RESUMO
The present work delves into the enigmatic world of mitochondrial alpha-keto acid dehydrogenase complexes discussing their metabolic significance, enzymatic operation, moonlighting activities, and pathological relevance with links to underlying structural features. This ubiquitous family of related but diverse multienzyme complexes is involved in carbohydrate metabolism (pyruvate dehydrogenase complex), the citric acid cycle (α-ketoglutarate dehydrogenase complex), and amino acid catabolism (branched-chain α-keto acid dehydrogenase complex, α-ketoadipate dehydrogenase complex); the complexes all function at strategic points and also participate in regulation in these metabolic pathways. These systems are among the largest multienzyme complexes with at times more than 100 protein chains and weights ranging up to ~10 million Daltons. Our chapter offers a wealth of up-to-date information on these multienzyme complexes for a comprehensive understanding of their significance in health and disease.
Assuntos
Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/enzimologia , Animais , Ciclo do Ácido Cítrico/fisiologia , Complexo Cetoglutarato Desidrogenase/metabolismo , Complexo Cetoglutarato Desidrogenase/químicaRESUMO
Pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) are vital entry points for monosaccharides and amino acids into the Krebs cycle and thus integral for mitochondrial bioenergetics. Both complexes produce mitochondrial hydrogen peroxide (mH2O2) and are deactivated by electrophiles. Here, we provide an update on the role of PDH and KGDH in mitochondrial redox balance and their function in facilitating metabolic reprogramming for the propagation of oxidative eustress signals in hepatocytes and how defects in these pathways can cause liver diseases. PDH and KGDH are known to account for â¼45% of the total mH2O2 formed by mitochondria and display rates of production several-fold higher than the canonical source complex I. This mH2O2 can also be formed by reverse electron transfer (RET) in vivo, which has been linked to metabolic dysfunctions that occur in pathogenesis. However, the controlled emission of mH2O2 from PDH and KGDH has been proposed to be fundamental for oxidative eustress signal propagation in several cellular contexts. Modification of PDH and KGDH with protein S-glutathionylation (PSSG) and S-nitrosylation (PSNO) adducts serves as a feedback inhibitor for mH2O2 production in response to glutathione (GSH) pool oxidation. PSSG and PSNO adduct formation also reprogram the Krebs cycle to generate metabolites vital for interorganelle and intercellular signaling. Defects in the redox modification of PDH and KGDH cause the over generation of mH2O2, resulting in oxidative distress and metabolic dysfunction-associated fatty liver disease (MAFLD). In aggregate, PDH and KGDH are essential platforms for emitting and receiving oxidative eustress signals.
Assuntos
Hepatócitos , Peróxido de Hidrogênio , Complexo Cetoglutarato Desidrogenase , Mitocôndrias Hepáticas , Complexo Piruvato Desidrogenase , Peróxido de Hidrogênio/metabolismo , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/metabolismo , Oxirredução , Estresse Oxidativo , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/metabolismo , Humanos , Hepatócitos/enzimologia , Mitocôndrias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Animais , CamundongosRESUMO
The oxoglutarate dehydrogenase complex (OGDHc) participates in the tricarboxylic acid cycle and, in a multi-step reaction, decarboxylates α-ketoglutarate, transfers succinyl to CoA, and reduces NAD+. Due to its pivotal role in metabolism, OGDHc enzymatic components have been studied in isolation; however, their interactions within the endogenous OGDHc remain elusive. Here, we discern the organization of a thermophilic, eukaryotic, native OGDHc in its active state. By combining biochemical, biophysical, and bioinformatic methods, we resolve its composition, 3D architecture, and molecular function at 3.35 Å resolution. We further report the high-resolution cryo-EM structure of the OGDHc core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of OGDHc participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-subunit communication, and the presence of a flexible subunit (E3BPo), connecting E2o and E3. This multi-scale analysis of a succinyl-CoA-producing native cell extract provides a blueprint for structure-function studies of complex mixtures of medical and biotechnological value.
