RESUMEN
Strategies for depleting carbon dioxide (CO2) from flue gases are urgently needed and carbonic anhydrases (CAs) can contribute to solving this problem. They catalyze the hydration of CO2 in aqueous solutions and therefore capture the CO2. However, the harsh conditions due to varying process temperatures are limiting factors for the application of enzymes. The current study aims to examine four recombinantly produced CAs from different organisms, namely CAs from Acetobacterium woodii (AwCA or CynT), Persephonella marina (PmCA), Methanobacterium thermoautotrophicum (MtaCA or Cab) and Sulphurihydrogenibium yellowstonense (SspCA). The highest expression yields and activities were found for AwCA (1814 WAU mg-1 AwCA) and PmCA (1748 WAU mg-1 PmCA). AwCA was highly stable in a mesophilic temperature range, whereas PmCA proved to be exceptionally thermostable. Our results indicate the potential to utilize CAs from anaerobic microorganisms to develop CO2 sequestration applications.
Asunto(s)
Acetobacterium/enzimología , Bacterias/enzimología , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/genética , Acetobacterium/genética , Anaerobiosis , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Estabilidad de Enzimas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
Acetobacterium woodii utilizes the Wood-Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non-acetogenic growth on 1,2-propanediol (1,2-PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2-PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC-encapsulated propanol-generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH-dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2-PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.
Asunto(s)
Acetatos/metabolismo , Acetobacterium/enzimología , Alcohol Deshidrogenasa/metabolismo , Proteínas Bacterianas/metabolismo , 1-Propanol/metabolismo , Acetaldehído/metabolismo , Acetobacterium/genética , Acetobacterium/crecimiento & desarrollo , Alcohol Deshidrogenasa/genética , Aldehídos/metabolismo , Proteínas Bacterianas/genética , Dióxido de Carbono/metabolismo , Etanol/metabolismo , Genoma Bacteriano , NAD/metabolismo , Oxidación-ReducciónRESUMEN
Flavin-based electron bifurcation is a long hidden mechanism of energetic coupling present mainly in anaerobic bacteria and archaea that suffer from energy limitations in their environment. Electron bifurcation saves precious cellular ATP and enables lithotrophic life of acetate-forming (acetogenic) bacteria that grow on H2 + CO2 by the only pathway that combines CO2 fixation with ATP synthesis, the Wood-Ljungdahl pathway. The energy barrier for the endergonic reduction of NADP+, an electron carrier in the Wood-Ljungdahl pathway, with NADH as reductant is overcome by an electron-bifurcating, ferredoxin-dependent transhydrogenase (Nfn) but many acetogens lack nfn genes. We have purified a ferredoxin-dependent NADH:NADP+ oxidoreductase from Sporomusa ovata, characterized the enzyme biochemically and identified the encoding genes. These studies led to the identification of a novel, Sporomusa type Nfn (Stn), built from existing modules of enzymes such as the soluble [Fe-Fe] hydrogenase, that is widespread in acetogens and other anaerobic bacteria.
Asunto(s)
Acetobacterium/enzimología , Proteínas Bacterianas/metabolismo , Ferredoxinas/metabolismo , Firmicutes/enzimología , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Acetobacterium/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Anaerobiosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Transporte de Electrón , Electrones , Firmicutes/genética , Hidrogenasas/genética , Hidrogenasas/aislamiento & purificación , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/aislamiento & purificación , Oxidación-Reducción , Homología de Secuencia de AminoácidoRESUMEN
rnf genes are widespread in anaerobic bacteria and hypothesized to encode a respiratory enzyme that couples exergonic reduction of NAD with reduced ferredoxin as a reductant to vectorial ion (Na+, H+) translocation across the cytoplasmic membrane. However, despite its importance for the physiology of these bacteria, little is known about the subunit composition and the function of subunits. Here, we have purified the entire Rnf complex from the acetogen Acetobacterium woodii or after its production in Escherichia coli. These studies revealed covalently bound flavin in RnfB and RnfD. Unfortunately, the complex did not catalyze electron transfer from reduced ferredoxin to NAD. We, therefore, concentrated on the two cytosolic subunits RnfC and RnfB. RnfC was produced in E. coli, purified and shown to have 8.3 mol iron and 8.6 mol sulfur per mol of the subunit, consistent with the presence of two [4Fe-4S] centers, which were verified by EPR analysis. Flavins could not be detected, but RnfC catalyzed NADH-dependent FMN reduction. These data confirm RnfC as NADH-binding subunit and FMN as an intermediate in the electron transport chain. RnfB could only be produced as a fusion to the maltose-binding protein. It contained 25 mol iron and 26 mol sulfur, consistent with the predicted six [4Fe4S] centers. The FeS centers in RnfB were reduced with reduced ferredoxin as reductant. These data are consistent with RnfB as the ferredoxin-binding subunit of the complex.
