RESUMEN
Interventions involving dietary fibers are known to benefit host health. A leading contribution of gut microbiota is commonly recognized with production of short chain fatty acids (SCFA) suspected to play a key role. However, the detailed mechanisms are largely unknown, and apart from a well-described bifidogenic effect of some fibers, results for other bacterial taxa are often incongruent between studies. We performed pooled analyses of 16S rRNA gene data derived from intervention studies (n = 14) based on three fibers, namely, inulin-type fructans (ITF), resistant starch (RS), and arabinoxylan-oligosaccharides (AXOS), harmonizing the bioinformatics workflow to reveal taxa stimulated by those substrates, specifically focusing on the SCFA-production potential. The results showed an increased butyrate production potential after ITF (p < 0.05) and RS (p < 0.1) treatment via an increase in bacteria exhibiting the enzyme butyryl-CoA:acetate CoA-transferase (but) that was governed by Faecalibacterium, Anaerostipes (ITF) and Agathobacter (RS) respectively. AXOS did not promote an increase in butyrate producers, nor were pathways linked to propionate production stimulated by any intervention. A bifidogenic effect was observed for AXOS and ITF, which was only partly associated with the behavior of but-containing bacteria and largely represented a separate response. Low and high Ruminococcus abundances pre-intervention for ITF and RS, respectively, promoted an increase in but-containing taxa (p < 0.05) upon interventions, whereas initial Prevotella abundance was negatively associated with responses of butyrate producers for both fibers. Collectively, our data demonstrate targeted stimulation of specific taxa by individual fibers increasing the potential to synthesize butyrate, where gut microbiota composition pre-intervention strongly controlled outcomes.
Asunto(s)
Bacterias , Butiratos , Fibras de la Dieta , Microbioma Gastrointestinal , ARN Ribosómico 16S , Xilanos , Fibras de la Dieta/metabolismo , Butiratos/metabolismo , Xilanos/metabolismo , ARN Ribosómico 16S/genética , Bacterias/genética , Bacterias/clasificación , Bacterias/metabolismo , Humanos , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Ácidos Grasos Volátiles/metabolismo , Inulina/metabolismo , Almidón/metabolismo , Oligosacáridos/metabolismo , Faecalibacterium/genética , Biología Computacional/métodosRESUMEN
Previously, we biosynthesized an evolved version of a bio-based polylactide (PLA) on microbial platforms using our engineered lactate-polymerizing enzyme (LPE). This lactate (LA)-based copolyester, LAHB, has advantages over PLA, including improved flexibility and biodegradability, and its properties can be regulated through the LA fraction. To expand the LA-incorporation capacity and improve polymer properties, in the state of in vivo LAHB production, propionyl-CoA transferases (PCTs) that exhibited enhanced production of LA-CoA than the conventional PCTs were selected. Here, the present study has demonstrated that the LA fraction of LAHB could be altered using various PCTs. Enhanced PCT performance was achieved by balancing polymer production and cell growth. Both events are governed by the use of acetyl-CoA, a commonly shared key metabolite. This could be attributed to the different reactivities of individual PCTs towards acetyl-CoA, which serves both as a CoA donor and a leading compound in the TCA cycle. Interestingly, we found complete sequence randomness in the LAHB copolymers, independent of the LA fraction. The mechanism of LA fraction-independent sequence randomness is discussed. This new PCT-based strategy synergistically combines with the evolution of LPE to advance the LAHB project, and enables us to perform advanced applications other than LAHB production utilizing CoA-linked substrates.