Assuntos
Ciclo do Ácido Cítrico , Complexo Cetoglutarato Desidrogenase , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/metabolismo , Acil Coenzima A/metabolismo , CitoplasmaRESUMO
5-fluorouracil (5-FU), which is a commonly used chemotherapy agent exerts undesired cardiac toxicity. Mitochondrial dysfunction is thought to be one of potentially important mechanisms of 5-FU- induced cardiotoxicity. α-ketoglutarate dehydrogenase (α-KGDHC) is the key regulatory enzyme of TCA cycle. The complex consists of multiple copies of three catalytic subunits: α-ketoglutarate dehydrogenase (E1), dihydrolipoamide succinyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). α-KGDHC together with branched chain α-ketoacid dehydrogenase (BCKDH) and pyruvate dehydrogenase (PDH), are the members of 2-oxoacid dehydrogenases family that share some structural and functional similarities. Recently, it has been found that 5-FU stimulates BCKDH in rat's cardiac muscle. Therefore, we hypothesize that 5-FU modifies α-KGDHC activity and affects cardiac muscle metabolism. The aim of this study was to determine the effect of 5-FU on α-KGDHC activity and protein levels of E1 and E2 subunits of the complex in rat's cardiac muscle. Wistar male rats were administered with 4 doses of 5-FU, 150 mg/ kg b.wt. each (study group) or 0.3% methylcellulose (control group). α-KGDHC activity was assayed spectrophotometrically. The E1 and E2 proteins levels were quantified by Western blot. 5-FU administration resulted in stimulation of myocardial α-KGDHC activity in rats. In addition, E2 protein level increased in response to 5-FU treatment, while the E1 protein level remained unchanged. Up-regulation of α-KGDHC appears to result from change in E2 subunit protein level. However, the effect of 5-FU on factors modifying α-KGDHC activity at post-translational level cannot be excluded.
Assuntos
Di-Hidrolipoamida Desidrogenase , Complexo Cetoglutarato Desidrogenase , Animais , Ratos , Masculino , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/metabolismo , Fluoruracila/farmacologia , Ratos Wistar , Miocárdio/metabolismo , Cetoácidos , Metilcelulose , PiruvatosRESUMO
The synthesis of parapyruvate is important for the analysis of the content in the pyruvate supplements and the study of aging-related neurodegenerative diseases. However, the pure parapyruvate crystal is not, as yet, commercially available. In this study, we applied the Taguchi's L9 orthogonal array to investigate the optimal conditions for the preparation of the pure parapyruvate by the alkaline treatment of the pyruvic acid and then followed it with the solvent crystallization steps. We were also interested in revealing the major factors that affect the yield for the synthesized pure parapyruvate crystals. In addition, the parapyruvate-inhibited enzyme kinetic of α-ketoglutarate dehydrogenase complex (KGDHC) was also investigated. We found that the pure parapyruvate could be obtained in combination with an alkaline treatment and two solvent crystallization steps. The main factors affecting the yield of the pure parapyruvate were the concentration of the pyruvic acid (the reactant), the pH of the alkali treatment, the type of solvent used for the crystallization and the volume ratio of solvent used for crystallization. Finally, the optimal conditions could prepare parapyruvate crystals with a high purity of 99.8% and a high yield of 72.8%. In addition, the results demonstrate that parapyruvate is a reversibly competitive inhibitor for KGDHC.
