RESUMO
Lipoic acid is synthesized by a remarkably atypical pathway in which the cofactor is assembled on its cognate proteins. An octanoyl moiety diverted from fatty acid synthesis is covalently attached to the acceptor protein, and sulfur insertion at carbons 6 and 8 of the octanoyl moiety form the lipoyl cofactor. Covalent attachment of this cofactor is required for function of several central metabolism enzymes, including the glycine cleavage H protein (GcvH). In Bacillus subtilis, GcvH is the sole substrate for lipoate assembly. Hence lipoic acid-requiring 2-oxoacid dehydrogenase (OADH) proteins acquire the cofactor only by transfer from lipoylated GcvH. Lipoyl transfer has been argued to be the primordial pathway of OADH lipoylation. The Escherichia coli pathway where lipoate is directly assembled on both its GcvH and OADH proteins, is proposed to have arisen later. Because roughly 3 billion years separate the divergence of these bacteria, it is surprising that E. coli GcvH functionally substitutes for the B. subtilis protein in lipoyl transfer. Known and putative GcvHs from other bacteria and eukaryotes also substitute for B. subtilis GcvH in OADH modification. Because glycine cleavage is the primary GcvH role in ancestral bacteria that lack OADH enzymes, lipoyl transfer is a "moonlighting" function: that is, development of a new function while retaining the original function. This moonlighting has been conserved in the absence of selection by some, but not all, GcvH proteins. Moreover, Aquifex aeolicus encodes five putative GcvHs, two of which have the moonlighting function, whereas others function only in glycine cleavage.
Assuntos
Proteínas de Bactérias/metabolismo , Ácido Tióctico/biossíntese , Aciltransferases/metabolismo , Aminoácido Oxirredutases/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Evolução Biológica , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Evolução Molecular , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Lipoilação , Complexos Multienzimáticos/metabolismo , Peptídeo Sintases/metabolismo , Processamento de Proteína Pós-Traducional , Ácido Tióctico/genética , Transferases/genética , Transferases/metabolismoRESUMO
Lipoyl synthase (LIAS) is an iron-sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe-4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron-sulfur (Fe-S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography-mass spectrometry (LC-MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS's two [4Fe-4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe-4S] center. These results detail the trafficking of Fe-S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe-S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe-4S] cluster reconstitution is evident.
Assuntos
Proteínas Ferro-Enxofre/metabolismo , Sulfurtransferases/metabolismo , Substituição de Aminoácidos , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Técnicas In Vitro , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise Espectral , Enxofre/metabolismo , Sulfurtransferases/química , Sulfurtransferases/genética , Ácido Tióctico/biossínteseRESUMO
In this work, we have identified a significantly improved variant (S131Y/Q252I) of the natural ϵ-keto ester reductase CpAR2 from Candida parapsilosis for efficiently manufacturing (R)-8-chloro-6-hydroxyoctanoic acid [(R)-ECHO] through co-evolution of activity and thermostability. The activity of the variant CpAR2S131Y/Q252I towards the ϵ-keto ester ethyl 8-chloro-6-oxooctanoate was improved to 214â U mg-1 -from 120â U mg-1 in the case of the wild-type enzyme (CpAR2WT )-and the half-deactivating temperature (T50 , for 15â min incubation) was simultaneously increased by 2.3 °C in relation to that of CpAR2WT . Consequently, only 2â g L-1 of lyophilized E.â coli cells harboring CpAR2S131Y/Q252I and a glucose dehydrogenase (GDH) were required in order to achieve productivity similar to that obtained in our previous work, under optimized reaction conditions (530â g L-1 d-1 ). This result demonstrated a more economical and efficient process for the production of the key (R)-α-lipoic acid intermediate ethyl 8-chloro-6-oxooctanoate.
