Branched-chain 2-oxo Acid Research Articles

Hyperglycemia Stimulates the Irreversible Catabolism of Branched-Chain Amino Acids and Generation of Ketone Bodies by Cultured Human Astrocytes.

Astrocytes are considered to possess a noticeable role in brain metabolism and, as a partners in neuron-glia cooperation, to contribute to the synthesis, bioconversion, and regulation of the flux of substrates for neuronal metabolism. With the aim of investigating to what extent human astrocytes are metabolizing amino acids and by which compounds are they enriching their surroundings, we employed a metabolomics analysis of their culture media by 1H-NMR. In addition, we compared the composition of media with either 5 mM or 25 mM glucose. The quantitative analysis of culture media by 1H-NMR revealed that astrocytes readily dispose from their milieu glutamine, branched-chain amino acids, and pyruvate with significantly high rates, while they enrich the culture media with lactate, branched-chain keto acids, citrate, acetate, ketone bodies, and alanine. Hyperglycemia suppressed the capacity of astrocytes to release branched-chain 2-oxo acids, while stimulating the generation of ketone bodies. Our results highlight the active involvement of astrocytes in the metabolism of several amino acids and the regulation of key metabolic intermediates. The observed metabolic activities of astrocytes provide valuable insights into their roles in supporting neuronal function, brain metabolism, and intercellular metabolic interactions within the brain. Understanding the complex metabolic interactions between astrocytes and neurons is essential for elucidating brain homeostasis and the pathophysiology of neurological disorders. The observed metabolic activities of astrocytes provide hints about their putative metabolic roles in brain metabolism.

Detection of anti-mitochondrial 2-oxoacid dehydrogenase complex subunit's antibodies for the diagnosis of Primary Biliary Cholangitis

Anti-mitochondrial antibodies (AMA), directed against the E2 subunits of the 2-oxo acid dehydrogenase complexes, are markers of Primary Biliary Cholangitis (PBC), a chronic autoimmune liver disease. However, it has not been stablished the clinical significance of subunits-specific AMA type PDC-E2 subunit of the pyruvate dehydrogenase complex-, BCOADC-E2 subunit of the branched-chain 2-oxo acid dehydrogenase complex-, OGDC-E2 subunit of the 2-oxo-glutarate dehydrogenase complex- and nPDC -native pyruvate dehydrogenase complex (M2-AMA), and not all AMA specificities are associated with PBC. The aim of the present study was to show the usefulness of the number and combination of subunits-specific AMA positive for the diagnosis of PBC. We detected AMA by indirect immunofluorescence (IIF-AMA) and M2-AMA by dot-blot. We studied the relationship of AMA with some clinical and laboratory variables in 307 patients (37% PBC) with positive dot-blot for M2-AMA. In PBC patients, we detected different E2 subunits of the 2-oxo acid dehydrogenase complexes antibodies (M2-AMA): 82.9% were specific for nPDC, 64.5% for PDC-E2, 44.4% for BCOADC-E2, and 9.6% for OGDC-E2. IIF and dot-blot tests achieved an Area Under the Receiver Operating Characteristic Curve (ROC AUC) of 0.674 (1:320 cut-off titer, Sensibility (Se) 64.7%, Specificity (Sp) 63.4%) and 0.663 (three specificities M2-AMA, Se 43%, Sp 81.2%), respectively. The detection of different E2 subunits of the 2-oxo acid dehydrogenase complexes antibodies (M2-AMA) by dot-blot showed different ROC AUC: anti-PDC-E2 showed an AUC of 0.610, a Se of 43.7%, and a Sp of 76.4%. Finally, the combined detection of nPDC/BCOADC-E2/PDC-E2 reached an AUC of 0.6095, a Se of 59.6%, and a Sp of 70.2%.The identification of two M2-AMA specificities through dot-blot increased PBC odds ratio (OR) by 2.05 (p:0.031), as compared to the identification of one specificity. Moreover, the identification of three and four specificities increased OR by 4.63 (p:0.000) and by 21.53 (p:0.006), respectively. nPDC/OGDC-E2/PDC-E2 and nPDC/OGDC-E2/BCOADC-E2/PDC-E2 combinations increased PBC OR by 10.04 (p:0.034), as compared to any other combination. 1:320 and 1:640 IIF-AMA increased PBC OR by 4.93 (p:0.009) and 7.67 (p:0.001), respectively, as compared to IIF-AMA titers equal to or less than 1:160. M2-AMA dot-blot was less sensitive but more specific than IIF-AMA, with similar predictive capacity for PBC. Increased numbers of M2-AMA specificities clearly increased the risk of PBC. Some combinations were strongly related to PBC (nPDC/BCOADC-E2/PDC-E2), but others were not (one single M2-AMA, and nPDC plus PDC-E2). M2-AMA dot-blot was less sensitive but more specific than IIF-AMA, with similar predictive capacity for PBC. Increased numbers of M2-AMA specificities clearly increased the risk of PBC, being some combinations, such as nPDC/BCOADC-E2/PDC-E2, more related to PBC than others. Finally, the determination of the number of M2-AMA specificities was more useful than the particular subunit target for PBC diagnosis. In conclusion, the study of the number of M2-AMA specificities by dot-blot should definitely be considered for PBC diagnosis.

