RESUMEN
The challenge of endergonic reduction of NADP+ using NADH is overcome by ferredoxin-dependent transhydrogenases that employ electron bifurcation for electron carrier adjustments in the ancient Wood-Ljungdahl pathway. Recently, an electron-bifurcating transhydrogenase with subunit compositions distinct from the well-characterized Nfn-type transhydrogenase was described: the Stn complex. Here, we present the single-particle cryo-EM structure of the Stn family transhydrogenase from the acetogenic bacterium Sporomusa ovata and functionally dissect its electron transfer pathway. Stn forms a tetramer consisting of functional heterotrimeric StnABC complexes. Our findings demonstrate that the StnAB subunits assume the structural and functional role of a bifurcating module, homologous to the HydBC core of the electron-bifurcating HydABC complex. Moreover, StnC contains a NuoG-like domain and a GltD-like NADPH binding domain that resembles the NfnB subunit of the NfnAB complex. However, in contrast to NfnB, StnC lost the ability to bifurcate electrons. Structural comparison allows us to describe how the same fold on one hand evolved bifurcation activity on its own while on the other hand combined with an associated bifurcating module, exemplifying modular evolution in anaerobic metabolism to produce activities critical for survival at the thermodynamic limit of life.
Asunto(s)
Electrones , Ferredoxinas , Anaerobiosis , Termodinámica , Extremidad SuperiorRESUMEN
Acetogenic bacteria are a group of strictly anaerobic bacteria that make a living from acetate formation from two molecules of CO2 via the Wood-Ljungdahl pathway (WLP). The free energy change of this reaction is very small and allows the synthesis of only a fraction of an ATP. How this pathway is coupled to energy conservation has been an enigma since its discovery ~90 years ago. Here, we describe an electron transport chain in the cytochrome- and quinone-containing acetogen Sporomusa ovata that leads from molecular hydrogen as an electron donor to an intermediate of the WLP, methylenetetrahydrofolate (methylene-tetrahydrofolate [THF]), as an electron acceptor. The catalytic site of the hydrogenase is periplasmic and likely linked cytochrome b to the membrane. We provide evidence that the MetVF-type methylenetetrahydrofolate reductase is linked proteins MvhD and HdrCBA to the cytoplasmic membrane. Membrane preparations catalyzed the H2-dependent reduction of methylene-THF to methyl-THF. In our model, a transmembrane electrochemical H+ gradient is established by both scalar and vectorial protons that leads to the synthesis of 0.5 mol ATP/mol methylene-THF by a H+-F1Fo ATP synthase. This H2- and methylene-THF-dependent electron transport chain may be present in other cytochrome-containing acetogens as well and represents a third way of chemiosmotic energy conservation in acetogens, but only in addition to the well-established respiratory enzymes Rnf and Ech. IMPORTANCE Acetogenic bacteria grow by making acetate from CO2 and are considered the first life forms on Earth since they couple CO2 reduction to the conservation of energy. How this is achieved has been an enigma ever since. Recently, two respiratory enzymes, a ferredoxin:NAD+ oxidoreductase (Rnf) and a ferredoxin:H+ oxidoreductase (Ech), have been found in cytochrome-free acetogenic model bacteria. However, some acetogens contain cytochromes in addition, and there has been a long-standing assumption of a cytochrome-containing electron transport chain in those acetogens. Here, we provide evidence for a respiratory chain in Sporomusa ovata that has a cytochrome-containing hydrogenase as the electron donor and a methylenetetrahydrofolate reductase as the terminal electron acceptor. This is the third way of chemiosmotic energy conservation found in acetogens.
Asunto(s)
Ferredoxinas , Hidrogenasas , Acetatos/metabolismo , Adenosina Trifosfato/metabolismo , Bacterias/genética , Bacterias/metabolismo , Dióxido de Carbono/metabolismo , Metabolismo Energético , Ferredoxinas/metabolismo , Firmicutes , Hidrogenasas/metabolismo , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Oxidorreductasas/metabolismoRESUMEN
Acetogenic bacteria such as Acetobacterium woodii use the Wood-Ljungdahl pathway (WLP) for fixation of CO2 and energy conservation. This pathway enables conversion of diverse substrates to the main product of acetogenesis, acetate. Methyl group containing substrates such as methanol or methylated compounds, derived from pectin, are abundant in the environment and a source for CO2 . Methyl groups enter the WLP at the level of methyltetrahydrofolic acid (methyl-THF). For methyl transfer from methanol to THF a substrate-specific methyltransferase system is required. In this study, we used genetic methods to identify mtaBC2A (Awo_c22760-Awo_c22740) as the methanol-specific methyltransferase system of A. woodii. After methyl transfer, methyl-THF serves as carbon and/or electron source and the respiratory Rnf complex is required for redox homeostasis if methanol + CO2 is the substrate. Resting cells fed with methanol + CO2 , indeed converted methanol to acetate in a 4:3 stoichiometry. When methanol was fed in combination with other electron sources such as H2 + CO2 or CO, methanol was converted Rnf-independently and the methyl group was condensed with CO to build acetate. When fed in combination with alternative electron sinks such as caffeate methanol was oxidized only and resulting electrons were used for non-acetogenic growth. These different pathways for the conversion of methyl-group containing substrates enable acetogens to adapt to various ecological niches and to syntrophic communities.