Assuntos
Aldo-Ceto Redutases/metabolismo , Candida parapsilosis/enzimologia , Mutação , Ácido Tióctico/biossíntese , Aldo-Ceto Redutases/química , Aldo-Ceto Redutases/genética , Conformação Proteica , Engenharia de Proteínas , Estereoisomerismo , TemperaturaRESUMO
Biotin, thiamine, and lipoic acid are industrially important molecules naturally synthesized by microorganisms via biosynthetic pathways requiring iron-sulfur (FeS) clusters. Current production is exclusively by chemistry because pathway complexity hinders development of fermentation processes. For biotin, the main bottleneck is biotin synthase, BioB, a S-adenosyl methionine-dependent radical enzyme that converts dethiobiotin (DTB) to biotin. BioB overexpression is toxic, though the mechanism remains unclear. We identified single mutations in the global regulator IscR that substantially improve cellular tolerance to BioB overexpression, increasing Escherichia coli DTB-to-biotin biocatalysis by more than 2.2-fold. Based on proteomics and targeted overexpression of FeS-cluster biosynthesis genes, FeS-cluster depletion is the main reason for toxicity. We demonstrate that IscR mutations significantly affect cell viability and improve cell factories for de novo biosynthesis of thiamine by 1.3-fold and lipoic acid by 1.8-fold. We illuminate a novel engineering target for enhancing biosynthesis of complex FeS-cluster-dependent molecules, paving the way for industrial fermentation processes.
Assuntos
Biotina/biossíntese , Proteínas de Escherichia coli/genética , Engenharia Metabólica/métodos , Tiamina/biossíntese , Ácido Tióctico/biossíntese , Fatores de Transcrição/genética , Biotina/análogos & derivados , Biotina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Proteômica , Sulfurtransferases/metabolismoRESUMO
Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C1 compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea.IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.
Assuntos
Proteínas Arqueais/genética , Sulfurtransferases/genética , Thermococcus/genética , Ácido Tióctico/biossíntese , Aminoácido Oxirredutases , Proteínas Arqueais/metabolismo , Complexos Multienzimáticos , Proteínas Recombinantes , Sulfurtransferases/metabolismo , Thermococcus/enzimologia , TransferasesRESUMO
Macrophages are critical mediators of innate immunity and must be overcome for bacterial pathogens to cause disease. The Gram-positive bacterium Staphylococcus aureus produces virulence factors that impede macrophages and other immune cells. We previously determined that production of the metabolic cofactor lipoic acid by the lipoic acid synthetase, LipA, blunts macrophage activation. A ΔlipA mutant was attenuated during infection and was more readily cleared from the host. We hypothesized that bacterial lipoic acid synthesis perturbs macrophage antimicrobial functions and therefore hinders the clearance of S. aureus Here, we found that enhanced innate immune cell activation after infection with a ΔlipA mutant was central to attenuation in vivo, whereas a growth defect imparted by the lipA mutation made a negligible contribution to overall clearance. Macrophages recruited to the site of infection with the ΔlipA mutant produced larger amounts of bactericidal reactive oxygen species (ROS) and reactive nitrogen species (RNS) than those recruited to the site of infection with the wild-type strain or the mutant strain complemented with lipA ROS derived from the NADPH phagocyte oxidase complex and RNS derived from the inducible nitric oxide synthetase, but not mitochondrial ROS, were critical for the restriction of bacterial growth under these conditions. Despite enhanced antimicrobial immunity upon primary infection with the ΔlipA mutant, we found that the host failed to mount an improved recall response to secondary infection. Our data suggest that lipoic acid synthesis in S. aureus promotes bacterial persistence during infection through limitation of ROS and RNS generation by macrophages. Broadly, this work furthers our understanding of the intersections between bacterial metabolism and immune responses to infection.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Macrófagos Peritoneais/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Ácido Tióctico/biossíntese , Animais , Proteínas de Bactérias/metabolismo , Feminino , Interações Hospedeiro-Patógeno/imunologia , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Masculino , Camundongos , Viabilidade Microbiana , Mutação , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Espécies Reativas de Nitrogênio/antagonistas & inibidores , Espécies Reativas de Nitrogênio/imunologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Ácido Tióctico/farmacologiaRESUMO