Effects of B-Group Vitamin Administration on Daily Change in Urine 2-Oxo Acids in Young Japanese Women.

B-group vitamins are required in amino acid catabolism, and recent findings suggest that urine 2-oxo acids, catabolites of amino acid, could be functional biomarkers indicating the nutritional status of B-group vitamins. To clarify the relationship between B-group vitamins and urine 2-oxo acids, we investigated the effects of B-group vitamin administration on daily changes in urinary amounts of 2-oxo acids in humans. Twenty-nine young Japanese women collected 24-h urine samples for 8 d, and took B-group vitamins for 7 d beginning on the second day of urine collection. The participants were divided into three groups on the basis of the amounts of total branched-chain 2-oxo acids, 2-oxoglutaric acid, 2-oxoadipic acid, and pyruvic acid excreted in urine. In the upper tertile, but not the middle and lower tertiles, each urine 2-oxo acid decreased from the first day of vitamin administration, and completely decreased to a normal level on the second day of administration. These results indicate that administration of B-group vitamins immediately affects 2-oxo acid metabolism in some young Japanese women. Thus, urinary 2-oxo acids could be useful and functional biomarkers for B-group vitamin status.

Glucose-Stimulated Insulin Secretion Fundamentally Requires H2O2 Signaling by NADPH Oxidase 4.

NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islets (PIs) of β-cells through an as yet unknown mechanism. We found NADPH oxidase isoform 4 (NOX4) to be the main producer of cytosolic H2O2, which is essential for GSIS; an increase in ATP alone was insufficient for GSIS. The fast GSIS phase was absent from PIs from NOX4-null, β-cell-specific knockout mice (NOX4βKO) (though not from NOX2 knockout mice) and from NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H2O2 rescued GSIS in PIs from NOX4βKO mice. NOX4 silencing suppressed Ca2+ oscillations, and the patch-clamped KATP channel opened more frequently when glucose was high. Mitochondrial H2O2, decreasing upon GSIS, provided alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxides through electron-transfer flavoprotein:Q-oxidoreductase. Unlike GSIS, such insulin secretion was blocked with mitochondrial antioxidant SkQ1. Both NOX4 knockout and NOX4βKO mice exhibited impaired glucose tolerance and peripheral insulin resistance. Thus, the redox signaling previously suggested to cause β-cells to self-check hypothetically induces insulin resistance when it is absent. In conclusion, increases in ATP and H2O2 constitute an essential signal that switches on insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (it does so partially for fatty acids). Redox signaling could be impaired by cytosolic antioxidants; hence, those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.