Asunto(s)
Acetobacterium/enzimología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Ferredoxinas/metabolismo , Mononucleótido de Flavina/metabolismo , NAD/metabolismo , Proteínas Bacterianas/genética , Transporte de Electrón , Oxidación-ReducciónRESUMEN
The Na+-translocating F1FO ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α3:ß3:γ:δ:ε:a:b2:(c2/3)9:c1 represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c1, c2 and c3, and an extra loop (γ195-211) within the rotary γ subunit. Here, the recombinant AwF-ATP synthase was subjected to negative stain electron microscopy and single particle analysis. The reference free 2D class averages revealed high flexibility of the enzyme, wherein the F1 and FO domains distinctively bended to adopt multiple conformations. Moreover, both the F1 and FO domains tilted relative to each other to a maximum extent of 28° and 30°, respectively. The first 3D reconstruction of the AwF-ATP synthase was determined which accommodates well the modelled structure of the AwF-ATP synthase as well as the γ195-211-loop. Molecular simulations of the enzyme underlined the bending features and flexibility observed in the electron micrographs, and enabled assessment of the dynamics of the extra γ195-211-loop.
Asunto(s)
Acetobacterium/enzimología , Proteínas Bacterianas/ultraestructura , ATPasas de Translocación de Protón Mitocondriales/ultraestructura , Acetobacterium/química , Acetobacterium/ultraestructura , Proteínas Bacterianas/análisis , Imagenología Tridimensional , Microscopía Electrónica , ATPasas de Translocación de Protón Mitocondriales/análisis , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/análisis , Proteínas Recombinantes/ultraestructuraRESUMEN
The advantage of using acetogens such as Acetobacterium woodii as biocatalysts converting the cheap substrate and greenhouse gas carbon dioxide (CO2) into value-added chemicals comes together with the disadvantage of a low overall ATP gain due to the bioenergetics associated with the Wood-Ljungdahl pathway. Expanding the product spectrum of recombinant A. woodii strains to compounds with high ATP-demanding biosynthesis is therefore challenging. As a least invasive strategy for improved ATP generation, the exploitation of the arginine deiminase pathway (ADI) was examined under native conditions and via using heterologously expressed genes in A. woodii. Several promoters were analyzed for application of different gene expression levels in A. woodii using ß-glucuronidase assays. Heterologous expression of the ADI pathway genes from Clostridium autoethanogenum was controlled using either the constitutive pta-ack promoter from Clostridium ljungdahlii or a tightly regulated tetracycline-inducible promoter Ptet. Unlike constitutive expression, only induced expression of the ADI pathway genes led to a 36% higher maximal OD600 when using arginine (OD600 3.4) as nitrogen source and a 52% lower acetate yield per biomass compared to cells growing with yeast extract as nitrogen source (OD600 2.5). In direct comparison, a 69% higher maximal OD600 and about 60% lower acetate yield per biomass in induced to non-induced recombinant A. woodii cells was noticed when using arginine. Our data suggests the application of the ADI pathway in A. woodii for expanding the product spectrum to compounds with high ATP-demanding biosynthesis.
Asunto(s)
Acetobacterium/enzimología , Acetobacterium/crecimiento & desarrollo , Expresión Génica , Hidrolasas/metabolismo , Ingeniería Metabólica , Redes y Vías Metabólicas/genética , Proteínas Recombinantes/metabolismo , Acetatos/metabolismo , Acetobacterium/genética , Arginina/metabolismo , Clostridium/enzimología , Clostridium/genética , Hidrolasas/genética , Nitrógeno/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Activación TranscripcionalRESUMEN
Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.
