a study of the kinetics of formation of the inducible enzymes, it was found that 3-hydroxyisobutyrate and methylmalonate semialdehyde dehydrogenases were coordinately induced. Induction of enzymes of the valine catabolic pathway was studied in a mutant that had lost the ability to grow on all three branched-chain amino acids. Strain PpM2106 had lowered levels of branched-chain amino acid transaminase and completely lacked branched-chain keto acid dehydrogenase when grown in medium which contained valine. Addition of 2-ketoisovalerate, 2-ketoisocaproate, or 2-keto-3-methylvalerate to the growth medium of strain PpM2106 resulted in induction of normal levels of branched-chain keto acid dehydrogenase; therefore, the branched-chain keto acids were the actual inducers of branched-chain keto acid dehydrogenase.     

Characterization of a fourth type of 2-keto acid-oxidizing enzyme from a hyperthermophilic archaeon: 2-ketoglutarate ferredoxin oxidoreductase from Thermococcus litoralis.

Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. It is known to contain three distinct ferredoxin-dependent, 2-keto acid oxidoreductases, which use pyruvate, aromatic 2-keto acids such as indolepyruvate, or branched-chain 2-keto acids such as 2-ketoisovalerate, as their primary substrates. We show here that T. litoralis also contains a fourth member of this family of enzymes, 2-ketoglutarate ferredoxin oxidoreductase (KGOR). In the presence of coenzyme A, KGOR catalyzes the oxidative decarboxylation of 2-ketoglutarate to succinyl coenzyme A and CO2 and reduces T. litoralis ferredoxin. The enzyme was oxygen sensitive (half-life of approximately 5 min) and was purified under anaerobic conditions. It had an M(r) of approximately 210,000 and appeared to be an octomeric enzyme (alpha2beta2gamma2delta2) with four different subunits with M(r)s of 43,000 (alpha), 29,000 (beta), 23,000 (gamma), and 10,000 (delta). The enzyme contained 0.9 mol of thiamine PPi and at least four [4Fe-4S] clusters per mol of holoenzyme as determined by metal analyses and electron paramagnetic resonance spectroscopy. Significant amounts of other metals (Cu, Zn, Mo, W, and Ni) were not present (<0.1 mol/mol of holoenzyme). Pure KGOR did not utilize other 2-keto acids, such as pyruvate, indolepyruvate, or 2-ketoisovalerate, as substrates, and the apparent Km values for 2-ketoglutarate, coenzyme A, T. litoralis ferredoxin, and thiamine PPi were approximately 250, 40, 8, and 9 microM, respectively. The enzyme was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.0 (at 80 degrees C). KGOR was quite thermostable, with a half-life at 80 degrees C (under anaerobic conditions) of about 2 days. An enzyme analogous to KGOR has been previously purified from a mesophilic archaeon, but the molecular properties of T. litoralis KGOR more closely resemble those of the other oxidoreductases from hyperthermophiles. In contrast to these enzymes, however, KGOR appears to have a biosynthetic function rather than a role in energy conservation.