Generation of superoxide and hydrogen peroxide by side reactions of mitochondrial 2-oxoacid dehydrogenase complexes in isolation and in cells.

Mitochondrial 2-oxoacid dehydrogenase complexes oxidize 2-oxoglutarate, pyruvate, branched-chain 2-oxoacids and 2-oxoadipate to the corresponding acyl-CoAs and reduce NAD+ to NADH. The isolated enzyme complexes generate superoxide anion radical or hydrogen peroxide in defined reactions by leaking electrons to oxygen. Studies using isolated mitochondria in media mimicking cytosol suggest that the 2-oxoacid dehydrogenase complexes contribute little to the production of superoxide or hydrogen peroxide relative to other mitochondrial sites at physiological steady states. However, the contributions may increase under pathological conditions, in accordance with the high maximum capacities of superoxide or hydrogen peroxide-generating reactions of the complexes, established in isolated mitochondria. We assess available data on the use of modulations of enzyme activity to infer superoxide or hydrogen peroxide production from particular 2-oxoacid dehydrogenase complexes in cells, and limitations of such methods to discriminate specific superoxide or hydrogen peroxide sources in vivo.

Biallelic Mutations in LIPT2 Cause a Mitochondrial Lipoylation Defect Associated with Severe Neonatal Encephalopathy

Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metabolism (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metabolism (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymatic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been associated with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metabolism. LIPT1 deficiency spares GCS deficiency and has been associated with a biochemical signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals' fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was observed after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clinical condition nor activities of PDHc, α-KGDHc, or leucine metabolism in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2.

Urinary Branched-Chain 2-Oxo Acids as a Biomarker for Function of B-Group Vitamins in Humans

To find a functional biomarker of B-group vitamins, we collected 24-h urine samples from young Japanese women who lived in the community (n=29) to measure branched-chain 2-oxo acids such as 2-oxo-3-methylbutanoic acid, 2-oxo-3-methylpentanoic acid, and 2-oxo-4-methylpentanoic acid because B-group vitamins are involved in the catabolism of branched-chain amino acids. The relationships between each pair of the three urinary 2-oxo acids were very high (2-oxo-3-methylbutanoic acid and 2-oxo-3-methylpentanoic acid, p<0.001; 2-oxo-3-methylbutanoic acid and 2-oxo-4-methylpentanoic acid, p<0.001; 2-oxo-3-methylpentanoic acid and 2-oxo-4-methylpentanoic acid, p<0.001). The participants were divided into three groups using the upper (n=10), middle (n=9), and lower tertiles (n=10) based on the urinary excretion amounts of the sum of the three branched-chain 2-oxo acids. The administration of capsules containing the daily necessary amounts of B-group vitamins led to a decrease in the urinary excretion of the sum of the three types of branched-chain 2-oxo acids in participants belonging to the upper tertile. A similar phenomenon was observed in the middle tertile, but not in the lower tertile. Intakes of B-group vitamins and the urinary excretion amounts of B-group vitamins were not observed to be significantly different among the upper, middle, and lower tertiles. These results indicate that some young Japanese women need much higher levels of B-group vitamins than the Dietary Reference Intakes for Japanese. Thus, urinary branched-chain 2-oxo acids are useful functional biomarkers for B-group vitamins in humans.

Comparative assessment of native and heterologous 2-oxo acid decarboxylases for application in isobutanol production by Saccharomyces cerevisiae.