LA (alpha-Lipoic acid) deficiency represents a risk factor in the pathogenesis of diabetic complications as synthetic LA is routinely used in the treatment of the complications in patients. The mechanism underlying LA deficiency remains elusive in the diabetic conditions. In the present study, we investigated the synthetic pathway of LA in both type 1 and 2 diabetic mice. LA deficiency was observed with a reduction in lipoylation of pyruvate dehydrogenase in the kidney of streptozocin-induced diabetic mice. Proteins of three enzymes (MCAT, OXSM and LIAS) in the LA synthetic pathway were examined in the kidney. A reduction was observed in OXSM, but not in the other two. In a 24h study in the cell culture, mRNA and protein of OXSM were transiently reduced by a high concentration of glucose (35â¯mM), and persistently decreased by TNF-α (20â¯nM). The high glucose effect was observed with the OXSM reduction in the kidney of db/db mice (type 2 diabetes model). The TNF-α effect was observed with OXSM reduction in the fat tissue of diet-induced obese mice. The result suggest that inhibition of OXSM by hyperglycemia and inflammation may contribute to the LA deficiency in the diabetic complications. The OXSM reduction suggests a new mechanism for the mitochondrial dysfunction in the pathogenesis of diabetic complications.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Diabetes Mellitus Experimental/metabolismo , Ácido Tióctico/deficiência , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Células 3T3-L1 , Animais , Vias Biossintéticas , Diabetes Mellitus Experimental/genética , Glucose/metabolismo , Hiperglicemia/genética , Hiperglicemia/metabolismo , Rim/metabolismo , Lipoilação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ácido Tióctico/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic deleterious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC50 values one order of magnitude lower (62-66⯵M). LA did not bypass the toxic effect of its analogues. Small monocarboxylic acids appear to be poorly internalized by T. cruzi: [14C]-octanoic acid was taken up 12 fold less efficiently than [14C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydrogenase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with BrO (1â¯mM) and MBrO (0.1â¯mM) completely inhibited E2 lipoylation and all three dehydrogenases activities. These observations indicate the lack of specific transporters for octanoic acid and most probably also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target for drug intervention. Moreover, the free LA available in the host would not interfere with such chemotherapeutic treatments.
Assuntos
Ácido Tióctico/metabolismo , Trypanosoma cruzi/metabolismo , Western Blotting , Caprilatos/metabolismo , Eletroforese em Gel de Poliacrilamida , Lipoilação/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Ácido Tióctico/análogos & derivados , Ácido Tióctico/biossíntese , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Lipoic acid (LA) is the cofactor of the E2 subunit of mitochondrial ketoacid dehydrogenases and plays a major role in oxidative decarboxylation. De novo LA biosynthesis is dependent on LIAS activity together with LIPT1 and LIPT2. LIAS is an ironsulfur (Fe-S) cluster-containing mitochondrial protein, like mitochondrial aconitase (mt-aco) and some subunits of respiratory chain (RC) complexes I, II and III. All of them harbor at least one [Fe-S] cluster and their activity is dependent on the mitochondrial [Fe-S] cluster (ISC) assembly machinery. Disorders in the ISC machinery affect numerous Fe-S proteins and lead to a heterogeneous group of diseases with a wide variety of clinical symptoms and combined enzymatic defects. Here, we present the biochemical profiles of several key mitochondrial [Fe-S]-containing proteins in fibroblasts from 13 patients carrying mutations in genes encoding proteins involved in either the lipoic acid (LIPT1 and LIPT2) or mitochondrial ISC biogenesis (FDX1L, ISCA2, IBA57, NFU1, BOLA3) pathway. Ten of them are new patients described for the first time. We confirm that the fibroblast is a good cellular model to study these deficiencies, except for patients presenting mutations in FDX1L and a muscular clinical phenotype. We find that oxidative phosphorylation can be affected by LA defects in LIPT1 and LIPT2 patients due to excessive oxidative stress or to another mechanism connecting LA and respiratory chain activity. We confirm that NFU1, BOLA3, ISCA2 and IBA57 operate in the maturation of [4Fe-4S] clusters and not in [2Fe-2S] protein maturation. Our work suggests a functional difference between IBA57 and other proteins involved in maturation of [Fe-S] proteins. IBA57 seems to require BOLA3, NFU1 and ISCA2 for its stability and NFU1 requires BOLA3. Finally, our study establishes different biochemical profiles for patients according to their mutated protein.
