2-oxo Acid Dehydrogenase Research Articles

Characterization of Autoantibodies against the E1α Subunit of Branched-Chain 2-Oxoacid Dehydrogenase in Patients with Primary Biliary Cirrhosis.

Primary biliary cirrhosis (PBC) is characterized by antimitochondrial antibodies (AMAs) that react with the lipoyl-containing E2 subunits of 2-oxoacid dehydrogenase complexes such as BCOADC and PDC. The lipoyl domains of E2 contain the major epitopes essential for immunopathology. However, the non-lipoyl-containing E1 subunits are also frequently targeted. Since anti-E1 antibodies always appear in combination with anti-E2 antibodies, the mechanisms underlying the autoimmunity against E1 may be linked to, but distinct from, those against E2. Here, we demonstrate that intermolecular and intramolecular determinant spreading underlies the autoimmunity against E1. We performed characterizations and epitope mapping for anti-BCOADC-E1α antibodies from both the intermolecular and intramolecular points of view. The antibody reactivities form a cluster against the BCOADC complex that is distinct from that against the PDC complex, and the anti-BCOADC-E1α antibodies arise as part of the cluster against the BCOADC complex. Multiple epitopes are present on the surface of the BCOADC-E1α molecule, and the major epitope overlaps with the active center. Sera with anti-BCOADC-E1α antibodies strongly inhibited the enzyme activity. These findings suggest that the E1α subunit as part of the native BCOADC complex is an immunogen, and that determinant spreading is involved in the pathogenesis of AMA production.

Mutations in Iron-Sulfur Cluster Scaffold Genes NFU1 and BOLA3 Cause a Fatal Deficiency of Multiple Respiratory Chain and 2-Oxoacid Dehydrogenase Enzymes

Severe combined deficiency of the 2-oxoacid dehydrogenases, associated with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, analysis of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs(∗)13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the production of [Fe-S] centers for the normal maturation of lipoate-containing 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.

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.

Protein-Protein Interactions in Assembly of Lipoic Acid on the 2-Oxoacid Dehydrogenases of Aerobic Metabolism

Lipoic acid is a covalently attached cofactor essential for the activity of 2-oxoacid dehydrogenases and the glycine cleavage system. In the absence of lipoic acid modification, the dehydrogenases are inactive, and aerobic metabolism is blocked. In Escherichia coli, two pathways for the attachment of lipoic acid exist, a de novo biosynthetic pathway dependent on the activities of the LipB and LipA proteins and a lipoic acid scavenging pathway catalyzed by the LplA protein. LipB is responsible for octanoylation of the E2 components of 2-oxoacid dehydrogenases to provide the substrates of LipA, an S-adenosyl-L-methionine radical enzyme that inserts two sulfur atoms into the octanoyl moiety to give the active lipoylated dehydrogenase complexes. We report that the intact pyruvate and 2-oxoglutarate dehydrogenase complexes specifically copurify with both LipB and LipA. Proteomic, genetic, and dehydrogenase activity data indicate that all of the 2-oxoacid dehydrogenase components are present. In contrast, LplA, the lipoate protein ligase enzyme of lipoate salvage, shows no interaction with the 2-oxoacid dehydrogenases. The interaction is specific to the dehydrogenases in that the third lipoic acid-requiring enzyme of Escherichia coli, the glycine cleavage system H protein, does not copurify with either LipA or LipB. Studies of LipB interaction with engineered variants of the E2 subunit of 2-oxoglutarate dehydrogenase indicate that binding sites for LipB reside both in the lipoyl domain and catalytic core sequences. We also report that LipB forms a very tight, albeit noncovalent, complex with acyl carrier protein. These results indicate that lipoic acid is not only assembled on the dehydrogenase lipoyl domains but that the enzymes that catalyze the assembly are also present "on site."

Lipoic Acid Synthesis: A New Family of Octanoyltransferases Generally Annotated as Lipoate Protein Ligases

Bacillus subtilis lacks a recognizable homologue of the LipB octanoyltransferase, an enzyme essential for lipoic acid synthesis in Escherichia coli. LipB transfers the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the 2-oxoacid dehydrogenases via a thioester-linked octanoyl-LipB intermediate. The octanoylated dehydrogenase is then converted to the enzymatically active lipoylated species by insertion of two sulfur atoms into the octanoyl moiety by the S-adenosyl-l-methionine radical enzyme, LipA (lipoate synthase). B. subtilis synthesizes lipoic acid and contains a LipA homologue that is fully functional in E. coli. Therefore, the lack of a LipB homologue presented the puzzle of how B. subtilis synthesizes the LipA substrate. We report that B. subtilis encodes an octanoyltransferase that has virtually no sequence resemblance to E. coli LipB but instead has a sequence that resembles that of the E. coli lipoate ligase, LplA. On the basis of this resemblance, these genes have generally been annotated as encoding a lipoate ligase, an enzyme that in E. coli scavenges lipoic acid from the environment but plays no role in de novo synthesis. We have named the B. subtilis octanoyltransferase LipM and find that, like LipB, the LipM reaction proceeds through a thioester-linked acyl enzyme intermediate. The LipM active site nucleophile was identified as C150 by the finding that this thiol becomes modified when LipM is expressed in E. coli. The level of the octanoyl-LipM intermediate can be significantly decreased by blocking fatty acid synthesis during LipM expression, and C150 was confirmed as an essential active site residue by site-directed mutagenesis. LipM homologues seem the sole type of octanoyltransferase present in the firmicutes and are also present in the cyanobacteria. LipM type octanoyltransferases represent a new clade of the PF03099 protein family, suggesting that octanoyl transfer activity has evolved at least twice within this superfamily.

Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.

Discovery of a putative acetoin dehydrogenase complex in the hyperthermophilic archaeon Sulfolobus solfataricus

Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2-oxoacid dehydrogenase complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α2β2 hetero-tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase multienzyme complex. This represents the first report of an acetoin multienzyme complex in archaea, and contrasts with the branched-chain 2-oxoacid dehydrogenase complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.

Genomic variants associated with primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune hepatobiliary disease characterized by immune-mediated injury of small and medium-sized bile ducts, eventually leading to liver cirrhosis. Several studies have addressed PBC immunopathology, and the data support an immune activation leading to autoantibodies and autoreactive T cells acting against the lipoylated 2-oxoacid dehydrogenase complexes. The causes of the disease remain unknown, but environmental factors and genetic susceptibility both contribute to its onset. Over the past two decades several association studies have addressed the role of genetic polymorphisms in PBC pathogenesis and have reported multiple associations. However, only a few studies had sufficient statistical power, and in most cases results were not independently validated. A genome-wide association study has recently been reported, but this too awaits independent confirmation. The aim of this present work is to critically review the numerous studies dedicated to revealing genetic associations in PBC, and to predict the potential for future studies based on these data.

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.

Scavenging of Cytosolic Octanoic Acid by Mutant LplA Lipoate Ligases Allows Growth of Escherichia coli Strains Lacking the LipB Octanoyltransferase of Lipoic Acid Synthesis

The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in Escherichia coli, transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid dehydrogenases of aerobic metabolism. Strains containing null mutations in lipB are auxotrophic for either lipoic acid or octanoic acid. We report the isolation of two spontaneously arising mutant strains that allow growth of lipB strains on glucose minimal medium; we determined that suppression was caused by single missense mutations within the coding sequence of the gene (lplA) that encodes lipoate-protein ligase. The LplA proteins encoded by the mutant genes have reduced K(m) values for free octanoic acid and thus are able to scavenge cytosolic octanoic acid for octanoylation of lipoyl domains.

A unique lipoylation system in the Archaea

Members of the 2-oxoacid dehydrogenase multienzyme complex family play a key role in the pathways of central metabolism. Post-translational lipoylation of the dihydrolipoyl acyltransferase component of these complexes is essential for their activity, the lipoyllysine moiety performing the transfer of substrates and intermediates between the different active sites within these multienzyme systems. We have previously shown that the thermophilic archaeon, Thermoplasma acidophilum, has a four-gene cluster encoding the components of such a complex, which, when recombinantly expressed in Escherichia coli, can be assembled into an active multienzyme in vitro. Crucially, the E. coli host carries out the required lipoylation of the archaeal dihydrolipoyl acyltransferase component. Because active 2-oxoacid dehydrogenase multienzyme complexes have never been detected in any archaeon, the question arises as to whether Archaea possess a functional lipoylation system. In this study, we report the cloning and heterologous expression of two genes from Tp. acidophilum whose protein products together show significant sequence identity with the single lipoate protein ligase enzyme of bacteria. We demonstrate that both recombinantly expressed Tp. acidophilum proteins are required for lipoylation of the acyltransferase, and that the two proteins associate together to carry out this post-translational modification. From the published DNA sequences, we suggest the presence of functional transcriptional and translational regulatory elements, and furthermore we present preliminary evidence that lipoylation occurs in vivo in Tp. acidophilum. This is the first report of the lipoylation machinery in the Archaea, which is unique in that the catalytic activity is dependent on two separate gene products.