Asunto(s)
Acetobacterium/enzimología , Proteínas Bacterianas/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Metiltransferasas/metabolismo , Moorella/enzimología , Acetobacterium/genética , Secuencias de Aminoácidos/genética , Proteínas Bacterianas/genética , Metilación de ADN , ADN Bacteriano/metabolismo , Epigénesis Genética , Genoma Bacteriano , Moorella/genética , Análisis de Secuencia de ADNRESUMEN
The Na+ translocating F1 FO ATP synthase from Acetobacterium woodii shows a subunit stoichiometry of α3 :ß3 :γ:δ:ε:a:b2 :(c2/3 )9 :c1 and reveals an evolutionary path between synthases and pumps involving adaptations in the rotor c-ring, which is composed of F- and vacuolar-type c subunits in a stoichiometry of 9 : 1. This hybrid turbine couples rotation with Na+ translocation in the FO part and rotation of the central stalk subunits γ-ε to drive ATP synthesis in the catalytic α3 :ß3 headpiece. Here, we isolated a highly pure recombinant A. woodii F-ATP synthase and present the first projected structure of this hybrid engine as determined by negative-stain electron microscopy and single-particle analysis. The uniqueness of the A. woodii F-ATP synthase is also reflected by an extra 17 amino acid residues loop (195 TSGKVKITEETKEEKSK211 ) in subunit γ. Deleting the loop-encoding DNA sequence (γΔ195-211 ) and purifying the recombinant F-ATP synthase γΔ195-211 mutant provided a platform to study its effect in enzyme stability and activity. The recombinant F-ATP synthase γΔ195-211 mutant revealed the same subunit composition as the wild-type enzyme and a minor reduction in ATP hydrolysis. When reconstituted into proteoliposomes ATP synthesis and Na+ transport were diminished, demonstrating the importance of the γ195-211 loop in both enzymatic processes. Based on a structural model, a coupling mechanism for this enzyme is proposed, highlighting the role of the γ-loop. Finally, the γ195-211 loop of A. woodii is discussed in comparison with the extra γ-loops of mycobacterial and chloroplasts F-ATP synthases described to be involved in species-specific regulatory mechanisms.
Asunto(s)
Acetobacterium/enzimología , Adenosina Trifosfato/biosíntesis , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Sodio/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microscopía Electrónica , Modelos Moleculares , Mutación , Conformación Proteica , Proteolípidos/metabolismo , ATPasas de Translocación de Protón/genéticaRESUMEN
The Rnf complex is a respiratory enzyme that catalyzes the oxidation of reduced ferredoxin to the reduction of NAD+, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic acetogenic bacteria, the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, Acetobacterium woodii, which enabled us to delete the rnf genes and to test their in vivo role. The rnf mutant did not grow on H2 plus CO2, nor did it produce acetate or ATP from H2 plus CO2, and ferredoxin:NAD+ oxidoreductase activity and Na+ translocation were also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low-energy substrates, such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of ferredoxin but only of NAD+, and we speculated that the growth phenotype is caused by a loss of reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. The electron-bifurcating hydrogenase of A. woodii reduces ferredoxin, and indeed, the addition of H2 to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex, which renders the Rnf complex essential also for growth on low-energy substrates.IMPORTANCE Ferredoxin and NAD+ are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of ferredoxin is lower than that of the NADH/NAD+ couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of ferredoxin and NAD+ When ferredoxin is greater than NADH, exergonic electron flow from ferredoxin to NAD+ generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria.
Asunto(s)
Acetobacterium/enzimología , Metabolismo Energético , Ferredoxinas/metabolismo , Complejos Multienzimáticos/metabolismo , NAD/metabolismo , Acetatos/metabolismo , Procesos Autotróficos , Transporte de Electrón , Mutagénesis , Mutación , Oxidación-ReducciónRESUMEN
Methanol derived from plant tissue is ubiquitous in anaerobic sediments and a good substrate for anaerobes growing on C1 compounds such as methanogens and acetogens. In contrast to methanogens little is known about the physiology, biochemistry and bioenergetics of methanol utilization in acetogenic bacteria. To fill this gap, we have used the model acetogen Acetobacterium woodii to study methanol metabolism using physiological and biochemical experiments paired with molecular studies and transcriptome analysis. These studies identified the genes and enzymes involved in acetogenesis from methanol and the redox carriers involved. We will present the first comprehensive model for carbon and electron flow from methanol in an acetogen and the bioenergetics of acetogenesis from methanol.
