RESUMEN

Per and polyfluoroalkyl substances (PFAS) are a large family of anthropogenic fluorinated chemicals of increasing environmental concern. Over recent years, numerous microbial communities have been found to be capable of metabolizing some polyfluoroalkyl substances, generating a range of low-molecular-weight PFAS metabolites. One proposed pathway for the microbial breakdown of fluorinated carboxylates includes ß-oxidation, this pathway is initiated by the formation of a CoA adduct. However, until recently no PFAS-CoA adducts had been reported. In a previous study, we were able to use a bacterial medium-chain acyl-CoA synthetase (mACS) to form CoA adducts of fluorinated adducts of propanoic acid and pentanoic acid but were not able to detect any products of fluorinated hexanoic acid analogues. Herein, we expressed and purified a long-chain acyl-CoA synthetase (lACS) and a A461K variant of mACS from the soil bacterium Gordonia sp. strain NB4-1Y and performed an analysis of substrate scope and enzyme kinetics using fluorinated and nonfluorinated carboxylates. We determined that lACS can catalyze the formation of CoA adducts of 1:5 fluorotelomer carboxylic acid (FTCA), 2:4 FTCA and 3:3 FTCA, albeit with generally low turnover rates (<0.02 s-1) compared with the nonfluorinated hexanoic acid (5.39 s-1). In addition, the A461K variant was found to have an 8-fold increase in selectivity toward hexanoic acid compared with wild-type mACS, suggesting that Ala-461 has a mechanistic role in selectivity toward substrate chain length. This provides further evidence to validate the proposed activation step involving the formation of CoA adducts in the enzymatic breakdown of PFAS.

Asunto(s)

Caproatos , Coenzima A Ligasas , Coenzima A Ligasas/metabolismo , Coenzima A Ligasas/genética , Coenzima A Ligasas/química , Caproatos/metabolismo , Caproatos/química , Bacteria Gordonia/metabolismo , Bacteria Gordonia/enzimología , Bacteria Gordonia/genética , Halogenación , Coenzima A/metabolismo , Coenzima A/química , Cinética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Acilcoenzima A/metabolismo , Acilcoenzima A/química , Especificidad por Sustrato

RESUMEN

Fusobacterium nucleatum, a Gram-negative obligate anaerobe, is common to the oral microbiota, but the species is known to infect other sites of the body where it is associated with a range of pathologies. At present, little is known about the mechanisms by which F. nucleatum mitigates against oxidative and nitrosative stress. Inspection of the F. nucleatum subsp. polymorphum ATCC 10953 genome reveals that it encodes a flavodiiron protein (FDP; FNP2073) that is known in other organisms to reduce NO to N2O and/or O2 to H2O. FNP2073 is dicistronic with a gene encoding a multicomponent enzyme termed BCR for butyryl-CoA reductase. BCR is composed of a butyryl-CoA dehydrogenase domain (BCD), the C-terminal domain of the α-subunit of the electron-transfer flavoprotein (Etfα), and a rubredoxin domain. We show that BCR and the FDP form an α4ß4 heterotetramic complex and use butyryl-CoA to selectively reduce O2 to H2O. The FAD associated with the Etfα domain (α-FAD) forms red anionic semiquinone (FAD•-), whereas the FAD present in the BCD domain (δ-FAD) forms the blue-neutral semiquinone (FADH•), indicating that both cofactors participate in one-electron transfers. This was confirmed in stopped-flow studies where the reduction of oxidized BCR with an excess of butyryl-CoA resulted in rapid (<1.6 ms) interflavin electron transfer evidenced by the formation of the FAD•-. Analysis of bacterial genomes revealed that the dicistron is present in obligate anaerobic gut bacteria considered to be beneficial by virtue of their ability to produce butyrate. Thus, BCR-FDP may help to maintain anaerobiosis in the colon.