Assuntos
Fibroblastos/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Mitocondriais/genética , Mutação , Ácido Tióctico/biossíntese , Aciltransferases/genética , Adolescente , Vias Biossintéticas/genética , Proteínas de Transporte/genética , Criança , Pré-Escolar , Feminino , Fibroblastos/química , Humanos , Lactente , Masculino , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo , Fenótipo , Proteínas/genética , Ácido Tióctico/genéticaRESUMO
For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicumIMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use an individual nonaggregating type II fatty acid synthase (FAS-II) system. In this study, we reported genetic evidence demonstrating that the FAS-I system is the source of the biotin precursor in vivo in the engineered biotin-prototrophic C. glutamicum strain. This study also uncovered the important physiological role of FasB in lipoic acid biosynthesis. Here, we present an FAS-I enzyme that functions in supplying the lipoic acid precursor, although its biosynthesis has been believed to exclusively depend on FAS-II in organisms. The findings obtained here provide new insights into the metabolic engineering of this industrially important microorganism to produce these compounds effectively.
Assuntos
Proteínas de Bactérias/metabolismo , Biotina/biossíntese , Corynebacterium glutamicum/enzimologia , Ácidos Graxos/biossíntese , Ácido Tióctico/biossíntese , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismoRESUMO
Lipoic acid synthase (LIAS) is an iron-sulfur cluster mitochondrial enzyme which catalyzes the final step in the de novo pathway for the biosynthesis of lipoic acid, a potent antioxidant. Recently there has been significant interest in its role in metabolic diseases and its deficiency in LIAS expression has been linked to conditions such as diabetes, atherosclerosis and neonatal-onset epilepsy, suggesting a strong inverse correlation between LIAS reduction and disease status. In this study we use a bioinformatics approach to predict its structure, which would be helpful to understanding its role. A homology model for LIAS protein was generated using X-ray crystallographic structure of Thermosynechococcus elongatus BP-1 (PDB ID: 4U0P). The predicted structure has 93% of the residues in the most favour region of Ramachandran plot. The active site of LIAS protein was mapped and docked with S-Adenosyl Methionine (SAM) using GOLD software. The LIAS-SAM complex was further refined using molecular dynamics simulation within the subsite 1 and subsite 3 of the active site. To the best of our knowledge, this is the first study to report a reliable homology model of LIAS protein. This study will facilitate a better understanding mode of action of the enzyme-substrate complex for future studies in designing drugs that can target LIAS protein.
Assuntos
Modelos Moleculares , Homologia de Sequência de Aminoácidos , Sulfurtransferases/química , Domínio Catalítico , Biologia Computacional/métodos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , S-Adenosilmetionina/química , Homologia Estrutural de Proteína , Ácido Tióctico/biossínteseRESUMO
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.
Assuntos
Metabolismo Energético/genética , Ácido Tióctico/biossíntese , Ácido Tióctico/genética , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , Aminoácido Oxirredutases/genética , Animais , Proteínas de Transporte/genética , Diagnóstico Diferencial , Humanos , Cetona Oxirredutases/genética , Mitocôndrias/genética , Complexos Multienzimáticos/genética , Transferases/genéticaRESUMO
4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) is a synthetic fatty-acid synthase (FASN) inhibitor with potential therapeutic effects in several cancer models. Human mitochondrial ß-ketoacyl-acyl carrier protein synthase (HsmtKAS) is a key enzyme in the newly discovered mitochondrial fatty acid synthesis pathway that can produce the substrate for lipoic acid (LA) synthesis. HsmtKAS shares conserved catalytic domains with FASN, which are responsible for binding to C75. In our study, we explored the possible effect of C75 on HsmtKAS and mitochondrial function. C75 treatment decreased LA content, impaired mitochondrial function, increased reactive oxygen species content, and reduced cell viability. HsmtKAS but not FASN knockdown had an effect that was similar to C75 treatment. In addition, an LA supplement efficiently inhibited C75-induced mitochondrial dysfunction and oxidative stress. Overexpression of HsmtKAS showed cellular protection against low dose C75 addition, whereas there was no protective effect upon high dose C75 addition. In summary, the mitochondrial fatty acid synthesis pathway has a vital role in mitochondrial function. Besides FASN, C75 might also inhibit HsmtKAS, thereby reducing LA production, impairing mitochondrial function, and potentially having toxic effects. LA supplements sufficiently ameliorated the toxicity of C75, showing that a combination of C75 and LA may be a reliable cancer treatment.