New ELISA for Detecting Primary Biliary Cirrhosis–Specific Antimitochondrial Antibodies

Antimitochondrial antibodies specific for primary biliary cirrhosis (PBC) target the E2 subunits of 2-oxo acid dehydrogenase complexes, in particular the pyruvate dehydrogenase complex (PDC)-E2. Their antigen-specific detection relies on conventional ELISA using purified PDC. More recent assays have employed a hybrid containing the 3 E2-subunits (MIT3). Some PBC sera react with one or the other preparation, suggesting the presence of nonoverlapping epitopes. We have developed an ELISA (anti-M2-3E) using a mixture of purified PDC and MIT3 as antigenic targets. We compared this assay to anti-MIT3 alone, conventional anti-PDC, and indirect immunofluorescence using 173 PBC and 247 disease controls. The anti-M2-3E ELISA showed a 93.6% diagnostic sensitivity compared with 91.3%, 83.8%, and 87.3% for MIT3, purified PDC, or indirect immunofluorescence, respectively, when all specificities are set to 98.8%. By immunoblotting, anti-M2-3E-positive sera unreactive to purified PDC recognized recombinant E2-subunits of the other 2 complexes, whereas those with no reactivity to MIT3 immunofixed PDC subunits E1alpha or E1beta. The diagnostic accuracy of the anti-M2-3E ELISA for detection of antibodies to 2-oxo acid dehydrogenase complexes exceeds that of conventional ELISA and IFL; its novelty derives from the combination of the MIT3 hybrid and purified PDC.

Detection of novel non-M2-related antimitochondrial antibodies in patients with anti-M2 negative primary biliary cirrhosis

Objective:In 95% of patients with primary biliary cirrhosis (PBC) antimitochondrial antibodies (AMAs) can be detected reacting with at least one of the five components of the M2 antigen identified as...

The titanium binding protein of Rhodococcus ruber GIN1 (NCIMB 40340) is a cell‐surface homolog of the cytosolic enzyme dihydrolipoamide dehydrogenase

Rhodococcus ruber GIN1 (formally Rh. strain GIN1) was previously isolated on the basis of its strong adherence to coal fly ash (CFA) and titanium dioxide particles from CFA sedimentation ponds of an electrical power plant in Israel. The interaction of the bacterium with oxides has been shown to be mediated by a cell surface protein designated TiBP (titanium binding protein) involving primarily strong, non-electrostatic forces. In this work, we set forward to identify this unique exocellular protein. Sequence analysis of the purified protein by mass spectrometry (LC/MS/MS) following trypsinization revealed 11 peptides. All of them showed >90% amino acid residues identity with sequences of one of the orthologs (dldh1) of the cytosolic enzyme dihydrolipoamide dehydrogenase (DLDH), based on the genome sequence of Rhodococcus strain RHA1. This genome was selected as a reference since currently it is the only sequenced Rhodococcal genome. Altogether, these peptides covered over 25% of the 52 kDa protein molecule. N- and C-termini primers were prepared and used to sequence the paralog gene from Rh. ruber GIN1 after polymerase chain reaction (PCR) amplification. All 11 peptides showed 100% identity with the sequence of this gene. The homology of TiBP with the supposedly cytosolic DLDH raised the question of whether the exocellular TiBP possesses DLDH activity. Indeed, intact late logarithmic phase Rh. ruber GIN1 cells, previously shown to express TiBP, were found to possess such activity, while very low activity was associated with stationary phase cells which possess diminished TiBP expression on their surface. Further evidence for the exocellular location of TiBP/DLDH was achieved using specific anti-TiBP polyclonal antibodies by whole cell and protein enzyme-linked immunosorbent assay (ELISA), showing high reactivity of the logarithmic phase cell surface and substantially lower reactivity with the stationary phase cells. As expected, logarithmic phase spheroplasts were not recognized by these antibodies. Similar results were obtained by fluorescence and scanning electron microscopy. Our postulation that DLDH is located on the surface of Rh. ruber GIN1, serving as a TiO2 binding protein, is in accordance with literary evidence on DLDH in other organisms, Bacteria, Archea, and Eukaryots that suggests it is associated with the outer membranes or cell surfaces. As an exocellular protein DLDH assumes various tasks which are not related to its classical role as a 2-oxoacid dehydrogenase, including serving as an adhesion/binding protein in certain bacteria.