Asunto(s)

Proteínas Bacterianas , Fusobacterium nucleatum , Oxidación-Reducción , Oxígeno , Fusobacterium nucleatum/metabolismo , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/enzimología , Oxígeno/metabolismo , Oxígeno/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Flavoproteínas Transportadoras de Electrones/metabolismo , Flavoproteínas Transportadoras de Electrones/genética , Flavoproteínas Transportadoras de Electrones/química , Transporte de Electrón , Acilcoenzima A/metabolismo , Butiril-CoA Deshidrogenasa/metabolismo , Butiril-CoA Deshidrogenasa/genética , Butiril-CoA Deshidrogenasa/química

RESUMEN

Phenolic compounds are commonly found in anoxic environments, where they serve as both carbon and energy sources for certain anaerobic bacteria. The anaerobic breakdown of m-cresol, catechol, and certain lignin-derived compounds yields the central intermediate 3-hydroxybenzoate/3-hydroxybenzoyl-CoA. In this study, we have characterized the transcription and regulation of the hbd genes responsible for the anaerobic degradation of 3-hydroxybenzoate in the ß-proteobacterium Aromatoleum sp. CIB. The hbd cluster is organized in three catabolic operons and a regulatory hbdR gene that encodes a dimeric transcriptional regulator belonging to the TetR family. HbdR suppresses the activity of the three catabolic promoters (PhbdN, PhbdE and PhbdH) by binding to a conserved palindromic operator box (ATGAATGAN4TCATTCAT). 3-Hydroxybenzoyl-CoA, the initial intermediate of the 3-hydroxybenzoate degradation pathway, along with benzoyl-CoA, serve as effector molecules that bind to HbdR inducing the expression of the hbd genes. Moreover, the hbd genes are subject to additional regulation influenced by the presence of non-aromatic carbon sources (carbon catabolite repression), and their expression is induced in oxygen-deprived conditions by the AcpR transcriptional activator. The prevalence of the hbd cluster among members of the Aromatoleum/Thauera bacterial group, coupled with its association with mobile genetic elements, suggests acquisition through horizontal gene transfer. These findings significantly enhance our understanding of the regulatory mechanisms governing the hbd gene cluster in bacteria, paving the way for further exploration into the anaerobic utilization/valorization of phenolic compounds derived from lignin.

Asunto(s)

Regulación Bacteriana de la Expresión Génica , Hidroxibenzoatos , Familia de Multigenes , Anaerobiosis , Hidroxibenzoatos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regiones Promotoras Genéticas , Redes y Vías Metabólicas/genética , Operón , Transcripción Genética , Acilcoenzima A/metabolismo , Acilcoenzima A/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Lignina/metabolismo

RESUMEN

The Burkholderia cepacia complex (Bcc) consists of opportunistic pathogens known to cause pneumonia in immunocompromised individuals, especially those with cystic fibrosis. Treating Bcc pneumonia is challenging due to the pathogens' high multidrug resistance. Therefore, inhalation therapy with tobramycin powder, which can achieve high antibiotic concentrations in the lungs, is a promising treatment option. In this study, we investigated potential mechanisms that could compromise the effectiveness of tobramycin therapy. By selecting for B. cenocepacia survivors against tobramycin, we identified three spontaneous mutations that disrupt a gene encoding a key enzyme in the biosynthesis of cobalamin (Vitamin B12). This disruption may affect the production of succinyl-CoA by methylmalonyl-CoA mutase, which requires adenosylcobalamin as a cofactor. The depletion of cellular succinyl-CoA may impact the tricarboxylic acid (TCA) cycle, which becomes metabolically overloaded upon exposure to tobramycin. Consequently, the mutants exhibited significantly reduced reactive oxygen species (ROS) production. Both the wild-type and mutants showed tolerance to tobramycin and various other bactericidal antibiotics under microaerobic conditions. This suggests that compromised ROS-mediated killing, due to the impacted TCA cycle, underlies the mutants' tolerance to bactericidal antibiotics. The importance of ROS-mediated killing and the potential emergence of mutants that evade it through the depletion of cobalamin (Vitamin B12) provide valuable insights for developing strategies to enhance antibiotic treatments of Bcc pneumonia.