Assuntos
4-Butirolactona/análogos & derivados , Inibidores Enzimáticos/farmacologia , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Ácido Tióctico/biossíntese , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , 4-Butirolactona/farmacologia , Sobrevivência Celular , Ácido Graxo Sintase Tipo I/metabolismo , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Lipoate is a covalently bound cofactor essential for five redox reactions in humans: in four 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). Two enzymes are from the energy metabolism, α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and three are from the amino acid metabolism, branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the GCS. All these enzymes consist of multiple subunits and share a similar architecture. Lipoate synthesis in mitochondria involves mitochondrial fatty acid synthesis up to octanoyl-acyl-carrier protein; and three lipoate-specific steps, including octanoic acid transfer to glycine cleavage H protein by lipoyl(octanoyl) transferase 2 (putative) (LIPT2), lipoate synthesis by lipoic acid synthetase (LIAS), and lipoate transfer by lipoyltransferase 1 (LIPT1), which is necessary to lipoylate the E2 subunits of the 2-oxoacid dehydrogenases. The reduced form dihydrolipoate is reactivated by dihydrolipoyl dehydrogenase (DLD). Mutations in LIAS have been identified that result in a variant form of nonketotic hyperglycinemia with early-onset convulsions combined with a defect in mitochondrial energy metabolism with encephalopathy and cardiomyopathy. LIPT1 deficiency spares the GCS, and resulted in a combined 2-oxoacid dehydrogenase deficiency and early death in one patient and in a less severely affected individual with a Leigh-like phenotype. As LIAS is an iron-sulphur-cluster-dependent enzyme, a number of recently identified defects in mitochondrial iron-sulphur cluster synthesis, including NFU1, BOLA3, IBA57, GLRX5 presented with deficiency of LIAS and a LIAS-like phenotype. As in DLD deficiency, a broader clinical spectrum can be anticipated for lipoate synthesis defects depending on which of the affected enzymes is most rate limiting.
Assuntos
Erros Inatos do Metabolismo Lipídico , Ácido Tióctico/biossíntese , Ácido Tióctico/deficiência , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , Aciltransferases/genética , Aminoácido Oxirredutases/genética , Animais , Proteínas de Transporte/genética , Di-Hidrolipoamida Desidrogenase/genética , Modelos Animais de Doenças , Humanos , Erros Inatos do Metabolismo Lipídico/enzimologia , Erros Inatos do Metabolismo Lipídico/genética , Complexos Multienzimáticos/genética , Sulfurtransferases/genética , Transferases/genéticaRESUMO
The covalent attachment of lipoate to the lipoyl domains (LDs) of the central metabolism enzymes pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) is essential for their activation and thus for respiratory growth in Saccharomyces cerevisiae. A third lipoate-dependent enzyme system, the glycine cleavage system (GCV), is required for utilization of glycine as a nitrogen source. Lipoate is synthesized by extraction of its precursor, octanoyl-acyl carrier protein (ACP), from the pool of fatty acid biosynthetic intermediates. Alternatively, lipoate is salvaged from previously modified proteins or from growth medium by lipoate protein ligases (Lpls). The first Lpl to be characterized, LplA of Escherichia coli, catalyses two partial reactions: activation of the acyl chain by formation of acyl-AMP, followed by transfer of the acyl chain to lipoyl domains (LDs). There is a surprising diversity within the Lpl family of enzymes, several of which catalyse reactions other than ligation reactions. For example, the Bacillus subtilis Lpl homologue LipM is an octanoyltransferase that transfers the octanoyl moiety from octanoyl-ACP to GCV. Another B. subtilis Lpl homologue, LipL, transfers octanoate from octanoyl-GCV to other LDs in an amido-transfer reaction. Study of eukaryotic Lpls has lagged behind studies of the bacterial enzymes. We report that the Lip3 Lpl homologue of the yeast S. cerevisiae has octanoyl-CoA-protein transferase activity, and discuss implications of this activity on the physiological role of Lip3 in lipoate synthesis.