Asunto(s)
Acetobacterium/metabolismo , Metanol/metabolismo , Acetobacterium/enzimología , Carbono/metabolismo , Metabolismo Energético , Oxidación-ReducciónRESUMEN
Flavin-based electron bifurcation (FBEB) is a recently discovered mode of energy coupling in anaerobic microorganisms. The electron-bifurcating caffeyl-CoA reductase (CarCDE) catalyzes the reduction of caffeyl-CoA and ferredoxin by oxidizing NADH. The 3.5 Å structure of the heterododecameric Car(CDE)4 complex of Acetobacterium woodii, presented here, reveals compared to other electron-transferring flavoprotein/acyl dehydrogenase family members an additional ferredoxin-like domain with two [4Fe-4S] clusters N-terminally fused to CarE. It might serve, in vivo, as specific adaptor for the physiological electron acceptor. Kinetic analysis of a CarCDE(∆Fd) complex indicates the bypassing of the ferredoxin-like domain by artificial electron acceptors. Site-directed mutagenesis studies substantiated the crucial role of the C-terminal arm of CarD and of ArgE203, hydrogen-bonded to the bifurcating FAD, for FBEB.
Asunto(s)
Acetobacterium/enzimología , Flavinas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Acetobacterium/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Flavoproteínas Transportadoras de Electrones/química , Flavoproteínas Transportadoras de Electrones/genética , Flavoproteínas Transportadoras de Electrones/metabolismo , Ferredoxinas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidorreductasas/genética , Conformación Proteica , Dominios ProteicosRESUMEN
Interconversion of CO2 and formic acid is an important reaction in bacteria. A novel enzyme complex that directly utilizes molecular hydrogen as electron donor for the reversible reduction of CO2 has recently been identified in the Wood-Ljungdahl pathway of an acetogenic bacterium. This pathway is utilized for carbon fixation as well as energy conservation. Here we describe the further characterization of the quaternary structure of this enzyme complex and the unexpected behavior of this enzyme in polymerizing into filamentous structures. Polymerization of metabolic enzymes into similar structures has been observed only in rare cases but the increasing number of examples point towards a more general characteristic of enzyme functioning. Polymerization of the purified enzyme into ordered filaments of more than 0.1 µm in length was only dependent on the presence of divalent cations. Polymerization was a reversible process and connected to the enzymatic activity of the oxygen-sensitive enzyme with the filamentous form being the most active state.
Asunto(s)
Proteínas Bacterianas/metabolismo , Dióxido de Carbono/metabolismo , Formiatos/metabolismo , Hidrógeno/metabolismo , Oxidorreductasas/metabolismo , Acetobacterium/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Biocatálisis , Estabilidad de Enzimas , Cinética , Sulfato de Magnesio/química , Microscopía Electrónica , Oxidorreductasas/química , Oxidorreductasas/ultraestructura , Multimerización de ProteínaRESUMEN
Ethanol is a common substrate for anaerobic microorganisms despite its high redox potential (E0' etha- nol/acetaldehyde = -190mV), which does not allow for NAD(+) reduction. How this thermodynamic barrier is overcome is largely unknown. The acetogenic bacterium Acetobacterium woodii can also grow on ethanol. The genome harbours 11 genes encoding putative alcohol dehydrogenases, but only one, adhE, was upregulated during growth on ethanol. The bifunctional acetaldehyde/ethanol dehydrogenase (AdhE) was purified from ethanol-grown cells. It catalysed the NAD(+) - and CoA-dependent oxidation of ethanol via acetaldehyde to acetyl-CoA. The enzyme was regulated by free coenzyme A: in the absence of coenzyme A, the Km value for ethanol was shifted from 3.4 to 40 mM. The alcohol dehydrogenase domain could also oxidize 1-propanol and 1-butanol; however, the aldehyde dehydrogenase domain was highly specific for acetaldehyde as substrate. Apparently, the bifunctional AdhE allows for NAD(+) reduction by lowering the concentration of acetaldehyde, which makes the first oxidation reaction thermodynamically feasible.