BackgroundDecarboxylation of α-ketoisovalerate to isobutyraldehyde is a key reaction in metabolic engineering of Saccharomyces cerevisiae for isobutanol production with published studies relying on overexpression of either the native ARO10 gene or of the Lactococcus lactis kivD decarboxylase gene resulting in low enzymatic activities. Here, we compare relevant properties for isobutanol production of Aro10, KivD and an additional, less studied, L. lactis decarboxylase KdcA.ResultsTo eliminate interference by native decarboxylases, each 2-oxo acid decarboxylase was overexpressed in a ‘decarboxylase-negative’ (pdc1Δ pdc5Δ pdc6Δ aro10Δ) S. cerevisiae background. Kinetic analyses in cell extracts revealed a superior Vmax/Km ratio of KdcA for α-ketoisovalerate and a wide range of linear and branched-chain 2-oxo acids. However, KdcA also showed the highest activity with pyruvate which, in engineered strains, can contribute to formation of ethanol as a by-product. Removal of native decarboxylase genes eliminated growth on valine as sole nitrogen source and subsequent complementation of this growth impairment by expression of each decarboxylase indicated that based on the increased growth rate, the in vivo activity of KdcA with α-ketoisovalerate was higher than that of KivD and Aro10. Moreover, during oxygen-limited incubation in the presence of glucose, strains expressing kdcA or kivD showed a ca. twofold higher in vivo rate of conversion of α-ketoisovalerate into isobutanol than an ARO10-expressing strain. Finally, cell extracts from cultures grown on different nitrogen sources revealed increased activity of constitutively expressed KdcA after growth on both valine and phenylalanine, while KivD and Aro10 activity was only increased after growth on phenylalanine suggesting a difference in the regulation of these enzymes.ConclusionsThis study illustrates important differences in substrate specificity, enzyme kinetics and functional expression between different decarboxylases in the context of isobutanol production and identifies KdcA as a promising alternative decarboxylase not only for isobutanol production but also for other branched-chain and linear alcohols.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0374-0) contains supplementary material, which is available to authorized users.

Anti-mitochondrial M2 antibody-positive autoimmune hepatitis.

Anti-mitochondrial M2 antibody (AMA-M2) is specific to primary biliary cirrhosis (PBC), but can also be found in certain patients with autoimmune hepatitis (AIH). Effective methods of differentiating between PBC and AIH are required, as their clinical course and management are different. Titers of AMA-M2 were analyzed before and after follow-up in patients with PBC or AIH. Patients who underwent liver biopsy and were diagnosed with either AIH (10 patients) or PBC (3 patients) were enrolled in the study. The AMA-M2 antibody titers of these patients were analyzed upon hospital admission. AMA-M2 reacted with the pyruvate dehydrogenase complex-E2, branched-chain 2-oxo acid dehydrogenase complex and 2-oxoglutaric acid dehydrogenase complex in the assay utilized for this study. The cut-off value for AMA-M2 was 5. Six AIH patients were AMA-M2(-) and 4 were AMA-M2(+). The titer for the AIH patients who were AMA-M2(+) was 24.8±14.8, compared with 324±174 in the patients with PBC (P=0.0138). Three AMA-M2(+) AIH patients were followed-up after liver biopsy. The AMA-M2 levels had decreased in all 3 patients, becoming undetectable in 2 of them. In conclusion, certain patients with AIH in this study were found to be AMA-M2(+), but the titers were significantly lower than those in the patients with PBC. At follow-up, the AIH patients exhibited decreased AMA-M2 titers.

Why are the 2-oxoacid dehydrogenase complexes so large? Generation of an active trimeric complex.

The four-component polypeptides of the 2-oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active mini-complex for this enzyme, and raises the question of why all 2-oxoacid dehydrogenase complexes assemble into such large structures.

Nuclear Magnetic Resonance Approaches in the Study of 2-Oxo Acid Dehydrogenase Multienzyme Complexes—A Literature Review

The 2-oxoacid dehydrogenase complexes (ODHc) consist of multiple copies of three enzyme components: E1, a 2-oxoacid decarboxylase; E2, dihydrolipoyl acyl-transferase; and E3, dihydrolipoyl dehydrogenase, that together catalyze the oxidative decarboxylation of 2-oxoacids, in the presence of thiamin diphosphate (ThDP), coenzyme A (CoA), Mg2+ and NAD+, to generate CO2, NADH and the corresponding acyl-CoA. The structural scaffold of the complex is provided by E2, with E1 and E3 bound around the periphery. The three principal members of the family are pyruvate dehydrogenase (PDHc), 2-oxoglutarate dehydrogenase (OGDHc) and branched-chain 2-oxo acid dehydrogenase (BCKDHc). In this review, we report application of NMR-based approaches to both mechanistic and structural issues concerning these complexes. These studies revealed the nature and reactivity of transient intermediates on the enzymatic pathway and provided site-specific information on the architecture and binding specificity of the domain interfaces using solubilized truncated domain constructs of the multi-domain E2 component in its interactions with the E1 and E3 components. Where studied, NMR has also provided information about mobile loops and the possible relationship of mobility and catalysis.

Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols.

Structural and Biochemical Characterization of Human Mitochondrial Branched-chain α-Ketoacid Dehydrogenase Phosphatase

The branched-chain α-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain α-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn(2+) ions for phosphatase activity, whereas Mg(2+) and Ca(2+) ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nm and 10 μm for Mn(2+) and Mg(2+) ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca(2+)-independent binding of BDP to the 24-meric transacylase (dihydrolipoyl transacylase; E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by x-ray crystallography to 2.4 Å resolution. The BDP structure is dominated by a central β-sandwich. There are two protrusions forming a narrow cleft ∼10 Å wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp-109, Asp-207, Asp-298, and Asp-337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg-104 increases the K(m) for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein.

Epithelial cell specificity and apotope recognition by serum autoantibodies in primary biliary cirrhosis

A major enigma of primary biliary cirrhosis (PBC) is the selective targeting of biliary cells. Our laboratory has reported that after apoptosis, human intrahepatic biliary epithelial cells (HiBECs) translocate the E2 subunit of the pyruvate dehydrogenase complex immunologically intact into apoptotic bodies, forming an apotope. However, the cell type and specificity of this reaction has not been fully defined. To address this issue, we investigated whether the E2 subunit of the pyruvate dehydrogenase complex, the E2 subunit of the branched chain 2-oxo acid dehydrogenase complex, the E2 subunit of the oxo-glutarate dehydrogenase complex, four additional inner mitochondrial enzymes, and four nuclear antigens remain immunologically intact with respect to postapoptotic translocation in HiBECs and three additional control epithelial cells. We report that all three 2-oxo acid dehydrogenase enzymes share the ability to remain intact within the apotope of HiBECs. Interestingly, the E2 subunit of the branched chain 2-oxo acid dehydrogenase complex also remained intact in the other cell types tested. We extended the data, using sera from 95 AMA-positive and 19 AMA-negative patients with PBC and 76 controls, by testing for reactivity against the seven mitochondrial proteins studied herein and also the ability of AMA-negative sera to react with HiBEC apotopes. Sera from 3 of 95 AMA-positive sera, but none of the controls, reacted with 2,4-dienoyl coenzyme A reductase 1, an enzyme also present intact only in the HiBEC apotope, but which has not been previously associated with any autoimmune disease. Finally, the specificity of HiBEC apotope reactivity was confined to AMA-positive sera. We submit that the biliary specificity of PBC is secondary to the unique processes of biliary apoptosis.

A 2-oxoacid dehydrogenase complex of Haloferax volcanii is essential for growth on isoleucine but not on other branched-chain amino acids