Asunto(s)
Coenzima A Transferasas , Ácido Láctico , Ácido Láctico/química , Coenzima A Transferasas/metabolismo , Coenzima A Transferasas/genética , Coenzima A Transferasas/química , Poliésteres/química , Acilcoenzima A/metabolismo , Acilcoenzima A/química , Polímeros/química , Acetilcoenzima A/metabolismo , Acetilcoenzima A/químicaRESUMEN
Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or nontoxic metabolites. Here, we report a putative novel target, succinyl-CoA:glutarate-CoA transferase (SUGCT), which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA and the derived 3-hydroxyglutaric acid. SUGCT is a type III CoA transferase that uses succinyl-CoA and glutaric acid as substrates. We report the structure of SUGCT, develop enzyme- and cell-based assays, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme in a high-throughput screen of FDA-approved compounds. The cocrystal structure of SUGCT with losartan carboxylic acid revealed a novel pocket in the active site and further validated the high-throughput screening approach. These results may form the basis for the future development of new pharmacological intervention to treat GA1.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Encefalopatías Metabólicas , Humanos , Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Encefalopatías Metabólicas/tratamiento farmacológico , Encefalopatías Metabólicas/metabolismo , Encefalopatías Metabólicas/enzimología , Glutaratos/metabolismo , Glutaratos/química , Losartán/farmacología , Losartán/química , Coenzima A Transferasas/metabolismo , Coenzima A Transferasas/antagonistas & inhibidores , Coenzima A Transferasas/genética , Coenzima A Transferasas/química , Valsartán , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Cristalografía por Rayos X , Dominio Catalítico , Acilcoenzima A/metabolismo , Acilcoenzima A/química , Modelos Moleculares , Ensayos Analíticos de Alto Rendimiento , Glutaril-CoA Deshidrogenasa/deficienciaRESUMEN
Oxalate degradation is one of lactic acid bacteria's desirable activities. It is achieved by two enzymes, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). The current study aimed to screen 15 locally isolated lactic acid bacteria to select those with the highest oxalate degradation ability. It also aimed to amplify the genes involved in degradation. MRS broth supplemented with 20 mM sodium oxalate was used to culture the tested isolates for 72 h. This was followed by an enzymatic assay to detect remaining oxalate. All isolates showed oxalate degradation activity to variable degrees. Five isolates demonstrated high oxalate degradation, 78 to 88%. To investigate the oxalate-degradation potential of the selected isolates, they have been further tested for the presence of genes that encode for enzymes involved in oxalate catabolism, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). Three strains showed bands with the specific OXC and FRC forward and reverse primers designated as (SA-5, 9 and 37). Species-level identification revealed Loigolactobacillus bifermentans, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum. Preliminary results revealed that the tested probiotic strains harbored both oxc and frc whose products are putatively involved in oxalate catabolism. The probiotic potential of the selected strains was evaluated, and they showed high survival rates to both simulated gastric and intestinal fluids and variable degrees of antagonism against the tested Gram-positive and negative pathogens and were sensitive to clarithromycin but resistant to both metronidazole and ceftazidime. Finally, these strains could be exploited as an innovative approach to establish oxalate homeostasis in humans and prevent kidney stone formation.
Asunto(s)
Acilcoenzima A , Carboxiliasas , Probióticos , Humanos , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Carboxiliasas/genética , Oxalatos/metabolismoRESUMEN
BACKGROUND: Subarachnoid hemorrhage (SAH) is a life-threatening neurological disease that usually has a poor prognosis. Neurogenesis is a potential therapeutic target for brain injury. Ketone metabolism also plays neuroprotective roles in many neurological disorders. OXCT1 (3-Oxoacid CoA-Transferase 1) is the rate-limiting enzyme of ketone body oxidation. In this study, we explored whether increasing ketone oxidation by upregulating OXCT1 in neurons could promote neurogenesis after SAH, and evaluated the potential mechanism involved in this process. METHODS: The ß-hydroxybutyrate content was measured using an enzymatic colorimetric assay. Adeno-associated virus targeting neurons was injected to overexpress OXCT1, and the expression and localization of proteins were evaluated by western blotting and immunofluorescence staining. Adult hippocampal neurogenesis was evaluated by dual staining with doublecortin and 5-Ethynyl-2'-Deoxyuridine. LY294002 was intracerebroventricularly administered to inhibit Akt activity. The Morris water maze and Y-maze tests were employed to assess cognitive function after SAH. RESULTS: The results showed that OXCT1 expression and hippocampal neurogenesis significantly decreased in the early stage of SAH. Overexpression of OXCT1 successfully increased hippocampal neurogenesis via activation of Akt/GSK-3ß/ß-catenin signaling and improved cognitive function, both of which were reversed by administration of LY294002. CONCLUSIONS: OXCT1 regulated hippocampal ketone body metabolism and increased neurogenesis through mechanisms mediated by the Akt/GSK-3ß/ß-catenin pathway, improving cognitive impairment after SAH.