Asunto(s)
Acetaldehído/metabolismo , Acetobacterium/enzimología , Alcohol Deshidrogenasa/metabolismo , Aldehído Oxidorreductasas/metabolismo , Etanol/metabolismo , Acetobacterium/genética , Acetilcoenzima A/metabolismo , Alcohol Deshidrogenasa/genética , Aldehído Oxidorreductasas/genética , Coenzima A/metabolismo , Oxidación-ReducciónRESUMEN
The ion-translocating c ring of the Na(+) F1 Fo ATP synthase of the anaerobic bacterium Acetobacterium woodii is the first heteromeric c ring found in nature that contains one V- (c1 ) and two F-type-like c subunits (c2 /c3 ), the latter of identical amino acid sequence. To address whether they are of equal or different importance for function, they were deleted in combination or individually. Deletion of c1 was compensated by incorporation of two c2 /c3 subunits but the enzyme was unstable and largely impaired in Na(+) transport. Deletion of c2 was compensated by incorporation of c3 but also led to a reduction of Na(+) transport. Deletion of c3 had no effect. In contrast, deletion of both c2 and c3 led to a complete loss of ATPase activity at the cytoplasmic membrane. Mass spectrometric analysis of c2 +1 Ala and c2 +2 Ala variants revealed a copy number of 8 : 1 for c2 /c3 which is consistent with the biochemical characteristics of the variants. These data indicate a role of c1 in assembly and a function of c2 as the predominant c ring constituent.
Asunto(s)
Acetobacterium/enzimología , ATPasas de Translocación de Protón/química , Secuencia de Aminoácidos , Eliminación de Gen , Espectrometría de Masas , Datos de Secuencia Molecular , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismoRESUMEN
The Wood-Ljungdahl pathway allows acetogenic bacteria to grow on a number of one-carbon substrates, such as carbon dioxide, formate, methyl groups, or even carbon monoxide. Since carbon monoxide alone or in combination with hydrogen and carbon dioxide (synthesis gas) is an increasingly important feedstock for third-generation biotechnology, we studied CO metabolism in the model acetogen Acetobacterium woodii. When cells grew on H2-CO2, addition of 5 to 15% CO led to higher final optical densities, indicating the utilization of CO as a cosubstrate. However, the growth rate was decreased by the presence of small amounts of CO, which correlated with an inhibition of H2 consumption. Experiments with resting cells revealed that the degree of inhibition of H2 consumption was a function of the CO concentration. Since the hydrogen-dependent CO2 reductase (HDCR) of A. woodii is known to be very sensitive to CO, we speculated that cells may be more tolerant toward CO when growing on formate, the product of the HDCR reaction. Indeed, addition of up to 25% CO did not influence growth rates on formate, while the final optical densities and the production of acetate increased. Higher concentrations (75 and 100%) led to a slight inhibition of growth and to decreasing rates of formate and CO consumption. Experiments with resting cells revealed that the HDCR is a site of CO inhibition. In contrast, A. woodii was not able to grow on CO as a sole carbon and energy source, and growth on fructose-CO or methanol-CO was not observed.
Asunto(s)
Acetobacterium/metabolismo , Monóxido de Carbono/metabolismo , Acetobacterium/enzimología , Acetobacterium/genética , Acetobacterium/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Formiatos/metabolismo , Hidrógeno/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismoRESUMEN
The acetogenic bacterium Acetobacterium woodii is able to reduce CO2 to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD(+)-dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO2 to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.
Asunto(s)
Acetobacterium/metabolismo , Butileno Glicoles/metabolismo , Acetaldehído/metabolismo , Acetatos/metabolismo , Acetobacterium/enzimología , Acetobacterium/genética , Acetobacterium/crecimiento & desarrollo , Acetoína/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dióxido de Carbono/metabolismoRESUMEN
The c ring of the Na+ F1F(o) ATP synthase from the anaerobic acetogenic bacterium Acetobacterium woodii is encoded by three different genes: atpE1, atpE2 and atpE3. Subunit c1 is similar to typical V-type c subunits and has four transmembrane helices with one ion binding site. Subunit c2 and c3 are identical at the amino acid level and are typical F-type c subunits with one ion binding site in two transmembrane helices. All three constitute a hybrid F(o)V(o) c ring, the first found in nature. To analyze whether other species may have similar hybrid rotors, we searched every genome sequence publicly available as of 23 February 2015 for F1F(o) ATPase operons that have more than one gene encoding the c subunit. This revealed no other species that has three different c subunit encoding genes but twelve species that encode one F(o)- and one V(o)-type c subunit in one operon. Their c subunits have the conserved binding motif for Na+. The organisms are all anaerobic. The advantage of hybrid c rings for the organisms in their environments is discussed.