Asunto(s)

Antibacterianos , Burkholderia cenocepacia , Mutación , Especies Reactivas de Oxígeno , Tobramicina , Vitamina B 12 , Vitamina B 12/farmacología , Vitamina B 12/metabolismo , Antibacterianos/farmacología , Burkholderia cenocepacia/efectos de los fármacos , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Tobramicina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Acilcoenzima A/metabolismo , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana/genética , Ciclo del Ácido Cítrico/efectos de los fármacos , Humanos , Metilmalonil-CoA Mutasa/genética , Metilmalonil-CoA Mutasa/metabolismo , Infecciones por Burkholderia/microbiología , Infecciones por Burkholderia/tratamiento farmacológico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo

RESUMEN

Nucleoside diphosphate (NDP) kinases 1 and 2 (NME1/2) are well-characterized enzymes known for their NDP kinase activity. Recently, these enzymes have been shown by independent studies to bind coenzyme A (CoA) or acyl-CoA. These findings suggest a hitherto unknown role for NME1/2 in the regulation of CoA/acyl-CoA-dependent metabolic pathways, in tight correlation with the cellular NTP/NDP ratio. Accordingly, the regulation of NME1/2 functions by CoA/acyl-CoA binding has been described, and additionally, NME1/2 have been shown to control the cellular pathways consuming acetyl-CoA, such as histone acetylation and fatty acid synthesis. NME1/2-controlled histone acetylation in turn mediates an important transcriptional response to metabolic changes, such as those induced following a high-fat diet (HFD). This review discusses the CoA/acyl-CoA-dependent NME1/2 activities and proposes that these enzymes be considered as the first identified carriers of CoA/short-chain acyl-CoAs.

Asunto(s)

Adenosina Trifosfato , Humanos , Animales , Adenosina Trifosfato/metabolismo , Acilcoenzima A/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Nucleósido Difosfato Quinasas NM23/genética , Nucleósido-Difosfato Quinasa/metabolismo , Nucleósido-Difosfato Quinasa/genética , Acetilación

RESUMEN

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2ß2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the ß-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.

Asunto(s)

Acilcoenzima A , Proteínas Bacterianas , Dominio Catalítico , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzimología , Cristalografía por Rayos X , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Acilcoenzima A/metabolismo , Acilcoenzima A/química , Especificidad por Sustrato , Sitios de Unión , Modelos Moleculares , Enoil-CoA Hidratasa/metabolismo , Enoil-CoA Hidratasa/química , Unión Proteica , 3-Hidroxiacil-CoA Deshidrogenasas/química , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo

RESUMEN

One of the hallmarks of living organisms is their capacity for self-organization and regeneration, which requires a tight integration of metabolic and genetic networks. We sought to construct a linked metabolic and genetic network in vitro that shows such lifelike behavior outside of a cellular context and generates its own building blocks from nonliving matter. We integrated the metabolism of the crotonyl-CoA/ethyl-malonyl-CoA/hydroxybutyryl-CoA cycle with cell-free protein synthesis using recombinant elements. Our network produces the amino acid glycine from CO2 and incorporates it into target proteins following DNA-encoded instructions. By orchestrating ~50 enzymes we established a basic cell-free operating system in which genetically encoded inputs into a metabolic network are programmed to activate feedback loops allowing for self-integration and (partial) self-regeneration of the complete system.

Asunto(s)

Dióxido de Carbono , Sistema Libre de Células , Glicina , Redes y Vías Metabólicas , Biosíntesis de Proteínas , Acilcoenzima A/metabolismo , Dióxido de Carbono/metabolismo , Retroalimentación Fisiológica , Redes Reguladoras de Genes , Glicina/biosíntesis , Glicina/genética