Asunto(s)
Coenzima A Transferasas , Disfunción Cognitiva , Hipocampo , Neurogénesis , Hemorragia Subaracnoidea , Ácido 3-Hidroxibutírico , beta Catenina , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hipocampo/crecimiento & desarrollo , Proteínas Proto-Oncogénicas c-akt , Animales , RatonesRESUMEN
Ketone bodies serve as an energy source, especially in the absence of carbohydrates or in the extended exercise. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a crucial energy sensor that regulates lipid and glucose metabolism. However, whether AMPK regulates ketone metabolism in whole body is unclear even though AMPK regulates ketogenesis in liver. Prolonged resulted in a significant increase in blood and urine levels of ketone bodies in wild-type (WT) mice. Interestingly, fasting AMPKα2-/- and AMPKα1-/- mice exhibited significantly higher levels of ketone bodies in both blood and urine compared to fasting WT mice. BHB tolerance assays revealed that both AMPKα2-/- and AMPKα1-/- mice exhibited slower ketone consumption compared to WT mice, as indicated by higher blood BHB or urine BHB levels in the AMPKα2-/- and AMPKα1-/- mice even after the peak. Interestingly, fasting AMPKα2-/- and AMPKα1-/- mice exhibited significantly higher levels of ketone bodies in both blood and urine compared to fasting WT mice. . Specifically, AMPKα2ΔMusc mice showed approximately a twofold increase in blood BHB levels, and AMPKα2ΔMyo mice exhibited a 1.5-fold increase compared to their WT littermates after a 48-h fasting. However, blood BHB levels in AMPKα1ΔMusc and AMPKα1ΔMyo mice were as same as in WT mice. Notably, AMPKα2ΔMusc mice demonstrated a slower rate of BHB consumption in the BHB tolerance assay, whereas AMPKα1ΔMusc mice did not show such an effect. Declining rates of body weights and blood glucoses were similar among all the mice. Protein levels of SCOT, the rate-limiting enzyme of ketolysis, decreased in skeletal muscle of AMPKα2-/- mice. Moreover, SCOT protein ubiquitination increased in C2C12 cells either transfected with kinase-dead AMPKα2 or subjected to AMPKα2 inhibition. AMPKα2 physiologically binds and stabilizes SCOT, which is dependent on AMPKα2 activity.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Cuerpos Cetónicos , Animales , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Ayuno , Cetonas , Ratones Noqueados , Ubiquitinación , Coenzima A Transferasas/metabolismoRESUMEN
Isopropanol has a good potential as a new fuel substitution. In the model biosynthesis pathway of isopropanol synthesis, acetoacetyl-CoA is converted to acetoacetate by acetoacetyl-CoA transferases, which requires an acetate molecule as a substrate. Herein, a novel isopropanol synthesis pathway based on mammalian ketone metabolic pathway was developed. In this pathway, acetoacetyl-CoA is condensed with acetyl-CoA to generate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase, and then catalyzed by HMG-CoA lyase to generate acetoacetate. This process is acetate-independent. Under the same experimental system using glycerol as carbon source, the E. coli strain MG::ISOP1 containing the novel pathway produced 11.7 times more isopropanol than the strain MG::ISOP0 containing the model pathway. The pta-ackA knockout mutant strain MG∆pta-ackA::ISOP1, which reduced the conversion of acetyl-CoA to acetate, further increased the production from 76 mg/L to 360 mg/L. In another strategy, knocking out atoDA to block the acetoacetate degradation pathway in strain MG∆atoDA::ISOP1 increased the production to 680 mg/L. By knocking out both of pta-ackA and atoDA, strain MGΔpta-ackAΔatoDA::ISOP1 produced 964 mg/L of isopropanol, which was 12.7 times that of MG::ISOP1. This study indicated that the novel pathway is competent for isopropanol synthesis, and provides a new perspective for biosynthesis of isopropanol.
Asunto(s)
2-Propanol , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , 2-Propanol/metabolismo , Acetoacetatos/metabolismo , Acetilcoenzima A/metabolismo , Coenzima A Transferasas/metabolismo , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , Glicerol/metabolismo , Acetatos/metabolismo , Carbono/metabolismoRESUMEN
This study aimed to characterize the prokaryotic community and putative microbial interactions involved in hydrogen (H2) production during the dark fermentation (DF) process, applying principal components analysis (PCA) to correlate changes in operational, physicochemical, and biological variables. For this purpose, a continuous stirred-tank reactor-type digester fed with tequila vinasses was operated at 24, 18, and 12 h of hydraulic retention times (HRTs) to apply organic loading rates of 20, 36, and 54 g-COD L-1 d-1, corresponding to stages I, II, and III, respectively. Results indicated high population dynamics for Archaea during the DF process toward a decrease in total sequences from 6299 to 99. Concerning the Bacteria community, lactic acid bacteria (LAB) were dominant reaching a relative abundance of 57.67%, while dominant H2-producing bacteria (HPB) decreased from 25.76% to 21.06% during stage III. Putative competitive exclusion mechanisms such as competition for substrates, bacteriocins production, and micronutrient depletion carried out by Archaea and non-H2-producing bacteria (non-HPB), especially LAB, could negatively impact the dominance of HPB such as Ethanoligenens harbinense and Clostridium tyrobutyricum. As a consequence, low maximal volumetric H2 production rate (672 mL-H2 L-1 d-1) and yield (3.88 mol-H2 assimilated sugars-1) were obtained. The global scenario obtained by PCA correlations suggested that C. tyrobutyricum positively impacted H2 molar yield through butyrate fermentation using the butyryl-CoA:acetate CoA transferase pathway, while the most abundant HPB E. harbinense decreased its relative abundance at the shortest HRT toward the dominance of non-HPB. This study provides new insights into the microbial interactions and helps to better understand the DF performance for H2 production using tequila vinasses as substrate. KEY POINTS: ⢠E. harbinense and C. tyrobutyricum were responsible for H2 production. ⢠Clostridiales used acetate and butyrate fermentations for H2 production. ⢠LAB won the competition for sugars against Clostridiales during DF. ⢠Putative bacteriocins production and micronutrients depletion could favor LAB.