Asunto(s)
Acetobacterium/enzimología , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , ATPasas de Translocación de Protón Mitocondriales/genética , Modelos Moleculares , Datos de Secuencia Molecular , Subunidades de Proteína/genética , Alineación de Secuencia , Sodio/química , Sodio/metabolismoRESUMEN
UNLABELLED: The methylenetetrahydrofolate reductase (MTHFR) of acetogenic bacteria catalyzes the reduction of methylene-THF, which is highly exergonic with NADH as the reductant. Therefore, the enzyme was suggested to be involved in energy conservation by reducing ferredoxin via electron bifurcation, followed by Na(+) translocation by the Rnf complex. The enzyme was purified from Acetobacterium woodii and shown to have an unprecedented subunit composition containing the three subunits RnfC2, MetF, and MetV. The stable complex contained 2 flavin mononucleotides (FMN), 23.5 ± 1.2 Fe and 24.5 ± 1.5 S, which fits well to the predicted six [4Fe4S] clusters in MetV and RnfC2. The enzyme catalyzed NADH:methylviologen and NADH:ferricyanide oxidoreductase activity but also methylene-tetrahydrofolate (THF) reduction with NADH as the reductant. The NADH:methylene-THF reductase activity was high (248 U/mg) and not stimulated by ferredoxin. Furthermore, reduction of ferredoxin, alone or in the presence of methylene-THF and NADH, was never observed. MetF or MetVF was not able to catalyze the methylene-THF-dependent oxidation of NADH, but MetVF could reduce methylene-THF using methyl viologen as the electron donor. The purified MTHFR complex did not catalyze the reverse reaction, the endergonic oxidation of methyl-THF with NAD(+) as the acceptor, and this reaction could not be driven by reduced ferredoxin. However, addition of protein fractions made the oxidation of methyl-THF to methylene-THF coupled to NAD(+) reduction possible. Our data demonstrate that the MTHFR of A. woodii catalyzes methylene-THF reduction according to the following reaction: NADH + methylene-THF â methyl-THF + NAD(+). The differences in the subunit compositions of MTHFRs of bacteria are discussed in the light of their different functions. IMPORTANCE: Energy conservation in the acetogenic bacterium Acetobacterium woodii involves ferredoxin reduction followed by a chemiosmotic mechanism involving Na(+)-translocating ferredoxin oxidation and a Na(+)-dependent F1Fo ATP synthase. All redox enzymes of the pathway have been characterized except the methylenetetrahydrofolate reductase (MTHFR). Here we report the purification of the MTHFR of A. woodii, which has an unprecedented heterotrimeric structure. The enzyme reduces methylene-THF with NADH. Ferredoxin did not stimulate the reaction; neither was it oxidized or reduced with NADH. Since the last enzyme with a potential role in energy metabolism of A. woodii has now been characterized, we can propose a quantitative bioenergetic scheme for acetogenesis from H2 plus CO2 in the model acetogen A. woodii.
Asunto(s)
Acetobacterium/enzimología , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , NAD/metabolismo , Multimerización de Proteína , Coenzimas/análisis , Mononucleótido de Flavina/análisis , Metilenotetrahidrofolato Reductasa (NADPH2)/química , Metilenotetrahidrofolato Reductasa (NADPH2)/aislamiento & purificación , Oxidación-Reducción , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Especificidad por SustratoRESUMEN
Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E0 ' = -190 mV excludes direct NAD(+) reduction (E0 ' = -320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron-transferring flavoprotein (Etf) that exhibited NAD(+) reduction only when reduced ferredoxin (Fd(2-) ) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin-based electron confurcation to drive endergonic lactate oxidation with NAD(+) as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E0 ' ≈ -500 mV) to NAD(+) according to: lactate + Fd(2-) + 2 NAD(+) â pyruvate + Fd + 2 NADH. The reduced Fd(2-) is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical ( Δ µ Ë Na + ) and finally redox energy (Fd(2-) from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.