RESUMEN

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/deficiencia

RESUMEN

Precursor supply plays a significant role in the production of secondary metabolites. In Streptomyces bacteria, propionyl-, malonyl-, and methylmalonyl-CoA are the most common precursors used for polyketide biosynthesis. Although propionyl-CoA synthetases participate in the propionate assimilation pathway and directly convert propionate into propionyl-CoA, malonyl- and methylmalonyl-CoA cannot be formed using common acyl-CoA synthetases. Therefore, both acetyl- and propionyl-CoA carboxylation, catalyzed by acyl-CoA carboxylases, should be considered when engineering a microorganism chassis to increase polyketide production. In this study, we identified a transcriptional regulator of the TetR family, BkdR, in Streptomyces albus B4, which binds directly to the promoter region of the neighboring pccAB operon. This operon encodes acetyl/propionyl-CoA carboxylase and negatively regulates its transcription. In addition to acetate and propionate, the binding of BkdR to pccAB is disrupted by acetyl- and propionyl-CoA ligands. We identified a 16-nucleotide palindromic BkdR-binding motif (GTTAg/CGGTCg/TTAAC) in the intergenic region between pccAB and bkdR. When bkdR was deleted, we found an enhanced supply of malonyl- and methylmalonyl-CoA precursors in S. albus B4. In this study, spinosad production was detected in the recombinant strain after introducing the entire artificial biosynthesized gene cluster into S. albus B4. When supplemented with propionate to provide propionyl-CoA, the novel bkdR-deleted strain produced 29.4% more spinosad than the initial strain in trypticase soy broth (TSB) medium. IMPORTANCE: In this study, we describe a pccAB operon involved in short-chain acyl-CoA carboxylation in S. albus B4 chassis. The TetR family regulator, BkdR, represses this operon. Our results show that BkdR regulates the precursor supply needed for heterologous spinosad biosynthesis by controlling acetyl- and propionyl-CoA assimilation. The deletion of the BkdR-encoding gene exerts an increase in heterologous spinosad yield. Our research reveals a regulatory mechanism in short-chain acyl-CoA metabolism and suggests new possibilities for S. albus chassis engineering to enhance heterologous polyketide yield.

Asunto(s)

Proteínas Bacterianas , Combinación de Medicamentos , Macrólidos , Streptomyces , Macrólidos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Ingeniería Metabólica , Operón , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Acilcoenzima A/metabolismo

RESUMEN

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/química

RESUMEN

Propionic acidemia (PA), resulting from Pcca or Pccb gene mutations, impairs propionyl-CoA metabolism and induces metabolic alterations. While speculation exists that fasting might exacerbate metabolic crises in PA patients by accelerating the breakdown of odd-chain fatty acids and amino acids into propionyl-CoA, direct evidence is lacking. Our investigation into the metabolic effects of fasting in Pcca-/-(A138T) mice, a PA model, reveals surprising outcomes. Propionylcarnitine, a PA biomarker, decreases during fasting, along with the C3/C2 (propionylcarnitine/acetylcarnitine) ratio, ammonia, and methylcitrate. Although moderate amino acid catabolism to propionyl-CoA occurs with a 23-h fasting, a significant reduction in microbiome-produced propionate and increased fatty acid oxidation mitigate metabolic alterations by decreasing propionyl-CoA synthesis and enhancing acetyl-CoA synthesis. Fasting-induced gluconeogenesis further facilitates propionyl-CoA catabolism without changing propionyl-CoA carboxylase activity. These findings suggest that fasting may alleviate metabolic alterations in Pcca-/-(A138T) mice, prompting the need for clinical evaluation of its potential impact on PA patients.

Asunto(s)

Ayuno , Metilmalonil-CoA Descarboxilasa , Mutación , Animales , Ratones , Metilmalonil-CoA Descarboxilasa/metabolismo , Metilmalonil-CoA Descarboxilasa/genética , Acidemia Propiónica/genética , Acidemia Propiónica/metabolismo , Masculino , Ratones Noqueados , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Acilcoenzima A/metabolismo

RESUMEN

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

Asunto(s)

Mycobacterium tuberculosis , Propionatos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Propionatos/farmacología , Propionatos/metabolismo , Virulencia , Lípidos/química , Ésteres del Colesterol/metabolismo , Tuberculosis/microbiología , Tuberculosis/prevención & control , Ácidos Grasos/metabolismo , Vitamina B 12/farmacología , Vitamina B 12/metabolismo , Humanos , Mutación , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Colesterol/metabolismo , Acilcoenzima A