Asunto(s)
Bacteriocinas , Reactores Biológicos , Acetatos/metabolismo , Archaea/metabolismo , Bacterias/genética , Bacterias/metabolismo , Bacteriocinas/metabolismo , Reactores Biológicos/microbiología , Butiratos/metabolismo , Coenzima A Transferasas/metabolismo , Fermentación , Hidrógeno/metabolismo , Interacciones Microbianas , Micronutrientes/metabolismo , Azúcares/metabolismoRESUMEN
AIM: To investigate cardiac signalling pathways connecting substrate utilization with left ventricular remodelling in a murine pressure overload model. METHODS: Cardiac hypertrophy was induced by transverse aortic constriction surgery in 20-week-old C57BL/6J mice treated with or without the sodium-glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin (225 mg kg-1 chow diet) for 10 weeks. RESULTS: Ertugliflozin improved left ventricular function and reduced myocardial fibrosis. This occurred simultaneously with a fasting-like response characterized by improved glucose tolerance and increased ketone body concentrations. While cardiac insulin signalling was reduced in response to SGLT2 inhibition, AMP-activated protein kinase (AMPK) signalling was increased with induction of the fatty acid transporter cluster of differentiation 36 and phosphorylation of acetyl-CoA carboxylase (ACC). Further, enzymes responsible for ketone body catabolism (ß-hydroxybutyrate dehydrogenase, succinyl-CoA:3-oxoacid-CoA transferase and acetyl-CoA acetyltransferase 1) were induced by SGLT2 inhibition. Ertugliflozin led to more cardiac abundance of fatty acids, tricarboxylic acid cycle metabolites and ATP. Downstream mechanistic target of rapamycin (mTOR) pathway, relevant for protein synthesis, cardiac hypertrophy and adverse cardiac remodelling, was reduced by SGLT2 inhibition, with alleviation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) providing a potential mechanism for abundant reduced left ventricular apoptosis and fibrosis. CONCLUSION: SGLT2 inhibition reduced left ventricular fibrosis in a murine model of cardiac hypertrophy. Mechanistically, this was associated with reduced cardiac insulin and increased AMPK signalling as a potential mechanism for less cardiac mTOR activation with alleviation of downstream ER stress, UPR and apoptosis.
Asunto(s)
Insulinas , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Proteínas Quinasas Activadas por AMP/metabolismo , Acetil-CoA C-Acetiltransferasa/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes , Cardiomegalia/metabolismo , Cardiomegalia/patología , Coenzima A Transferasas/metabolismo , Estrés del Retículo Endoplásmico , Ácidos Grasos/metabolismo , Fibrosis , Glucosa/metabolismo , Hidroxibutirato Deshidrogenasa/metabolismo , Cetoácidos/metabolismo , Cetonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Sirolimus/metabolismo , Sodio/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome.