Assuntos
Complexo Cetoglutarato Desidrogenase , Complexo Cetoglutarato Desidrogenase/química , Ácido Pirúvico , SolventesRESUMO
BACKGROUND: The human mitochondrial alpha-ketoglutarate dehydrogenase complex (hKGDHc) converts KG to succinyl-CoA and NADH. Malfunction of and reactive oxygen species generation by the hKGDHc as well as its E1-E2 subcomplex are implicated in neurodegenerative disorders, ischemia-reperfusion injury, E3-deficiency and cancers. METHODS: We performed cryo-EM, cross-linking mass spectrometry (CL-MS) and molecular modeling analyses to determine the structure of the E2 component of the hKGDHc (hE2k); hE2k transfers a succinyl group to CoA and forms the structural core of hKGDHc. We also assessed the overall structure of the hKGDHc by negative-stain EM and modeling. RESULTS: We report the 2.9â¯Å resolution cryo-EM structure of the hE2k component. The cryo-EM map comprises density for hE2k residues 151-386 - the entire (inner) core catalytic domain plus a few additional residues -, while residues 1-150 are not observed due to the inherent flexibility of the N-terminal region. The structure of the latter segment was also determined by CL-MS and homology modeling. Negative-stain EM on in vitro assembled hKGDHc and previous data were used to build a putative overall structural model of the hKGDHc. CONCLUSIONS: The E2 core of the hKGDHc is composed of 24 hE2k chains organized in octahedral (8â¯×â¯3 type) assembly. Each lipoyl domain is oriented towards the core domain of an adjacent chain in the hE2k homotrimer. hE1k and hE3 are most likely tethered at the edges and faces, respectively, of the cubic hE2k assembly. GENERAL SIGNIFICANCE: The revealed structural information will support the future pharmacologically targeting of the hKGDHc.
Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Reagentes de Ligações Cruzadas/química , Microscopia Crioeletrônica/métodos , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/metabolismo , Espectrometria de Massas/métodos , Acil Coenzima A/metabolismo , Humanos , Ácidos Cetoglutáricos/metabolismo , Modelos Moleculares , NAD/metabolismo , Conformação ProteicaRESUMO
Unlike the OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH), which is an essential enzyme present in all animal tissues, expression of the DHTKD1-encoded isoenzyme, 2-oxoadipate dehydrogenase (OADH), depends on a number of factors, and mutant DHTKD1 phenotypes are rarely manifested. Physiological significance of OADH is also obscured by the fact that both isoenzymes transform 2-oxoglutarate and 2-oxoadipate. By analogy with other members of the 2-oxo acid dehydrogenases family, OADH is assumed to be a component of the multienzyme complex that catalyzes oxidative decarboxylation of 2-oxoadipate. This study aims at molecular characterization of OADH from animal tissues. Phylogenetic analysis of 2-oxo acid dehydrogenases reveals OADH only in animals and Dictyostelium discoideum slime mold, within a common branch with bacterial OGDH. Examination of partially purified animal OADH by immunoblotting and mass spectrometry identifies two OADH isoforms with molecular weights of about 130 and 70 kDa. These isoforms are not observed upon the expression of human DHTKD1 protein in either bacterial or yeast system, where the synthesized OADH is of expected molecular weight (about 100 kDa). Thus, the OADH isoforms present in animal tissues, may result from the animal-specific regulation of the DHTKD1 expression and/or posttranslational modifications of the encoded protein. Mapping of the peptides identified in the OADH preparations, onto the protein structure suggests that the 70-kDa isoform is truncated at the N-terminus, but retains the active site. Since the N-terminal domain of OGDH is required for the formation of the multienzyme complex, it is possible that the 70-kDa isoform catalyzes non-oxidative transformation of dicarboxylic 2-oxo acids that does not require the multienzyme structure. In this case, the ratio of the OADH isoforms in animal tissues may correspond to the ratio between the oxidative and non-oxidative decarboxylation of 2-oxoadipate.