RESUMEN

A comprehensive pangenomic approach was employed to analyze the genomes of 75 type II methylotrophs spanning various genera. Our investigation revealed 256 exact core gene families shared by all 75 organisms, emphasizing their crucial role in the survival and adaptability of these organisms. Additionally, we predicted the functionality of 12 hypothetical proteins. The analysis unveiled a diverse array of genes associated with key metabolic pathways, including methane, serine, glyoxylate, and ethylmalonyl-CoA (EMC) metabolic pathways. While all selected organisms possessed essential genes for the serine pathway, Methylooceanibacter marginalis lacked serine hydroxymethyltransferase (SHMT), and Methylobacterium variabile exhibited both isozymes of SHMT, suggesting its potential to utilize a broader range of carbon sources. Notably, Methylobrevis sp. displayed a unique serine-glyoxylate transaminase isozyme not found in other organisms. Only nine organisms featured anaplerotic enzymes (isocitrate lyase and malate synthase) for the glyoxylate pathway, with the rest following the EMC pathway. Methylovirgula sp. 4MZ18 stood out by acquiring genes from both glyoxylate and EMC pathways, and Methylocapsa sp. S129 featured an A-form malate synthase, unlike the G-form found in the remaining organisms. Our findings also revealed distinct phylogenetic relationships and clustering patterns among type II methylotrophs, leading to the proposal of a separate genus for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129. This pangenomic study unveils remarkable metabolic diversity, unique gene characteristics, and distinct clustering patterns of type II methylotrophs, providing valuable insights for future carbon sequestration and biotechnological applications. IMPORTANCE: Methylotrophs have played a significant role in methane-based product production for many years. However, a comprehensive investigation into the diverse genetic architectures across different genera of methylotrophs has been lacking. This study fills this knowledge gap by enhancing our understanding of core hypothetical proteins and unique enzymes involved in methane oxidation, serine, glyoxylate, and ethylmalonyl-CoA pathways. These findings provide a valuable reference for researchers working with other methylotrophic species. Furthermore, this study not only unveils distinctive gene characteristics and phylogenetic relationships but also suggests a reclassification for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129 into separate genera due to their unique attributes within their respective genus. Leveraging the synergies among various methylotrophic organisms, the scientific community can potentially optimize metabolite production, increasing the yield of desired end products and overall productivity.

Asunto(s)

Genoma Bacteriano , Filogenia , Genoma Bacteriano/genética , Redes y Vías Metabólicas/genética , Glioxilatos/metabolismo , Genómica , Evolución Molecular , Serina/metabolismo , Serina/genética , Acilcoenzima A/metabolismo , Acilcoenzima A/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Metano/metabolismo

RESUMEN

Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl-coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, which activates their ability to stimulate the DRP1 GTPase. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51. In cells, this LCACA binding mutant does not assemble into puncta on mitochondria or rescue MiD49/51 knockdown effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with BSA-bound oleic acid, which causes increased LCACA, promotes mitochondrial fission in an MiD49/51-dependent manner. These results suggest that LCACA is an endogenous ligand for MiDs, inducing mitochondrial fission and providing a potential mechanism for fatty-acid-induced mitochondrial division. Finally, MiD49 or MiD51 oligomers synergize with Mff, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation.

Asunto(s)

Acilcoenzima A , Dinaminas , GTP Fosfohidrolasas , Mitocondrias , Dinámicas Mitocondriales , Proteínas Mitocondriales , Dinámicas Mitocondriales/efectos de los fármacos , Dinaminas/metabolismo , Dinaminas/genética , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Acilcoenzima A/metabolismo , Multimerización de Proteína , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Animales , Unión Proteica , Células HeLa , Células HEK293 , Ácido Oléico/farmacología , Ácido Oléico/metabolismo , Proteínas de la Membrana , Factores de Elongación de Péptidos

Asunto(s)

Mitocondrias , Dinámicas Mitocondriales , Humanos , Mitocondrias/metabolismo , Animales , Acilcoenzima A/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética

RESUMEN

BACKGROUND: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs. RESULT: In this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b in planta, through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool). CONCLUSIONS: This set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that PtDGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.

Asunto(s)

Diacilglicerol O-Acetiltransferasa , Diatomeas , Nicotiana , Diatomeas/genética , Diatomeas/enzimología , Diatomeas/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Nicotiana/genética , Nicotiana/enzimología , Nicotiana/metabolismo , Acilcoenzima A/metabolismo , Plantas Modificadas Genéticamente , Triglicéridos/biosíntesis , Triglicéridos/metabolismo , Ácido Eicosapentaenoico/biosíntesis , Ácido Eicosapentaenoico/metabolismo , Ácidos Grasos Omega-3/biosíntesis , Ácidos Grasos Omega-3/metabolismo , Ingeniería Metabólica

RESUMEN

Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 µg/mL - 4.36-fold higher than WT value (48.9 µg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 µg/mL. Overexpression of fatty acid ß-oxidation pathway genes in the strain further increased B1a titer to 452.8 µg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 µg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides.

Asunto(s)

Acilcoenzima A , Ivermectina , Ivermectina/análogos & derivados , Ingeniería Metabólica , Redes y Vías Metabólicas , Sintasas Poliquetidas , Streptomyces , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Acilcoenzima A/metabolismo , Acilcoenzima A/genética , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces/enzimología , Redes y Vías Metabólicas/genética , Ivermectina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo

RESUMEN

Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3-mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development.

Asunto(s)

Biogénesis de Organelos , Succinato-CoA Ligasas , Animales , Humanos , Ratones , Acilcoenzima A/metabolismo , Acilcoenzima A/genética , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , ADN Mitocondrial/metabolismo , ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Leucemia/metabolismo , Leucemia/genética , Leucemia/patología , Mitocondrias/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Succinato-CoA Ligasas/metabolismo , Succinato-CoA Ligasas/genética

RESUMEN

Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol-3-phosphate, wherein the second acylation step is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane-bound enzyme, and functions as 1-acylglycerol-3-phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol-3-phosphate (LPA) from monounsaturated fatty acyl-coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant in vivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA in vitro and were not able to rescue the growth defect of E. coli PlsC mutant in vivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.

Asunto(s)

Mycobacterium tuberculosis , Triglicéridos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Triglicéridos/biosíntesis , Triglicéridos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/genética , Aciltransferasas/metabolismo , Aciltransferasas/genética , Acilación , Ácidos Grasos/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Fosfatidicos/metabolismo , Ácidos Fosfatidicos/biosíntesis , Acilcoenzima A/metabolismo

RESUMEN

Acyl-Coenzyme A binding domain containing proteins (ACBDs) are ubiquitous in nearly all eukaryotes. They can exist as a free protein, or a domain of a large, multidomain, multifunctional protein. Besides modularity, ACBDs also display multiplicity. The same organism may have multiple ACBDs, differing in sequence and organization. By virtue of this diversity, ACBDs perform functions ranging from transport, synthesis, trafficking, signal transduction, transcription, and gene regulation. In plants and some microorganisms, these ACBDs are designated ACBPs (acyl-CoA binding proteins). The simplest ACBD/ACBP is a small, ∼10 kDa, soluble protein, comprising the acyl-CoA binding (ACB) domain. Most of these small ACBDs exist as monomers, while a few show a tendency to oligomerize. In sync with those studies, we report the crystal structure of two ACBDs from Leishmania major, named ACBP103, and ACBP96 based on the number of residues present. Interestingly, ACBP103 crystallized as a monomer and a dimer under different crystallization conditions. Careful examination of the dimer disclosed an exposed 'AXXA' motif in the helix I of the two ACBP103 monomers, aligned in a head-to-tail arrangement in the dimer. Glutaraldehyde cross-linking studies confirm that apo-ACBP103 can self-associate in solution. Isothermal titration calorimetry studies further show that ACBP103 can bind ligands ranging from C8 - to C20-CoA, and the data could be best fit to a 'two sets of sites'/sequential binding site model. Taken together, our studies show that Leishmania major ACBP103 can self-associate in the apo-form through a unique dimerization motif, an interaction that may play an important role in its function.

Asunto(s)

Secuencias de Aminoácidos , Leishmania major , Multimerización de Proteína , Leishmania major/metabolismo , Leishmania major/genética , Acilcoenzima A/metabolismo , Acilcoenzima A/química , Cristalografía por Rayos X , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Modelos Moleculares , Sitios de Unión