Asunto(s)
Acetobacterium , Metanol , Acetatos/metabolismo , Acetobacterium/metabolismo , Dióxido de Carbono/metabolismo , Metanol/metabolismo , Metiltransferasas/metabolismoRESUMEN
The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.(AU)
Asunto(s)
Humanos , Ferredoxinas , Metilenotetrahidrofolato Deshidrogenasa (NAD+) , Acetogeninas , Microbiología , Pruebas de HipótesisRESUMEN
The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.
Asunto(s)
Electrones , FerredoxinasRESUMEN
Methanol is the simplest of all alcohols, is universally distributed in anoxic sediments as a result of plant material decomposition and is constantly attracting attention as an interesting substrate for anaerobes like acetogens that can convert bio-renewable methanol into value-added chemicals. A major drawback in the development of environmentally friendly but economically attractive biotechnological processes is the present lack of information on biochemistry and bioenergetics during methanol conversion in these bacteria. The mesophilic acetogen Eubacterium callanderi KIST612 is naturally able to consume methanol and produce acetate as well as butyrate. To grasp the full potential of methanol-based production of chemicals, we analysed the genes and enzymes involved in methanol conversion to acetate and identified the redox carriers involved. We will display a complete model for methanol-derived acetogenesis and butyrogenesis in Eubacterium callanderi KIST612, tracing the electron transfer routes and shed light on the bioenergetics during the process.
Asunto(s)
Eubacterium , Metanol , Butiratos , Eubacterium/genética , Oxidación-ReducciónRESUMEN
The production of bulk chemicals mostly depends on exhausting petroleum sources and leads to emission of greenhouse gases. Within the last decades the urgent need for alternative sources has increased and the development of bio-based processes received new attention. To avoid the competition between the use of sugars as food or fuel, other feedstocks with high availability and low cost are needed, which brought acetogenic bacteria into focus. This group of anaerobic organisms uses mixtures of CO2, CO and H2 for the production of mostly acetate and ethanol. Also methanol, a cheap and abundant bulk chemical produced from methane, is a suitable substrate for acetogenic bacteria. In methylotrophic acetogens the methyl group is transferred to the Wood-Ljungdahl pathway, a pathway to reduce CO2 to acetate via a series of C1-intermediates bound to tetrahydrofolic acid. Here we describe the biochemistry and bioenergetics of methanol conversion in the biotechnologically interesting group of anaerobic, acetogenic bacteria. Further, the bioenergetics of biochemical production from methanol is discussed.
Asunto(s)
Bacterias/metabolismo , Tecnología Química Verde , Metanol/metabolismo , Acetatos/metabolismo , Metabolismo Energético , Etanol/metabolismo , Metanol/químicaRESUMEN
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
The quarternary, trimethylated amine glycine betaine (GB) is widespread in nature but its fate under anoxic conditions remains elusive. It can be used by some acetogenic bacteria as carbon and energy source but the pathway of GB metabolism has not been elucidated. We have identified a gene cluster involved in GB metabolism and studied acetogenesis from GB in the model acetogen Acetobacterium woodii. GB is taken up by a secondary active, Na+ coupled transporter of the betaine-choline-carnitine (BCC) family. GB is demethylated to dimethylglycine, the end product of the reaction, by a methyltransferase system. Further conversion of the methyl group requires CO2 as well as Na+ indicating that GB metabolism involves the Wood-Ljungdahl pathway. These studies culminate in a model for the path of carbon and electrons during acetogenensis from GB and a model for the bioenergetics of acetogenesis from GB.
Asunto(s)
Acetobacterium/metabolismo , Betaína/metabolismo , Acetobacterium/genética , Carbono/metabolismo , Metabolismo Energético/genética , Genes Bacterianos , Familia de MultigenesRESUMEN
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
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.