Assuntos
Encéfalo/metabolismo , Escherichia coli/metabolismo , Complexo Cetoglutarato Desidrogenase/química , Fígado/metabolismo , Miocárdio/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Domínio Catalítico , Dictyostelium/genética , Dictyostelium/metabolismo , Escherichia coli/genética , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Complexo Cetoglutarato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/metabolismo , Masculino , Oxirredução , Filogenia , Ratos , Ratos Wistar , Saccharomyces cerevisiae/genéticaRESUMO
Pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) are critical enzymes in central carbon metabolism. In Corynebacterium glutamicum, an unusual hybrid complex consisting of CgE1p (thiamine diphosphate-dependent pyruvate dehydrogenase, AceE), CgE2 (dihydrolipoamide acetyltransferase, AceF), CgE3 (dihydrolipoamide dehydrogenase, Lpd), and CgE1o (thiamine diphosphate-dependent 2-oxoglutarate dehydrogenase, OdhA) has been suggested. Here, we elucidated that the PDH-ODH hybrid complex in C. glutamicum probably consists of six copies of CgE2 in its core, which is rather compact compared with PDH and ODH in other microorganisms that have twenty-four copies of E2. We found that CgE2 formed a stable complex with CgE3 (CgE2-E3 subcomplex) in vitro, hypothetically comprised of two CgE2 trimers and four CgE3 dimers. We also found that CgE1o exists mainly as a hexamer in solution and is ready to form an active ODH complex when mixed with the CgE2-E3 subcomplex. Our in vitro reconstituted system showed CgE1p- and CgE1o-dependent inhibition of ODH and PDH, respectively, actively supporting the formation of the hybrid complex, in which both CgE1p and CgE1o associate with a single CgE2-E3. In gel filtration chromatography, all the subunits of CgODH were eluted in the same fraction, whereas CgE1p was eluted separately from CgE2-E3, suggesting a weak association of CgE1p with CgE2 compared with that of CgE1o. This study revealed the unique molecular architecture of the hybrid complex from C. glutamicum and the compact-sized complex would provide an advantage to determine the whole structure of the unusual hybrid complex.
Assuntos
Proteínas de Bactérias/química , Corynebacterium glutamicum/enzimologia , Complexo Cetoglutarato Desidrogenase/química , Complexo Piruvato Desidrogenase/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Complexo Cetoglutarato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/metabolismo , Cinética , Ligação Proteica , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismoRESUMO
Metabolites produced via traditional biochemical processes affect intracellular communication, inflammation, and malignancy. Unexpectedly, acetyl-CoA, α-ketoglutarate and palmitic acid, which are chemical species of reactions catalyzed by highly abundant, gigantic enzymatic complexes, dubbed as "metabolons", have broad "nonmetabolic" signaling functions. Conserved unstructured regions within metabolons determine the yield of these metabolites. Unstructured regions tether functional protein domains, act as spatial constraints to confine constituent enzyme communication, and, in the case of acetyl-CoA production, tend to be regulated by intricate phosphorylation patterns. This review presents the multifaceted roles of these three significant metabolites and describes how their perturbation leads to altered or transformed cellular function. Their dedicated enzymatic systems are then introduced, namely, the pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) complexes, and the fatty acid synthase (FAS), with a particular focus on their structural characterization and the localization of unstructured regions. Finally, upstream metabolite regulation, in which spatial occupancy of unstructured regions within dedicated metabolons may affect metabolite availability and subsequently alter cell functions, is discussed. Video abstract.
Assuntos
Ácido Graxo Sintases/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Transdução de Sinais , Acetilcoenzima A/metabolismo , Animais , Ácido Graxo Sintases/química , Humanos , Proteínas Intrinsicamente Desordenadas/química , Complexo Cetoglutarato Desidrogenase/química , Ácidos Cetoglutáricos/metabolismo , Modelos Moleculares , Ácido Palmítico/metabolismo , Complexo Piruvato Desidrogenase/químicaRESUMO
DHTKD1 is the E1 component of the 2-oxoadipate dehydrogenase complex, which is an enzyme involved in the catabolism of (hydroxy-)lysine and tryptophan. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q and eosinophilic esophagitis, but the pathophysiology of these clinically distinct disorders remains elusive. Here, we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.25 Å. We also report the impact of 10 disease-associated missense mutations on DHTKD1. Whereas the majority of the DHTKD1 variants displayed impaired folding or reduced thermal stability in combination with absent or reduced enzyme activity, three variants showed no abnormalities. Our work provides chemical and structural tools for further understanding of the function of DHTKD1 and its role in several human pathologies.