Assuntos
Peptídeo Sintases/metabolismo , Saccharomyces cerevisiae/enzimologia , Ácido Tióctico/biossíntese , Aciltransferases/genética , Aciltransferases/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Complexo Cetoglutarato Desidrogenase/metabolismo , Peptídeo Sintases/genética , Complexo Piruvato Desidrogenase/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Scale up studies for production of lipoic acid (LA) from Saccharomyces cerevisiae have been reported in this paper for the first time. LA production in batch mode was carried out in a stirred tank bioreactor at varying agitation and aeration with maximum LA production of 512 mg/L obtained at 350 rpm and 25 % dissolved oxygen in batch culture conditions. Thus, LA production increased from 352 mg/L in shake flask to 512 mg/L in batch mode in a 5 L stirred tank bioreactor. Biomass production under these conditions was mathematically explained using logistic equation and data obtained for LA production and substrate utilization were successfully fitted using Luedeking-Piret and Mercier's models. The kinetic studies showed LA production to be growth associated. Further enhancement of LA production was carried out using fed-batch (variable volume) and semi-continuous modes of fermentation. Semi-continuous fermentation with three feeding cycles of sucrose effectively increased the production of LA from 512 to 725 mg/L.
Assuntos
Reatores Biológicos , Microbiologia Industrial/instrumentação , Saccharomyces cerevisiae/metabolismo , Ácido Tióctico/biossíntese , Biomassa , Biotecnologia , Fermentação , Cinética , Modelos Teóricos , Oxigênio/química , Espectrofotometria , Sacarose/química , Fatores de TempoRESUMO
In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl-GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester-linked acyl-enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys-Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Vias Biossintéticas/genética , Genes Bacterianos , Ácido Tióctico/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deleção de Genes , Complexo Piruvato Desidrogenase/metabolismoRESUMO
The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM and ywfL, which we have renamed lplJ, lipM and lipL respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to Escherichia coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulphur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilisΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coliΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion article provide evidence that LipM and LipL catalyse sequential reactions in a novel pathway for lipoic acid biosynthesis.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Vias Biossintéticas/genética , Genes Bacterianos , Ácido Tióctico/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Modelos BiológicosRESUMO
Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function. Curiously, the deficiency in mitochondrial FAS cannot be complemented by inclusion of fatty acids in the culture medium or by products of the cytosolic FAS complex. Defects in mitochondrial FAS in yeast result in the inability to grow on nonfermentable carbon sources, the loss of mitochondrial cytochromes a/a3 and b, mitochondrial RNA processing defects, and loss of cellular lipoic acid. Eukaryotic FAS II generates octanoyl-ACP, a substrate for mitochondrial lipoic acid synthase. Endogenous lipoic acid synthesis challenges the hypothesis that lipoic acid can be provided as an exogenously supplied vitamin. Purified eukaryotic FAS II enzymes are catalytically active in vitro using substrates with an acyl chain length of up to 16 carbon atoms. However, with the exception of 3-hydroxymyristoyl-ACP, a component of respiratory complex I in higher eukaryotes, the fate of long-chain fatty acids synthesized by the mitochondrial FAS pathway remains an enigma. The linkage of FAS II genes to published animal models for human disease supports the hypothesis that mitochondrial FAS dysfunction leads to the development of disorders in mammals.
Assuntos
Ácidos Graxos/biossíntese , Mitocôndrias/metabolismo , Animais , Respiração Celular , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Humanos , Lipoilação , Modelos Biológicos , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mitocondrial , Ácido Tióctico/biossínteseRESUMO
Isolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and can fundamentally change the outcome of the associated infections. Bacterial SCVs often emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The basis of SCV emergence in vivo is not well understood in Gram-negative bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of NCV and SCV co-isolates of Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei. We confirmed SCV emergence was attributed to limited genomic mutations: 4 single nucleotide variants in the E. coli SCV, 5 in C. freundii, and 8 in E. hormaechei. In addition, a 10.2 kb chromosomal segment containing 11 genes was deleted in the E. hormaechei SCV isolate. Each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved in iron capture. Chemical and genetic rescue confirmed defects in heme biosynthesis for E. coli and C. freundii and lipoic acid biosynthesis in E. hormaachei were responsible for the SCV phenotype. Prototrophic growth in all 3 SCV Enterobacteriaceae species was unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo.