2-oxo Acid Dehydrogenase Complex Research Articles

Dldhcri3 zebrafish exhibit altered mitochondrial ultrastructure, morphology, and dysfunction partially rescued by probucol or thiamine.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto (or 2-oxo) acid dehydrogenase complexes. DLD disease symptoms are multisystemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue, and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption, and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated that probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.

Association of 2-oxoacid dehydrogenase complexes with respirasomes in mitochondria.

In the present study, cryo-electron tomography was used to investigate the localization of 2-oxoacid dehydrogenase complexes (OADCs) in cardiac mitochondria and mitochondrial inner membrane samples. Two classes of ordered OADC inner cores with different symmetries were distinguished and their quaternary structures modeled. One class corresponds to pyruvate dehydrogenase complexes and the other to dehydrogenase complexes of α-ketoglutarate and branched-chain α-ketoacids. OADCs were shown to be localized in close proximity to membrane-embedded respirasomes, as observed both in densely packed lamellar cristae of cardiac mitochondria and in ruptured mitochondrial samples where the dense packing is absent. This suggests the specificity of the OADC-respirasome interaction, which allows localized NADH/NAD+ exchange between OADCs and complex I of the respiratory chain. The importance of this local coupling is based on OADCs being the link between respiration, glycolysis and amino acid metabolism. The coupling of these basic metabolic processes can vary in different tissues and conditions and may be involved in the development of various pathologies. The present study shows that this important and previously missing parameter of mitochondrial complex coupling can be successfully assessed using cryo-electron tomography.

Detection of M2-Type Anti-Mitochondrial Autoantibodies against Specific Subunits in the Diagnosis of Primary Biliary Cholangitis in Patients with Discordant Results.

M2-type anti-mitochondrial autoantibodies are considered the hallmark of primary biliary cholangitis and are directed mainly against the E2 subunits of the 2-oxo acid dehydrogenase complex enzymes (PDC, BCOADC and OGDC). The aim of this study was to determine whether a Dot-blot that includes these E2 subunits separately could confirm the results of methods with non-separated subunits in patients with low positive or discordant results between techniques. Sera of 24 patients with low positive or discordant results and of 10 patients with clear positive results by non-separated subunits methods were analyzed by Dot-blot with separated subunits. Autoantibodies against E2 subunits of PDC, BCOADC or OGDC were detected in all patients, except in one case from the low positive or discordant results group, by Dot-blot with separated subunits. It would be advisable to use methods that include the three E2 subunits, and a Dot-blot with separated subunits could confirm doubtful cases by non-separated assays.

A Lipoate-Protein Ligase Is Required for De Novo Lipoyl-Protein Biosynthesis in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Lipoic acid is an organosulfur cofactor essential for several key enzyme complexes in oxidative and one-carbon metabolism. It is covalently bound to the lipoyl domain of the E2 subunit in some 2-oxoacid dehydrogenase complexes and the H-protein in the glycine cleavage system. Lipoate-protein ligase (Lpl) is involved in the salvage of exogenous lipoate and attaches free lipoate to the E2 subunit or the H-protein in an ATP-dependent manner. In the hyperthermophilic archaeon Thermococcus kodakarensis, TK1234 and TK1908 are predicted to encode the N- and C-terminal regions of Lpl, respectively. TK1908 and TK1234 recombinant proteins form a heterodimer and together displayed significant ligase activity toward octanoate in addition to lipoate when a chemically synthesized octapeptide was used as the acceptor. The proteins also displayed activity toward other fatty acids, indicating broad fatty acid specificity. On the other hand, lipoyl synthase from T. kodakarensis only recognized octanoyl-peptide as a substrate. Examination of individual proteins indicated that the TK1908 protein alone was able to catalyze the ligase reaction although with a much lower activity. Gene disruption of TK1908 led to lipoate/serine auxotrophy, whereas TK1234 gene deletion did not. Acyl carrier protein homologs are not found on the archaeal genomes, and the TK1908/TK1234 protein complex did not utilize octanoyl-CoA, raising the possibility that the substrate of the ligase reaction is octanoic acid itself. Although Lpl has been considered as an enzyme involved in lipoate salvage, the results imply that in T. kodakarensis, the TK1908 and TK1234 proteins function in de novo lipoyl-protein biosynthesis. IMPORTANCE Based on previous studies in bacteria and eukaryotes, lipoate-protein ligases (Lpls) have been considered to be involved exclusively in lipoate salvage. The genetic analyses in this study on the lipoate-protein ligase in T. kodakarensis, however, suggest otherwise and that the enzyme is additionally involved in de novo protein lipoylation. We also provide biochemical evidence that the lipoate-protein ligase displays broad substrate specificity and is capable of ligating acyl groups of various chain-lengths to the peptide substrate. We show that this apparent ambiguity in Lpl is resolved by the strict substrate specificity of the lipoyl synthase LipS in this organism, which only recognizes octanoyl-peptide. The results provide relevant physiological insight into archaeal protein lipoylation.