The halophilic archaeon Haloferax volcanii contains three operons encoding 2-oxoacid dehydrogenase complexes (OADHCs) OADHC1-OADHC3. However, the biological role of these OADHCs is not known as previous studies have demonstrated that they cannot use any of the known OADHC substrates. Even the construction of single mutants in all three oadhc operons, reported recently, could not identify a substrate. Therefore, all three possible double mutants and a triple mutant were generated, and single, double and triple mutants were compared to the wild-type. The four mutants devoid of a functional OADHC1 had a reduced growth yield during nitrate-respirative growth on tryptone. A metabolome analysis of the medium after growth of the triple mutant in comparison to the wild-type revealed that the mutant was unable to degrade isoleucine and leucine, in contrast to the wild-type. It was shown that oadhc1 mutants were unable to grow in synthetic medium on isoleucine, in contrast to the other mutants and the isogenic parent strain. However, all strains grew indistinguishably on valine and leucine. The transcript of the oadhc1 operon was highly induced during growth on isoleucine. However, attempts to detect enzymic activity were unsuccessful, while the branched-chain OADHC (BCDHC) of Pseudomonas putida could be measured easily. Therefore, the growth capability of the triple mutant and the wild-type on the two first degradation intermediates of isoleucine was tested and provided further evidence that OADHC is involved in isoleucine degradation. Taken together, the results indicate that OADHC1 is a specialized BCDHC that uses only one (or maximally two) of the three branched-chain 2-oxoacids, in contrast to BCDHCs from other species.

Branched-chain 2-oxoacid transamination increases GABA-shunt metabolism and insulin secretion in isolated islets

We have previously shown that oxo-4-methylpentanoate promotes islet GABA (gamma-aminobutyric acid) metabolism and stimulates insulin secretion. The main aim of this work was to explore the participation of the transamination of branched-chain 2-oxoacids in these processes with the aid of several inhibitors of this enzyme activity. No correlation was found between the transamination of branched-chain 2-oxoacids in islet homogenates and insulin secretion. However, in vivo transamination rates correlated better with the secretion capacity of the different branched-chain 2-oxoacids. Gabapentin, a specific inhibitor of the cytosolic isoenzyme, showed greater potential to decrease the in vitro transamination rates of oxo-3-methylbutyrate and oxo-3-methylpentanoate than those of oxo-4-methylpentanoate and oxohexanoate; this correlated with its capacity to decrease insulin secretion. 4-Methylvaleric acid very strongly inhibited the transamination of all the branched-chain 2-oxoacids and blocked their capacity to decrease islet GABA and to stimulate insulin secretion. KCl at 70 mM at stimulated islet GABA release, subsequently decreasing its tissue concentration. This 'non-metabolic' decrease of GABA suppressed the second phase of insulin secretion triggered by oxo-4-methylpentanoate and oxohexanoate. Oxo-4-methylpentanoate and oxo-3-methylpentanoate suppressed dose-dependent 2-oxoglutarate dehydrogenase activity in islet homogenates. In conclusion, the transamination of branchedchain 2-oxoacids is more important to the stimulation of insulin secretion than their catabolism, and transamination decreases islet GABA concentrations by promoting GABA metabolism. Also, inhibition of 2-oxoglutarate dehydrogenase by branched-chain 2-oxoacids may increase metabolic flux in the 'GABA-shunt' at the expense of reduced tricarboxylic acid cycle flux.

Recall rate and positive predictive value of MSUD screening is not influenced by hydroxyproline

Maple syrup urine disease (MSUD) is an autosomal recessive inherited disorder caused by the deficiency of the branched-chain 2-oxo acid dehydrogenase (BCOA-DH) complex. The worldwide incidence is approximately 1 in 185,000. MSUD is integrated in many "expanded" newborn screening (NBS) programs that use electrospray ionization tandem mass spectrometry (ESI-MS/MS). Elevated leucine, isoleucine, and alloisoleucine in the dried blood samples (DBS) of newborns are diagnostic parameters. However, with the applied method, it is not possible to distinguish the amino acids from each other, and also not from the other isobaric amino acid, hydroxyproline. While the branched chain amino acids (BCAA) leucine, isoleucine, and alloisoleucine are no diagnostic problem, because they are all elevated in MSUD patients, and, rather, increase the diagnostic sensitivity and specificity, hydroxyproline may cause false-positive screening results. Hydroxyproline is elevated in the benign familial condition hyperhydroxyprolinemia, which needs no medical intervention. The detection of cases with hyperhydroxyprolinemia have formerly been reported from screening programs that used thin-layer chromatography for phenylketonuria (PKU) screening, and, recently, two more cases have been reported, detected by ESI-MS/MS-based NBS. However, the detection of non-diseases is a heavy burden for screening programs and should be avoided if possible. With optimal settings for the quantitation of BCAAs and interpretation rules, elevated hydroxyproline should not cause false-positive screening results.