Leishmania infantum: Provision of reducing equivalents to the mitochondrial tryparedoxin/tryparedoxin peroxidase system

Within the mitochondrion of Leishmania infantum, hydroperoxide metabolism relies on the activity of tryparedoxin-dependent peroxidases (TXNPxs). Tryparedoxins (TXNs) are thioredoxin-related oxidoreductases, which in vitro are reduced by the trypanothione reductase/trypanothione [TR/T(SH) 2] redox couple. Still, there is no evidence that this actually occurs in the mitochondrion. This communication addresses the question of how the mitochondrial TXN/TXNPx system is reduced. First, using a digitonin fractionation assay, we show that TR activity is absent from the L. infantum mitochondrion. The possibility that this organelle possesses alternative electron sources for TXN/TXNPx is then investigated. Biochemical assays performed with purified recombinant enzymes, revealed that TR and T(SH) 2 can be replaced, albeit less efficiently, by the dihydrolipoamide dehydrogenase/lipoamide redox system as TXN/TXNPx electron donor. This result challenges the classical view that T(SH) 2 is the only reductant for TXNs and add new prospects regarding the involvement of 2-oxo acid dehydrogenase complexes in L. infantum mitochondrial hydroperoxide metabolism.

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.

Primary biliary cirrhosis following lactobacillus vaccination for recurrent vaginitis

Antimitochondrial antibodies directed against the E2 subunit of the pyruvate dehydrogenase complex, PDC-E2, and other mitochondrial 2-oxoacid dehydrogenases (AMA-M2) are the hallmark for diagnosis of primary biliary cirrhosis (PBC). AMA-M2 formation as an early step in the pathogenesis of PBC has recently been assumed to be triggered by bacterial mimics of the E2 subunit and certain reactant xenobiotics. We report a case of symptomatic PBC diagnosed after sequential immunization with a lactobacillus vaccine for recurrent vaginitis over years. Serum AMA-M2 specificity of the patient was evaluated by indirect immunofluorescence, immunoblotting and ELISA. Serum antibody responses against pyruvate dehydrogenase complex-E2 subunit (PDC-E2(212-226)), the major PBC-specific mitochondrial autoepitope, and microbial mimics revealed cross-reactivity with beta-galactosidase of Lactobacillus delbrueckii (LACDE BGAL(266-280)) which shows a high local homology with that of Lactobacillus species administered via the vaccine. The relative affinity of antibody reactivity to LACDE BGAL(266-280) was significantly higher than that against human PDC-E2(212-226). We conclude that lactobacillus vaccination therapy may be another culprit for the development of PBC in genetically susceptible women.

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.

Three 2-oxoacid dehydrogenase operons in Haloferax volcanii: expression, deletion mutants and evolution

Two unrelated protein families catalyse the oxidative decarboxylation of 2-oxoacids, i.e. the 2-oxoacid dehydrogenase complexes (OADHCs) and the 2-oxoacid ferredoxin oxidoreductases (OAFORs). In halophilic archaea, OAFORs were found to be responsible for decarboxylation of pyruvate and 2-oxoglutarate. Nevertheless, two gene clusters encoding OADHCs were found previously in Haloferax volcanii, but their biological function remained obscure. Here a third oadhc gene cluster of H. volcanii is presented. To characterize the function, the genes encoding the E1 subunit were inactivated in all three gene clusters by in-frame deletions. Under aerobic conditions none of the three mutants showed any phenotypic difference from the wild-type in various media. However, growth yields of two mutants were considerably lower than that of wild-type under nitrate-respirative conditions in complex medium. Northern blot analyses revealed (1) that polycistronic transcripts are formed and all three gene clusters are bona fide operons and (2) that transcription of all three operons is induced under anaerobic conditions compared to aerobic conditions. Taken together, the three H. volcanii enzymes do not fulfil one of the 'usual' aerobic functions of typical OADHCs, but decarboxylate an as-yet-unidentified novel substrate under anaerobic conditions. A survey of all 28 fully sequenced archaeal genomes revealed that nearly all archaea contain several OAFORs (three to four on average), suggesting that this protein family was already present in their last common ancestor. In contrast, only nine archaea encode one or two OADHCs, indicating that this protein family entered archaea by lateral transfer of the cognate genes from bacteria. This view is underscored by a phylogenetic tree of 33 archaeal and bacterial OADHCs.