Engineering the 2-Oxoglutarate Dehydrogenase Complex to Understand Catalysis and Alter Substrate Recognition

The E. coli 2-oxoglutarate dehydrogenase complex (OGDHc) is a multienzyme complex in the tricarboxylic acid cycle, consisting of multiple copies of three components, 2-oxoglutarate dehydrogenase (E1o), dihydrolipoamide succinyltransferase (E2o) and dihydrolipoamide dehydrogenase (E3), which catalyze the formation of succinyl-CoA and NADH (+H+) from 2-oxoglutarate. This review summarizes applications of the site saturation mutagenesis (SSM) to engineer E. coli OGDHc with mechanistic and chemoenzymatic synthetic goals. First, E1o was engineered by creating SSM libraries at positions His260 and His298.Variants were identified that: (a) lead to acceptance of substrate analogues lacking the 5-carboxyl group and (b) performed carboligation reactions producing acetoin-like compounds with good enantioselectivity. Engineering the E2o catalytic (core) domain enabled (a) assignment of roles for pivotal residues involved in catalysis, (b) re-construction of the substrate-binding pocket to accept substrates other than succinyllysyldihydrolipoamide and (c) elucidation of the mechanism of trans-thioesterification to involve stabilization of a tetrahedral oxyanionic intermediate with hydrogen bonds by His375 and Asp374, rather than general acid–base catalysis which has been misunderstood for decades. The E. coli OGDHc is the first example of a 2-oxo acid dehydrogenase complex which was evolved to a 2-oxo aliphatic acid dehydrogenase complex by engineering two consecutive E1o and E2o components.

Antimitochondrial Antibodies: from Bench to Bedside.

Anti-mitochondrial antibodies (AMA) are directed against the E2 subunits of the 2-oxo acid dehydrogenase complexes (PDC-E2) and are the typical biomarkers of primary biliary cholangitis (PBC), being present in 90–95% of patients, with increasing sensitivity at increasing titers. Albeit being highly specific for PBC diagnosis, AMA can be detected in less than 1% of healthy subjects, and thus the management subjects with no sign or symptom of liver disease is still a challenge and data concerning clinical risk of developing PBC in this subgroup of patients are controversial. Moreover, AMA can also be detected in patients affected by overlap syndrome, as well as hepatic diseases (i.e., NASH and viral hepatitis), while the association with autoimmune diseases, in particular Sjögren’s syndrome, systemic sclerosis, and systemic lupus erythematosus, is well established. Furthermore, new associations are being identified with inflammatory myositis and heart disease. AMA are directed towards the pyruvate dehydrogenase multi enzyme complex (PDC-E2) subunit, which represents an epithelial specific autoantigen for PBC. This review focuses on the main characteristics of AMA, their association with autoimmune diseases and liver diseases.