Structural bases for the specific interactions between the E2 and E3 components of the Thermus thermophilus 2-oxo acid dehydrogenase complexes

Pyruvate dehydrogenase (PDH), branched-chain 2-oxo acid dehydrogenase (BCDH) and 2-oxoglutarate dehydrogenase (OGDH) are multienzyme complexes that play crucial roles in several common metabolic pathways. These enzymes belong to a family of 2-oxo acid dehydrogenase complexes that contain multiple copies of three different components (E1, E2 and E3). For the Thermus thermophilus enzymes, depending on its substrate specificity (pyruvate, branched-chain 2-oxo acid or 2-oxoglutarate), each complex has distinctive E1 (E1p, E1b or E1o) and E2 (E2p, E2b or E2o) components and one of the two possible E3 components (E3b and E3o). (The suffixes, p, b and o identify their respective enzymes, PDH, BCDH and OGDH.) Our biochemical characterization demonstrates that only three specific E3*E2 complexes can form (E3b*E2p, E3b*E2b and E3o*E2o). X-ray analyses of complexes formed between the E3 components and the peripheral subunit-binding domains (PSBDs), derived from the corresponding E2-binding partners, reveal that E3b interacts with E2p and E2b in essentially the same manner as observed for Geobacillus stearothermophilus E3*E2p, whereas E3o interacts with E2o in a novel fashion. The buried intermolecular surfaces of the E3b*PSBDp/b and E3o*PSBDo complexes differ in size, shape and charge distribution and thus, these differences presumably confer the binding specificities for the complexes.

Structure–function relationships in the 2‐oxo acid dehydrogenase family: Substrate‐specific signatures and functional predictions for the 2‐oxoglutarate dehydrogenase‐like proteins

Structural relationship within the family of the thiamine diphosphate-dependent 2-oxo acid dehydrogenases was analyzed by combining different methods of sequence alignment with crystallographic and enzymological studies of the family members. For the first time, the sequence similarity of the homodimeric 2-oxoglutarate dehydrogenase to heterotetrameric 2-oxo acid dehydrogenases is established. The presented alignment of the catalytic domains of the dehydrogenases of pyruvate, branched-chain 2-oxo acids and 2-oxoglutarate unravels the sequence markers of the substrate specificity and the essential residues of the family members without the 3D structures resolved. Predicted dual substrate specificity of some of the 2-oxo acid dehydrogenases was confirmed experimentally. The results were used to decipher functions of the two hypothetical proteins of animal genomes, OGDHL and DHTKD1, similar to the 2-oxoglutarate dehydrogenase. Conservation of all the essential residues confirmed their catalytic competence. Sequence analysis indicated that OGDHL represents a previously unknown isoform of the 2-oxoglutarate dehydrogenase, whereas DHTKD1 differs from the homologs at the N-terminus and substrate binding pocket. The differences suggest changes in heterologous protein interactions and accommodation of more polar and/or bulkier structural analogs of 2-oxoglutarate, such as 2-oxoadipate, 2-oxo-4-hydroxyglutarate, or products of the carboligase reaction between a 2-oxodicarboxylate and glyoxylate or acetaldehyde. The signatures of the Ca2+-binding sites were found in the Ca2+-activated 2-oxoglutarate dehydrogenase and OGDHL, but not in DHTKD1. Mitochondrial localization was predicted for OGDHL and DHTKD1, with DHTKD1 probably localized also to nuclei. Medical implications of the obtained results are discussed in view of the possible associations of the 2-oxo acid dehydrogenases and DHTKD1 with neurodegeneration and cancer.

The 2‐oxoacid dehydrogenase multi‐enzyme complex of the archaeon Thermoplasma acidophilum − recombinant expression, assembly and characterization

The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.