Asunto(s)
Microbioma Gastrointestinal , Metaboloma , Polisacáridos/metabolismo , Acilcoenzima A/metabolismo , Secuencia de Aminoácidos , Aminoácidos de Cadena Ramificada/metabolismo , Bacteroidetes/efectos de los fármacos , Bacteroidetes/genética , Bacteroidetes/crecimiento & desarrollo , Butiratos/química , Butiratos/farmacología , Coenzima A Transferasas/química , Coenzima A Transferasas/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Variación Genética/efectos de los fármacos , Concentración de Iones de Hidrógeno , Metaboloma/efectos de los fármacos , Metaboloma/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Especificidad de la Especie , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Transcripción Genética/efectos de los fármacosRESUMEN
The coenzyme A (CoA) transferases are a superfamily of proteins central to the metabolism of acetyl-CoA and other CoA thioesters. They are diverse group, catalyzing over a 100 biochemical reactions and spanning all three domains of life. A deeply rooted idea, proposed two decades ago, is these enzymes fall into three families (I, II, and III). Here we find they fall into different families, which we achieve by analyzing all CoA transferases characterized to date. We manually annotated 94 CoA transferases with functional information (including rates of catalysis for 208 reactions) from 97 publications. This represents all enzymes we could find in the primary literature, and it is double the number annotated in four protein databases (BRENDA, KEGG, MetaCyc, UniProt). We found family I transferases are not closely related to each other in terms of sequence, structure, and reactions catalyzed. This family is not even monophyletic. These problems are solved by regrouping the three families into six, including one family with many non-CoA transferases. The problem (and solution) became apparent only by analyzing our large set of manually annotated proteins. It would have been missed if we had used the small number of proteins annotated in UniProt and other databases. Our work is important to understanding the biology of CoA transferases. It also warns investigators doing phylogenetic analyses of proteins to go beyond information in databases.
Asunto(s)
Proteínas Bacterianas , Coenzima A Transferasas , Proteínas Bacterianas/química , Catálisis , Coenzima A , Coenzima A Transferasas/química , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Bases de Datos de Proteínas , Humanos , FilogeniaRESUMEN
Lactobacillus paragasseri was identified as a novel sister taxon of L. gasseri in 2018. Since the reclassification of L. paragasseri, there has been hardly any report describing the probiotic properties of this species. In this study, an L. paragasseri strain UBLG-36 was sequenced and analyzed to determine the molecular basis that may confer the bacteria with probiotic potential. UBLG-36 was previously documented as an L. gasseri strain. Average nucleotide identity and phylogenomic analysis allowed accurate taxonomic identification of UBLG-36 as an L. paragasseri strain. Analysis of the draft genome (~1.94 Mb) showed that UBLG-36 contains 5 contigs with an average G+C content of 34.85%. Genes essential for the biosynthesis of bacteriocins, adhesion to host epithelium, stress resistance, host immunomodulation, defense, and carbohydrate metabolism were identified in the genome. Interestingly, L. paragasseri UBLG-36 also harbored genes that code for enzymes involved in oxalate catabolism, such as formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). In vitro oxalate degradation assay showed that UBLG-36 is highly effective in degrading oxalate (averaging more than 45% degradation), a feature that has not been reported before. As a recently identified bacterium, there are limited genomic reports on L. paragasseri, and our draft genome sequence analysis is the first to describe and emphasize the probiotic potential and oxalate degrading ability of this species. With results supporting the probiotic functionalities and oxalate catabolism of UBLG-36, we propose that this strain is likely to have immense biotechnological applications upon appropriate characterization.
Asunto(s)
Lactobacillus/genética , Lactobacillus/metabolismo , Oxalatos/metabolismo , Bacteriocinas/metabolismo , Composición de Base/genética , Carboxiliasas/metabolismo , Coenzima A Transferasas/metabolismo , Genómica , Inmunomodulación , Nucleótidos , Filogenia , Probióticos/metabolismo , Análisis de Secuencia , Secuenciación Completa del Genoma/métodosRESUMEN
Lack of glucose uptake compromises metabolic flexibility and reduces energy efficiency in the diabetes mellitus (DM) heart. Although increased use of fatty acid to compensate glucose substrate has been studied, less is known about ketone body metabolism in the DM heart. Ketogenic diet reduces obesity, a risk factor for T2DM. How ketogenic diet affects ketone metabolism in the DM heart remains unclear. At the metabolic level, the DM heart differs from the non-DM heart because of altered metabolic substrate and the T1DM heart differs from the T2DM heart because of insulin levels. How these changes affect ketone body metabolism in the DM heart are poorly understood. Ketogenesis produces ketone bodies by using acetyl-CoA, whereas ketolysis consumes ketone bodies to produce acetyl-CoA, showing their opposite roles in the ketone body metabolism. Cardiac-specific transgenic upregulation of ketogenesis enzyme or knockout of ketolysis enzyme causes metabolic abnormalities leading to cardiac dysfunction. Empirical evidence demonstrates upregulated transcription of ketogenesis enzymes, no change in the levels of ketone body transporters, very high levels of ketone bodies, and reduced expression and activity of ketolysis enzymes in the T1DM heart. Based on these observations, I hypothesize that increased transcription and activity of cardiac ketogenesis enzyme suppresses ketolysis enzyme in the DM heart, which decreases cardiac energy efficiency. The T1DM heart exhibits highly upregulated ketogenesis compared with the T2DM heart because of the lack of insulin, which inhibits ketogenesis enzyme.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Cardiomiopatías Diabéticas/etiología , Metabolismo Energético , Insulina/metabolismo , Cuerpos Cetónicos/metabolismo , Miocardio/metabolismo , Animales , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatías Diabéticas/metabolismo , Cetoacidosis Diabética/etiología , Cetoacidosis Diabética/metabolismo , Dieta Cetogénica , Femenino , Humanos , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , MasculinoRESUMEN