Assuntos
Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Tiamina Pirofosfato/química , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/genética , Estrutura Molecular , Mutação de Sentido IncorretoRESUMO
Query fever is a zoonotic disease caused by Coxiella burnetii. There is no universal method for the prevention of this disease. Recombinant vaccine is a potent strategy that can be utilized for this purpose. The current study was conducted to develop a multi-epitope vaccine against Coxiella burnetii. Hence, OmpA, Tuf2, GroEL, Mip and sucB antigens were used for the prediction of epitopes. Then, a multi-epitope vaccine was developed based on a molecular adjuvant and fragments that contained the best MHCI, B cell, MHCII and IFN-γ epitopes. The features of the developed vaccine including physicochemical parameters, antigenicity and protein structures were assessed. Also, interaction between the developed vaccine and TLR4/MD2 receptor along with molecular dynamics of the ligand-receptor complex were investigated. Finally, the codon adaptation and cloning were conducted for the developed vaccine. According to the results, molecular weight, instability index, antigenicity and random coil percentage of the developed vaccine were 54.4â¯kDa, 32.84, 1.1936 and 34.92%, respectively. Besides, residues distribution in core region of the refined model was 85%. The results demonstrated that the developed vaccine could dock to its receptor with the lowest energy of -976.7 as well as RMSD value of the complex was between 0.15 and 0.22â¯nm. Also, the results showed that CIA index of the codon adapted sequence was 0.95. Finally, cloning results revealed that nucleotide sequence of the developed vaccine could be successfully cloned into pET-21a (+). Based on these results, it seems that the developed vaccine can be a suitable candidate to prevent Coxiella burnetii.
Assuntos
Vacinas Bacterianas/imunologia , Zoonoses Bacterianas/prevenção & controle , Coxiella burnetii/imunologia , Epitopos de Linfócito B/imunologia , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Febre Q/prevenção & controle , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/química , Chaperonina 60/química , Chaperonina 60/imunologia , Epitopos de Linfócito B/química , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/imunologia , Antígeno 96 de Linfócito/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Febre Q/imunologia , Febre Q/microbiologia , Receptor 4 Toll-Like/químicaRESUMO
Mycobacterial KGD, the thiamine diphosphate (ThDP)-dependent E1o component of the 2-oxoglutarate dehydrogenase complex (OGDHC), is known to undergo significant conformational changes during catalysis with two distinct conformational states, previously named as the early and late state. In this work, we employ two phosphonate analogues of 2-oxoglutarate (OG), i.e. succinyl phosphonate (SP) and phosphono ethyl succinyl phosphonate (PESP), as tools to isolate the first catalytic steps and understand the significance of conformational transitions for the enzyme regulation. The kinetics showed a more efficient inhibition of mycobacterial E1o by SP (Ki 0.043⯱â¯0.013â¯mM) than PESP (Ki 0.88⯱â¯0.28â¯mM), consistent with the different circular dichroism spectra of the corresponding complexes. PESP allowed us to get crystallographic snapshots of the Michaelis-like complex, the first one for 2-oxo acid dehydrogenases, followed by the covalent adduction of the inhibitor to ThDP, mimicking the pre-decarboxylation complex. In addition, covalent ThDP-phosphonate complexes obtained with both compounds by co-crystallization were in the late conformational state, probably corresponding to slowly dissociating enzyme-inhibitor complexes. We discuss the relevance of these findings in terms of regulatory features of the mycobacterial E1o enzymes, and in the perspective of developing tools for species-specific metabolic regulation.