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.

Toward an Understanding of the Structural and Mechanistic Aspects of Protein-Protein Interactions in 2-Oxoacid Dehydrogenase Complexes.

The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxoacid dehydrogenase complexes, their function and regulation.

DHTKD1 and OGDH display substrate overlap in cultured cells and form a hybrid 2-oxo acid dehydrogenase complex in vivo.

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by a specific encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. Substrate reduction through inhibition of DHTKD1, an enzyme upstream of the defective glutaryl-CoA dehydrogenase, has been investigated as a potential therapy, but revealed the existence of an alternative enzymatic source of glutaryl-CoA. Here, we show that loss of DHTKD1 in glutaryl-CoA dehydrogenase-deficient HEK-293 cells leads to a 2-fold decrease in the established GA1 clinical biomarker glutarylcarnitine and demonstrate that oxoglutarate dehydrogenase (OGDH) is responsible for this remaining glutarylcarnitine production. We furthermore show that DHTKD1 interacts with OGDH, dihydrolipoyl succinyltransferase and dihydrolipoamide dehydrogenase to form a hybrid 2-oxoglutaric and 2-oxoadipic acid dehydrogenase complex. In summary, 2-oxoadipic acid is a substrate for DHTKD1, but also for OGDH in a cell model system. The classical 2-oxoglutaric dehydrogenase complex can exist as a previously undiscovered hybrid containing DHTKD1 displaying improved kinetics towards 2-oxoadipic acid.

Correction to: Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance by Victoria I. Bunik. Antioxid Redox Signal 30:1911-1947, 2019. DOI: 10.1089/ars.2017.7311.

Antioxidants & Redox SignalingVol. 31, No. 9 CorrectionFree AccessCorrection to: Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance by Victoria I. Bunik. Antioxid Redox Signal 30:1911–1947, 2019. DOI: 10.1089/ars.2017.7311is erratum ofRedox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic ImbalancePublished Online:25 Jul 2019https://doi.org/10.1089/ars.2017.7311.correxAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail In the June 1, 2019 issue of Antioxidants & Redox Signaling (vol. 30, no. 16; 1911–1947) the article entitled “Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance” by Victoria I. Bunik requires correction.Dr. Schirris was not listed as a reviewing editor.The list of reviewing editors originally read:Reviewing Editors: Miguel A. Aon, Rui Benfeitas, Enrique Cadenas, Jose Manuel Cuezva, Arne Holmgren, Saijaliisa Kangasjärvi, John Mieyal, and Andras PerlDr. Schirris has now been added to the list:Reviewing Editors: Miguel A. Aon, Rui Benfeitas, Enrique Cadenas, Jose Manuel Cuezva, Arne Holmgren, Saijaliisa Kangasjärvi, John Mieyal, Andras Perl, and Tom J.J. SchirrisThe online version has been corrected to reflect this.FiguresReferencesRelatedDetailsRelated articlesRedox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance11 Apr 2019Antioxidants & Redox Signaling Volume 31Issue 9Sep 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Correction to: Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance by Victoria I. Bunik. Antioxid Redox Signal 30:1911–1947, 2019. DOI: 10.1089/ars.2017.7311.Antioxidants & Redox Signaling.Sep 2019.671-671.http://doi.org/10.1089/ars.2017.7311.correxPublished in Volume: 31 Issue 9: July 25, 2019PDF download

Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance.

This article develops a holistic view on production of reactive oxygen species (ROS) by 2-oxo acid dehydrogenase complexes. Recent Advances: Catalytic and structural properties of the complexes and their components evolved to minimize damaging effects of side reactions, including ROS generation, simultaneously exploiting the reactions for homeostatic signaling. Side reactions of the complexes, characterized in vitro, are analyzed in view of protein interactions and conditions in vivo. Quantitative data support prevalence of the forward 2-oxo acid oxidation over the backward NADH oxidation in feeding physiologically significant ROS production by the complexes. Special focus on interactions between the active sites within 2-oxo acid dehydrogenase complexes highlights the central relevance of the complex-bound thiyl radicals in regulation of and signaling by complex-generated ROS. The thiyl radicals arise when dihydrolipoyl residues of the complexes regenerate FADH2 from the flavin semiquinone coproduced with superoxide anion radical in 1e- oxidation of FADH2 by molecular oxygen. Interaction of 2-oxo acid dehydrogenase complexes with thioredoxins (TRXs), peroxiredoxins, and glutaredoxins mediates scavenging of the thiyl radicals and ROS generated by the complexes, underlying signaling of disproportional availability of 2-oxo acids, CoA, and NAD+ in key metabolic branch points through thiol/disulfide exchange and medically important hypoxia-inducible factor, mammalian target of rapamycin (mTOR), poly (ADP-ribose) polymerase, and sirtuins. High reactivity of the coproduced ROS and thiyl radicals to iron/sulfur clusters and nitric oxide, peroxynitrite reductase activity of peroxiredoxins and transnitrosylating function of thioredoxin, implicate the side reactions of 2-oxo acid dehydrogenase complexes in nitric oxide-dependent signaling and damage.

Catalysis of transthiolacylation in the active centers of dihydrolipoamide acyltransacetylase components of 2‐oxo acid dehydrogenase complexes

The Escherichia coli 2‐oxoglutarate dehydrogenase complex (OGDHc) comprises multiple copies of three enzymes—E1o, E2o, and E3—and transthioesterification takes place within the catalytic domain of E2o. The succinyl group from the thiol ester of S8‐succinyldihydrolipoyl‐E2o is transferred to the thiol group of coenzyme A (CoA), forming the all‐important succinyl‐CoA. Here, we report mechanistic studies of enzymatic transthioesterification on OGDHc. Evidence is provided for the importance of His375 and Asp374 in E2o for the succinyl transfer reaction. The magnitude of the rate acceleration provided by these residues (54‐fold from each with alanine substitution) suggests a role in stabilization of the symmetrical tetrahedral oxyanionic intermediate by formation of two hydrogen bonds, rather than in acid–base catalysis. Further evidence ruling out a role in acid–base catalysis is provided by site‐saturation mutagenesis studies at His375 (His375Trp substitution with little penalty) and substitutions to other potential hydrogen bond participants at Asp374. Taking into account that the rate constant for reductive succinylation of the E2o lipoyl domain (LDo) by E1o and 2‐oxoglutarate (99 s−1) was approximately twofold larger than the rate constant for k cat of 48 s−1 for the overall reaction (NADH production), it could be concluded that succinyl transfer to CoA and release of succinyl‐CoA, rather than reductive succinylation, is the rate‐limiting step. The results suggest a revised mechanism of catalysis for acyl transfer in the superfamily of 2‐oxo acid dehydrogenase complexes, thus provide fundamental information regarding acyl‐CoA formation, so important for several biological processes including post‐translational succinylation of protein lysines.Enzymes2‐oxoglutarate dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/2.html); dihydrolipoamide succinyltransferase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/61.html); dihydrolipoamide dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/8/1/4.html); pyruvate dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/1.html); dihydrolipoamide acetyltransferase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/12.html).

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.

The human Krebs cycle 2-oxoglutarate dehydrogenase complex creates an additional source of superoxide/hydrogen peroxide from 2-oxoadipate as alternative substrate

Recently, we reported that the human 2-oxoglutarate dehydrogenase (hE1o) component of the 2-oxoglutarate dehydrogenase complex (OGDHc) could produce the reactive oxygen species superoxide and hydrogen peroxide (detected by chemical means) from its substrate 2-oxoglutarate (OG), most likely concurrently with one-electron oxidation by dioxygen of the thiamin diphosphate (ThDP)-derived enamine intermediate to a C2α-centered radical (detected by Electron Paramagnetic Resonance) [Nemeria et al., 2014 [17]; Ambrus et al. 2015 [18]]. We here report that hE1o can also utilize the next higher homologue of OG, 2-oxoadipate (OA) as a substrate according to multiple criteria in our toolbox: (i) Both E1o-specific and overall complex activities (NADH production) were detected using OA as a substrate; (ii) Two post-decarboxylation intermediates were formed by hE1o from OA, the ThDP-enamine and the C2α-hydroxyalkyl-ThDP, with nearly identical rates for OG and OA; (iii) Both OG and OA could reductively acylate lipoyl domain created from dihydrolipoyl succinyltransferase (E2o); (iv) Both OG and OA gave α-ketol carboligaton products with glyoxylate, but with opposite chirality; a finding that could be of utility in chiral synthesis; (v) Dioxygen could oxidize the ThDP-derived enamine from both OG and OA, leading to ThDP-enamine radical and generation of superoxide and H2O2. While the observed oxidation-reduction with dioxygen is only a side reaction of the predominant physiological product glutaryl-CoA, the efficiency of superoxide/ H2O2 production was 7-times larger from OA than from OG, making the reaction of OGDHc with OA one of the important superoxide/ H2O2 producers among 2-oxo acid dehydrogenase complexes in mitochondria.

The Pyruvate Dehydrogenase Complex and Related Assemblies in Health and Disease.

The family of 2-oxoacid dehydrogenase complexes (2-OADC), typified by the pyruvate dehydrogenase multi-enzyme complex (PDC) as its most prominent member, are massive molecular machines (Mr, 4-10 million) controlling key steps in glucose homeostasis (PDC), citric acid cycle flux (OGDC, 2-oxoglutarate dehydrogenase) and the metabolism of the branched-chain amino acids, leucine, isoleucine and valine (BCOADC, branched-chain 2-OADC). These highly organised mitochondrial arrays, composed of multiple copies of three separate enzymes, have been widely studied as paradigms for the analysis of enzyme cooperativity, substrate channelling, protein-protein interactions and the regulation of activity by phosphorylation . This chapter will highlight recent advances in our understanding of the structure-function relationships, the overall organisation and the transport and assembly of PDC in particular, focussing on both native and recombinant forms of the complex and their individual components or constituent domains. Biophysical approaches, including X-ray crystallography (MX), nuclear magnetic resonance spectroscopy (NMR), cryo-EM imaging, analytical ultracentrifugation (AUC) and small angle X-ray and neutron scattering (SAXS and SANS), have all contributed significant new information on PDC subunit organisation, stoichiometry, regulatory mechanisms and mode of assembly. Moreover, the recognition of specific genetic defects linked to PDC deficiency, in combination with the ability to analyse recombinant PDCs housing both novel naturally-occurring and engineered mutations, have all stimulated renewed interest in these classical metabolic assemblies. In addition, the role played by PDC, and its constituent proteins, in certain disease states will be briefly reviewed, focussing on the development of new and exciting areas of medical and immunological research.

Directed Regulation of Multienzyme Complexes of 2-Oxo Acid Dehydrogenases Using Phosphonate and Phosphinate Analogs of 2-Oxo Acids.

2-Oxo acid dehydrogenase complexes are important metabolic checkpoints functioning at the intercept of sugar and amino acid degradation. This review presents a short summary of architectural, catalytic, and regulatory principles of the complexes structure and function, based on recent advances in studies of well-characterized family members. Special attention is given to use of synthetic phosphonate and phosphinate analogs of 2-oxo acids as selective and efficient inhibitors of the cognate complexes in biological systems of bacterial, plant, and animal origin. We summarize our own results concerning the application of synthetic analogs of 2-oxo acids in situ and in vivo to reveal functional interactions between 2-oxo acid dehydrogenase complexes and other components of metabolic networks specific to different cells and tissues. Based on our study of glutamate excitotoxicity in cultured neurons, we show how a modulation of metabolism by specific inhibition of its key reaction may be employed to correct pathologies. This approach is further developed in our study on the action of the phosphonate analog of 2-oxoglutarate in animals. The study revealed that upregulation of 2-oxoglutarate dehydrogenase complex is involved in animal stress response and may provide increased resistance to damaging effects, underlying so-called preconditioning. The presented analysis of published data suggests synthetic inhibitors of metabolic checkpoints as promising tools to solve modern challenges of systems biology, metabolic engineering, and medicine.

Differential diagnosis of lipoic acid synthesis defects.

Lipoic acid (LA) is an essential cofactor required for the activity of five multienzymatic complexes that play a central role in the mitochondrial energy metabolism: four 2-oxoacid dehydrogenase complexes [pyruvate dehydrogenase (PDH), branched-chain ketoacid dehydrogenase (BCKDH), 2-ketoglutarate dehydrogenase (2-KGDH), and 2-oxoadipate dehydrogenase (2-OADH)] and the glycine cleavage system (GCS). LA is synthesized in a complex multistep process that requires appropriate function of the mitochondrial fatty acid synthesis (mtFASII) and the biogenesis of iron-sulphur (Fe-S) clusters. Defects in the biosynthesis of LA have been reported to be associated with multiple and severe defects of the mitochondrial energy metabolism. In recent years, disease-causing mutations in genes encoding for proteins involved in LA metabolism have been reported: NFU1, BOLA3, IBA57, LIAS, GLRX5, LIPT1, ISCA2, and LIPT2. These studies represented important progress in understanding the pathophysiology and molecular bases underlying these disorders. Here we review current knowledge regarding involvement of LA synthesis defects in human diseases with special emphasis on the diagnostic strategies for these disorders. The clinical and biochemical characteristics of patients with LA synthesis defects are discussed and a workup for the differential diagnosis proposed.

Production of superoxide/hydrogen peroxide by the mitochondrial 2-oxoadipate dehydrogenase complex

In humans, mutations in dehydrogenase E1 and transketolase domain containing 1 (DHTKD1) are associated with neurological abnormalities and accumulation of 2-oxoadipate, 2-aminoadipate, and reactive oxygen species. The protein encoded by DHTKD1 has sequence and structural similarities to 2-oxoglutarate dehydrogenase, and the 2-oxoglutarate dehydrogenase complex can produce superoxide/H2O2 at high rates. The DHTKD1 enzyme is hypothesized to catalyze the oxidative decarboxylation of 2-oxoadipate, a shared intermediate of the degradative pathways for tryptophan, lysine and hydroxylysine. Here, we show that rat skeletal muscle mitochondria can produce superoxide/H2O2 at high rates when given 2-oxoadipate. We identify the putative mitochondrial 2-oxoadipate dehydrogenase complex as one of the sources and characterize the conditions that favor its superoxide/H2O2 production. Rates increased at higher NAD(P)H/NAD(P)+ ratios and were higher at each NAD(P)H/NAD(P)+ ratio when 2-oxoadipate was present, showing that superoxide/H2O2 was produced during the forward reaction from 2-oxoadipate, but not in the reverse reaction from NADH in the absence of 2-oxoadipate. The maximum capacity of the 2-oxoadipate dehydrogenase complex for production of superoxide/H2O2 is comparable to that of site IF of complex I, and seven, four and almost two-fold lower than the capacities of the 2-oxoglutarate, pyruvate and branched-chain 2-oxoacid dehydrogenase complexes, respectively. Regulation by ADP and ATP of H2O2 production driven by 2-oxoadipate was very different from that driven by 2-oxoglutarate, suggesting that site AF of the 2-oxoadipate dehydrogenase complex is a new source of superoxide/H2O2 associated with the NADH isopotential pool in mitochondria.

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.