Emerging research evidence has established the critical role of the gut-liver axis in the development of alcohol-associated liver disease (ALD). The present study employed 16S rRNA gene and whole genome shotgun (WGS) metagenomic analysis in combination with a revised microbial dataset to comprehensively detail the butyrate-producing microbial communities and the associated butyrate metabolic pathways affected by chronic ethanol feeding. Specifically, the data demonstrated that a decrease in several butyrate-producing bacterial genera belonging to distinct families within the Firmicutes phyla was a significant component of ethanol-induced dysbiosis. WGS analysis of total bacterial genomes encompassing butyrate synthesizing pathways provided the functional characteristics of the microbiome associated with butyrate synthesis. The data revealed that in control mice microbiome, the acetyl-coenzyme A (CoA) butyrate synthesizing pathway was the most prevalent and was significantly and maximally decreased by chronic ethanol feeding. Further WGS analysis i) validated the ethanol-induced decrease in the acetyl-CoA pathway by identifying the decrease in two critical genes but - (butyryl-CoA: acetate CoA transferase) and buk - (butyrate kinase) that encode the terminal condensing enzymes required for converting butyryl-CoA to butyrate and ii) detection of specific taxa of butyrate-producing bacteria containing but and buk genes. Notably, the administration of tributyrin (Tb) - a butyrate prodrug - significantly prevented ethanol-induced decrease in butyrate-producing bacteria, hepatic steatosis, inflammation, and injury. Taken together, our findings strongly suggest that the loss of butyrate-producing bacteria using the acetyl-CoA pathway is a significant pathogenic feature of ethanol-induced microbial dysbiosis and ALD and can be targeted for therapy.
Asunto(s)
Butiratos/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Coenzima A Transferasas/metabolismo , Disbiosis/inducido químicamente , Etanol/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Ruminococcus/metabolismo , Animales , Modelos Animales de Enfermedad , Disbiosis/fisiopatología , Humanos , Redes y Vías Metabólicas , RatonesRESUMEN
Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation, contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In the present study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. Enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA into butyrate and caproyl-CoA into caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8-times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse ß-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center. Single point mutations in the conserved motif of CPB6-CoAT (Asp346 and Ala351) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT and that Asp346 and Ala351 have a significant impact on the enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves understanding of the chain elongation pathway for MCFA production.
Asunto(s)
Proteínas Bacterianas/metabolismo , Butiratos/metabolismo , Caproatos/metabolismo , Clonación Molecular , Clostridium tyrobutyricum/enzimología , Coenzima A Transferasas/metabolismo , Acilcoenzima A/metabolismo , Proteínas Bacterianas/genética , Clostridium tyrobutyricum/genética , Coenzima A Transferasas/química , Coenzima A Transferasas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Modelos Moleculares , Mutación , Oxidación-Reducción , Filogenia , Conformación Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Many bacteria and other organisms carry out fermentations forming acetate. These fermentations have broad importance for foods, agriculture, and industry. They also are important for bacteria themselves because they often generate ATP. Here, we found a biochemical pathway for forming acetate and synthesizing ATP that was unknown in fermentative bacteria. We found that the bacterium Cutibacterium granulosum formed acetate during fermentation of glucose. It did not use phosphotransacetylase or acetate kinase, enzymes found in nearly all acetate-forming bacteria. Instead, it used a pathway involving two different enzymes. The first enzyme, succinyl coenzyme A (succinyl-CoA):acetate CoA-transferase (SCACT), forms acetate from acetyl-CoA. The second enzyme, succinyl-CoA synthetase (SCS), synthesizes ATP. We identified the genes encoding these enzymes, and they were homologs of SCACT and SCS genes found in other bacteria. The pathway resembles one described in eukaryotes, but it uses bacterial, not eukaryotic, gene homologs. To find other instances of the pathway, we analyzed sequences of all biochemically characterized homologs of SCACT and SCS (103 enzymes from 64 publications). Homologs with similar enzymatic activity had similar sequences, enabling a large-scale search for them in genomes. We searched nearly 600 genomes of bacteria known to form acetate, and we found that 6% encoded homologs with SCACT and SCS activity. This included >30 species belonging to 5 different phyla, showing that a diverse range of bacteria encode the SCACT/SCS pathway. This work suggests the SCACT/SCS pathway is important for acetate formation in many branches of the tree of life. IMPORTANCE Pathways for forming acetate during fermentation have been studied for over 80 years. In that time, several pathways in a range of organisms, from bacteria to animals, have been described. However, one pathway (involving succinyl-CoA:acetate CoA-transferase and succinyl-CoA synthetase) has not been reported in prokaryotes. Here, we discovered enzymes for this pathway in the fermentative bacterium Cutibacterium granulosum. We also found >30 other fermentative bacteria that encode this pathway, demonstrating that it could be common. This pathway represents a new way for bacteria to form acetate from acetyl-CoA and synthesize ATP via substrate-level phosphorylation. It could be a target for controlling yield of acetate during fermentation, with relevance for foods, agriculture, and industry.