Assuntos
Complexo Cetoglutarato Desidrogenase/metabolismo , Mycobacterium/enzimologia , Domínio Catalítico , Complexo Cetoglutarato Desidrogenase/química , Ácidos Cetoglutáricos/metabolismo , Cinética , Mycobacterium/metabolismo , Organofosfonatos/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Succinatos/metabolismo , Tiamina Pirofosfato/metabolismoRESUMO
The human 2-oxoglutaric acid dehydrogenase complex (hOGDHc) plays a pivotal role in the tricarboxylic acid (TCA) cycle, and its diminished activity is associated with neurodegenerative diseases. The hOGDHc comprises three components, hE1o, hE2o, and hE3, and we recently reported functionally active E1o and E2o components, enabling studies on their assembly. No atomic-resolution structure for the hE2o component is currently available, so here we first studied the interactions in the binary subcomplexes (hE1o-hE2o, hE1o-hE3, and hE2o-hE3) to gain insight into the strength of their interactions and to identify the interaction loci in them. We carried out multiple physico-chemical studies, including fluorescence, hydrogen-deuterium exchange MS (HDX-MS), and chemical cross-linking MS (CL-MS). Our fluorescence studies suggested a strong interaction for the hE1o-hE2o subcomplex, but a much weaker interaction in the hE1o-hE3 subcomplex, and failed to identify any interaction in the hE2o-hE3 subcomplex. The HDX-MS studies gave evidence for interactions in the hE1o-hE2o and hE1o-hE3 subcomplexes comprising full-length components, identifying: (i) the N-terminal region of hE1o, in particular the two peptides 18YVEEM22 and 27ENPKSVHKSWDIF39 as constituting the binding region responsible for the assembly of the hE1o with both the hE2o and hE3 components into hOGDHc, an hE1 region absent in available X-ray structures; and (ii) a novel hE2o region comprising residues from both a linker region and from the catalytic domain as being a critical region interacting with hE1o. The CL-MS identified the loci in the hE1o and hE2o components interacting with each other.
Assuntos
Complexo Cetoglutarato Desidrogenase/metabolismo , Mapeamento de Interação de Proteínas/métodos , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Complexo Cetoglutarato Desidrogenase/química , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica em alfa-HéliceRESUMO
Commercial dietary supplements of calcium pyruvate claim to be beneficial for losing weight, increasing muscle endurance, and regulating metabolism. Most industrial preparations have some impurities, including parapyruvate. Parapyruvate is an inhibitor of the α-ketoglutarate dehydrogenase complex (KGDHC). However, the effect and mechanism of parapyruvate on cell senescence and the content of parapyruvate in the dietary supplements of calcium pyruvate are unknown. In this study, we prepared pure parapyruvate with a purity of 99.8 ± 0.1% and investigated its ability to inhibit KGDHC activity and affect fibroblast senescence. Parapyruvate dose-dependently decreased KGDHC activity, with an IC50 of 4.13 mM and induced Hs68 cell senescence. Calcium ions, a KGDHC activator, antagonized the senescent effects of parapyruvate. The parapyruvate content was 1.4 ± 0.1% to 10.6 ± 0.2% in five brands of calcium pyruvate supplements. In this study, we showed that parapyruvate strongly induces Hs68 cell senescence by inhibiting KGDHC activity. Because of its KGDHC inhibition activity, the parapyruvate content should be an important issue for the food safety of calcium pyruvate supplements.
Assuntos
Envelhecimento/efeitos dos fármacos , Suplementos Nutricionais/análise , Contaminação de Medicamentos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Ácido Pirúvico/farmacologia , Linhagem Celular , Suplementos Nutricionais/efeitos adversos , Fibroblastos/química , Fibroblastos/enzimologia , Humanos , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/metabolismo , Ácido Pirúvico/químicaRESUMO
α-Ketoglutarate decarboxylase (α-KGD), one member of α-keto acid decarboxylases, catalyzing non-oxidative decarboxylation of α-ketoglutarate to form succinic semialdehyde, was proposed to play critical role in completing tricarboxylic acid (TCA) cycle of cyanobacteria. Although the catalytic function of α-KGD from Synechococcus sp. PCC7002 was demonstrated previously, there was no detailed biochemical characterization of α-KGD from Synechococcus sp. PCC7002 yet. In this study, the gene encoding α-KGD from Synechococcus sp. PCC7002 was amplified and soluble expression of recombinant α-KGD was achieved by coexpressing with pTf16 chaperone plasmid in E. coli BL21 (DE3). Kinetic analysis showed that the activity of α-KGD was dependent on cofactors of thiamine pyrophosphate and divalent cation. Meanwhile this α-KGD was specific for α-ketoglutarate with respect to the decarboxylation activity despite of the pretty low activity of acetolactate synthase. The catalytic efficiency of α-KGD (the values of kcat and kcat/Km for α-ketoglutarate were 1.2s-1 and 6.3×103M-1s-1, respectively) might provide evidence for its physiological role in TCA cycle of Synechococcus sp. PCC7002.
Assuntos
Proteínas de Bactérias , Expressão Gênica , Complexo Cetoglutarato Desidrogenase , Synechococcus/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Complexo Cetoglutarato Desidrogenase/biossíntese , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Synechococcus/genéticaRESUMO
Herein are reported unique properties of the novel human thiamin diphosphate (ThDP)-dependent enzyme 2-oxoadipate dehydrogenase (hE1a), known as dehydrogenase E1 and transketolase domain-containing protein 1 that is encoded by the DHTKD1 gene. It is involved in the oxidative decarboxylation of 2-oxoadipate (OA) to glutaryl-CoA on the final degradative pathway of L-lysine and is critical for mitochondrial metabolism. Functionally active recombinant hE1a has been produced according to both kinetic and spectroscopic criteria in our toolbox leading to the following conclusions: (i) The hE1a has recruited the dihydrolipoyl succinyltransferase (hE2o) and the dihydrolipoyl dehydrogenase (hE3) components of the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex (OGDHc) for its activity. (ii) 2-Oxoglutarate (OG) and 2-oxoadipate (OA) could be oxidized by hE1a, however, hE1a displays an approximately 49-fold preference in catalytic efficiency for OA over OG, indicating that hE1a is specific to the 2-oxoadipate dehydrogenase complex. (iii) The hE1a forms the ThDP-enamine radical from OA according to electron paramagnetic resonance detection in the oxidative half reaction, and could produce superoxide and H2O2 from decarboxylation of OA in the forward physiological direction, as also seen with the 2-oxoglutarate dehydrogenase hE1o component. (iv) Once assembled to complex with the same hE2o and hE3 components, the hE1o and hE1a display strikingly different regulation: both succinyl-CoA and glutaryl-CoA significantly reduced the hE1o activity, but not the activity of hE1a.
Assuntos
Adipatos/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mitocôndrias/metabolismo , Oxirredutases/metabolismo , Acil Coenzima A/metabolismo , Adipatos/química , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Metabolismo Energético , Humanos , Complexo Cetoglutarato Desidrogenase/química , Ácidos Cetoglutáricos/química , Cetona Oxirredutases/genética , Cetona Oxirredutases/metabolismo , Oxirredução , Oxirredutases/química , Domínios Proteicos/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase (ODH) complex is negatively regulated by the unphosphorylated form of OdhI protein, which is critical for L-glutamate overproduction. We examined the potential impact of protein acylation at lysine (K)-132 of OdhI in C. glutamicum ATCC13032. The K132E succinylation-mimic mutation reduced the ability of OdhI to bind OdhA, the catalytic subunit of the ODH complex, which reduced the inhibition of ODH activity. In vitro succinylation of OdhI protein also reduced the ability to inhibit ODH, and the K132R mutation blocked the effect. These results suggest that succinylation at K132 may attenuate the OdhI function. Consistent with these results, the C. glutamicum mutant strain with OdhI-K132E showed decreased L-glutamate production. Our results indicated that not only phosphorylation but also succinylation of OdhI protein may regulate L-glutamate production in C. glutamicum.
Assuntos
Corynebacterium glutamicum/metabolismo , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/biossíntese , Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Complexo Cetoglutarato Desidrogenase/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Corynebacterium glutamicum/enzimologia , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/genética , Modelos Moleculares , Mutação , Fosforilação , Domínios Proteicos , Ácido Succínico/metabolismoRESUMO
Apart from its vital role as the terminal electron acceptor in oxidative phosphorylation in nature, dioxygen also serves as a universal agent which diversifies natural products by oxidative transformations. Ferrous iron and α-ketoglutarate (αKG)-dependent dioxygenases (αKGDs) are versatile enzymes that use dioxygen as an oxidant to catalyse various reactions via CH bond activation, including hydroxylation, dealkylation, desaturation, epoxidation, epimerisation, halogenation, cyclisation, peroxide formation, and ring expansion/contraction reactions. This review updates the reported αKGDs that catalyse reactions related to microbial natural product biosynthesis in the past 10 years. We hope that the versatility of αKGDs shown here can serve as an inspiration for future engineering and catalyst design, which could provide alternative methods to meet the on-going demand for fine chemicals and pharmaceutics.
Assuntos
Bactérias/enzimologia , Produtos Biológicos/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Oxirredução , Produtos Biológicos/química , Catálise , Cristalografia por Raios X , Compostos Ferrosos/metabolismo , Hidroxilação , Ferro/metabolismo , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/genética , Oxigênio/metabolismoRESUMO
Bacimethrin (4-amino-5-hydroxymethyl-2-methoxypyrimidine), a natural product isolated from some bacteria, has been implicated as an inhibitor of bacterial and yeast growth, as well as in inhibition of thiamin biosynthesis. Given that thiamin biosynthetic enzymes could convert bacimethrin to 2'-methoxythiamin diphosphate (MeOThDP), it is important to evaluate the effect of this coenzyme analogue on thiamin diphosphate (ThDP)-dependent enzymes. The potential functions of MeOThDP were explored on five ThDP-dependent enzymes: the human and Escherichia coli pyruvate dehydrogenase complexes (PDHc-h and PDHc-ec, respectively), the E. coli 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), and the human and E. coli 2-oxoglutarate dehydrogenase complexes (OGDHc-h and OGDHc-ec, respectively). Using several mechanistic tools (fluorescence, circular dichroism, kinetics, and mass spectrometry), it was demonstrated that MeOThDP binds in the active centers of ThDP-dependent enzymes, however, with a binding mode different from that of ThDP. While modest activities resulted from addition of MeOThDP to E. coli PDHc (6-11%) and DXPS (9-14%), suggesting that MeOThDP-derived covalent intermediates are converted to the corresponding products (albeit with rates slower than that with ThDP), remarkably strong activity (up to 75%) resulted upon addition of the coenzyme analogue to PDHc-h. With PDHc-ec and PDHc-h, the coenzyme analogue could support all reactions, including communication between components in the complex. No functional substitution of MeOThDP for ThDP was in evidence with either OGDH-h or OGDH-ec, shown to be due to tight binding of ThDP.
Assuntos
Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Complexo Piruvato Desidrogenase/metabolismo , Tiamina Pirofosfato/análogos & derivados , Tiamina Pirofosfato/metabolismo , Transferases/metabolismo , Substituição de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Ligação Competitiva , Biocatálise , Domínio Catalítico , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/metabolismo , Mutação , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pirimidinas/química , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transferases/químicaRESUMO
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.