Asunto(s)
Acetatos/metabolismo , Adenosina Trifosfato/metabolismo , Propionibacteriaceae/metabolismo , Succinato-CoA Ligasas/metabolismo , Acetilcoenzima A/metabolismo , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Fermentación , Genoma Bacteriano , Propionibacteriaceae/genética , Succinato-CoA Ligasas/genéticaRESUMEN
Polyhydroxyalkanoates (PHAs) provide biodegradable and bio-based alternatives to conventional plastics. Incorporation of 2-hydroxy acid monomers into polymer, in addition to 3-hydroxy acids, offers possibility to tailor the polymer properties. In this study, poly(D-lactic acid) (PDLA) and copolymer P(LA-3HB) were produced and characterized for the first time in the yeast Saccharomyces cerevisiae. Expression of engineered PHA synthase PhaC1437Ps6-19, propionyl-CoA transferase Pct540Cp, acetyl-CoA acetyltransferase PhaA, and acetoacetyl-CoA reductase PhaB1 resulted in accumulation of 3.6% P(LA-3HB) and expression of engineered enzymes PhaC1Pre and PctMe resulted in accumulation of 0.73% PDLA of the cell dry weight (CDW). According to NMR, P(LA-3HB) contained D-lactic acid repeating sequences. For reference, expression of PhaA, PhaB1, and PHA synthase PhaC1 resulted in accumulation 11% poly(hydroxybutyrate) (PHB) of the CDW. Weight average molecular weights of these polymers were comparable to similar polymers produced by bacterial strains, 24.6, 6.3, and 1 130 kDa for P(LA-3HB), PDLA, and PHB, respectively. The results suggest that yeast, as a robust and acid tolerant industrial production organism, could be suitable for production of 2-hydroxy acid containing PHAs from sugars or from 2-hydroxy acid containing raw materials. Moreover, the wide substrate specificity of PHA synthase enzymes employed increases the possibilities for modifying copolymer properties in yeast in the future.
Asunto(s)
Ácido Láctico/metabolismo , Polihidroxialcanoatos/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Escherichia coli/metabolismo , Ingeniería Genética , Hidroxibutiratos/metabolismo , Microbiología Industrial , Redes y Vías Metabólicas , Polihidroxialcanoatos/químicaRESUMEN
Succinyl-CoA:3-oxoacid coenzyme A transferase deficiency (SCOTD) is a rare autosomal recessive disorder of ketone body utilization caused by mutations in OXCT1. We performed a systematic literature search and evaluated clinical, biochemical and genetic data on 34 previously published and 10 novel patients with SCOTD. Structural mapping and in silico analysis of protein variants is also presented. All patients presented with severe ketoacidotic episodes. Age at first symptoms ranged from 36 h to 3 years (median 7 months). About 70% of patients manifested in the first year of life, approximately one quarter already within the neonatal period. Two patients died, while the remainder (95%) were alive at the time of the report. Almost all the surviving patients (92%) showed normal psychomotor development and no neurologic abnormalities. A total of 29 missense mutations are reported. Analysis of the published crystal structure of the human SCOT enzyme, paired with both sequence-based and structure-based methods to predict variant pathogenicity, provides insight into the biochemical consequences of the reported variants. Pathogenic variants cluster in SCOT protein regions that affect certain structures of the protein. The described pathogenic variants can be viewed in an interactive map of the SCOT protein at https://michelanglo.sgc.ox.ac.uk/r/oxct. This comprehensive data analysis provides a systematic overview of all cases of SCOTD published to date. Although SCOTD is a rather benign disorder with often favourable outcome, metabolic crises can be life-threatening or even fatal. As the diagnosis can only be made by enzyme studies or mutation analyses, SCOTD may be underdiagnosed.
Asunto(s)
Acidosis , Encefalopatías Metabólicas Innatas , Coenzima A Transferasas/deficiencia , Mutación Missense , Trastornos del Neurodesarrollo , Acidosis/enzimología , Acidosis/genética , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/genética , Coenzima A Transferasas/química , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Cristalografía por Rayos X , Humanos , Cuerpos Cetónicos/química , Cuerpos Cetónicos/genética , Cuerpos Cetónicos/metabolismo , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Dominios ProteicosRESUMEN
1,3-Propanediol (1,3-PDO) is a promising platform chemical used to manufacture various polyesters, polyethers, and polyurethanes. Microbial production of 1,3-PDO using non-natural producers often requires adding expensive cofactors such as vitamin B12, which increases the whole production cost. In this study, we proposed and engineered a non-natural 1,3-PDO synthetic pathway derived from acetyl-CoA, enabling efficient accumulation of 1,3-PDO in Escherichia coli without adding expensive cofactors. This functional pathway was established by introducing the malonyl-CoA-dependent 3-hydroxypropionic acid (3-HP) module and screening the key enzymes to convert 3-HP to 1,3-PDO. The best engineered strain can produce 2.93 g/L 1,3-PDO with a yield of 0.35 mol/mol glucose in shake flask cultivation (and 7.98 g/L in fed-batch fermentation), which is significantly higher than previous reports based on homoserine- or malate-derived non-natural pathways. We also demonstrated for the first time the feasibility of producing 1,3-PDO from diverse carbohydrates including xylose, glycerol, and acetate based on the same pathway. Thus, this study provides an alternative route for 1,3-PDO production.
Asunto(s)
Escherichia coli/metabolismo , Glucosa/metabolismo , Ácido Láctico/análogos & derivados , Ingeniería Metabólica , Glicoles de Propileno/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Glicerol/metabolismo , Ácido Láctico/química , Ácido Láctico/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Glicoles de Propileno/química , Vitamina B 12/química , Xilosa/metabolismoRESUMEN
Karst caves are widely distributed subsurface systems, and the microbiomes therein are proposed to be the driving force for cave evolution and biogeochemical cycling. In past years, culture-independent studies on the microbiomes of cave systems have been conducted, yet intensive microbial cultivation is still needed to validate the sequence-derived hypothesis and to disclose the microbial functions in cave ecosystems. In this study, the microbiomes of two karst caves in Guizhou Province in southwest China were examined. A total of 3,562 bacterial strains were cultivated from rock, water, and sediment samples, and 329 species (including 14 newly described species) of 102 genera were found. We created a cave bacterial genome collection of 218 bacterial genomes from a karst cave microbiome through the extraction of 204 database-derived genomes and de novo sequencing of 14 new bacterial genomes. The cultivated genome collection obtained in this study and the metagenome data from previous studies were used to investigate the bacterial metabolism and potential involvement in the carbon, nitrogen, and sulfur biogeochemical cycles in the cave ecosystem. New N2-fixing Azospirillum and alkane-oxidizing Oleomonas species were documented in the karst cave microbiome. Two pcaIJ clusters of the ß-ketoadipate pathway that were abundant in both the cultivated microbiomes and the metagenomic data were identified, and their representatives from the cultivated bacterial genomes were functionally demonstrated. This large-scale cultivation of a cave microbiome represents the most intensive collection of cave bacterial resources to date and provides valuable information and diverse microbial resources for future cave biogeochemical research.IMPORTANCE Karst caves are oligotrophic environments that are dark and humid and have a relatively stable annual temperature. The diversity of bacteria and their metabolisms are crucial for understanding the biogeochemical cycling in cave ecosystems. We integrated large-scale bacterial cultivation with metagenomic data mining to explore the compositions and metabolisms of the microbiomes in two karst cave systems. Our results reveal the presence of a highly diversified cave bacterial community, and 14 new bacterial species were described and their genomes sequenced. In this study, we obtained the most intensive collection of cultivated microbial resources from karst caves to date and predicted the various important routes for the biogeochemical cycling of elements in cave ecosystems.