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1.
Thomas Rydzak Marina Grigoryan Zack J. Cunningham Oleg V. Krokhin Peyman Ezzati Nazim Cicek David B. Levin John A. Wilkins Richard Sparling 《Applied microbiology and biotechnology》2014,98(14):6497-6510
While annotation of the genome sequence of Clostridium thermocellum has allowed predictions of pathways catabolizing cellobiose to end products, ambiguities have persisted with respect to the role of various proteins involved in electron transfer reactions. A combination of growth studies modulating carbon and electron flow and multiple reaction monitoring (MRM) mass spectrometry measurements of proteins involved in central metabolism and electron transfer was used to determine the key enzymes involved in channeling electrons toward fermentation end products. Specifically, peptides belonging to subunits of ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase (NFOR) were low or below MRM detection limits when compared to most central metabolic proteins measured. The significant increase in H2 versus ethanol synthesis in response to either co-metabolism of pyruvate and cellobiose or hypophosphite mediated pyruvate:formate lyase inhibition, in conjunction with low levels of ferredoxin-dependent hydrogenase and NFOR, suggest that highly expressed putative bifurcating hydrogenases play a substantial role in reoxidizing both reduced ferredoxin and NADH simultaneously. However, product balances also suggest that some of the additional reduced ferredoxin generated through increased flux through pyruvate:ferredoxin oxidoreductase must be ultimately converted into NAD(P)H either directly via NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (NfnAB) or indirectly via NADPH-dependent hydrogenase. While inhibition of hydrogenases with carbon monoxide decreased H2 production 6-fold and redirected flux from pyruvate:ferredoxin oxidoreductase to pyruvate:formate lyase, the decrease in CO2 was only 20 % of that of the decrease in H2, further suggesting that an alternative redox system coupling ferredoxin and NAD(P)H is active in C. thermocellum in lieu of poorly expressed ferredoxin-dependent hydrogenase and NFOR. 相似文献
2.
Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405 总被引:1,自引:0,他引:1
Thomas Rydzak David B. Levin Nazim Cicek Richard Sparling 《Journal of biotechnology》2009,140(3-4):169-175
End-product synthesis and enzyme activities involved in pyruvate catabolism, H2 synthesis, and ethanol production in mid-log (OD600 0.25), early stationary (OD600 0.5), and stationary phase (OD600 0.7) cell extracts were determined in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose. Carbon dioxide, hydrogen, ethanol, acetate and formate were major end-products and their production paralleled growth and cellobiose consumption. Lactate dehydrogenase, pyruvate:formate lyase, pyruvate:ferredoxin oxidoreductase, methyl viologen-dependant hydrogenase, ferredoxin-dependant hydrogenase, NADH-dependant hydrogenase, NADPH-dependant hydrogenase, NADH-dependant acetaldehyde dehydrogenase, NADH-dependant alcohol dehydogenase, and NADPH-dependant alcohol dehydrogenase activities were detected in all extracts, while pyruate dehydrogenase and formate dehydrogenase activities were not detected. All hydrogenase activities decreased (2–12-fold) as growth progressed from early exponential to stationary phase. Alcohol dehydrogenase activities fluctuated only marginally (<45%), while lactate dehydrogenase, pyruvate:formate lyase, and pyruvate:ferredoxin oxidoreductase remained constant in all cell extracts. We have proposed a pathway involved in pyruvate catabolism and end-product formation based on enzyme activity profiles in conjunction with bioinformatics analysis. 相似文献
3.
When attempting to increase yields of desirable end-products during fermentation, there is the possibility that increased
concentrations of one product redirects metabolism towards the synthesis of less desired products. Changes in growth, final
end-product concentrations, and activities of enzymes involved in pyruvate catabolism and fermentative end-product formation
were studied in Clostridium thermocellum in response to the addition of individual end-products (H2, acetate, ethanol, formate, and lactate) to the growth medium. These were added to the growth medium at concentrations ten
times greater than those found at the end of growth in cultures grown under carbon-limited conditions using cellobiose (1.1 g l−1) as model soluble substrate. Although growth rate and final cell biomass decreased significantly with the addition of all
end-products, addition of individual end-products had less pronounced effects on growth. Metabolic shifts, represented by
changes in final end-product concentrations, were observed; H2 and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence
of exogenous hydrogen (H2), acetate, and lactate. Late exponential phase enzyme activity data of enzymes involved in pyruvate catabolism and end-product
formation revealed no changes in enzyme levels greater than 2-fold in response to the presence of any given end-product, with
the exception of pyruvate:formate lyase (PFL), ferredoxin-dependent hydrogenase (Fd-H2ase), and pyruvate:ferredoxin oxidoreductase (PFO): PFL and Fd-H2ase activities increased 2-fold in the presence of ethanol, while PFO activity decreased by 57% in the presence of sodium
formate. Changes in enzyme levels did not necessarily correlate with changes in final end-product yields, suggesting that
changes in final end-product yields may be governed by thermodynamic considerations rather than levels of enzyme expressed
under the conditions tested. We demonstrate that bacterial metabolism may be manipulated in order to selectively improve desired
product yields. 相似文献
4.
5.
Collet C Girbal L Péringer P Schwitzguébel JP Soucaille P 《Archives of microbiology》2006,185(5):331-339
The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l−1) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h−1. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h−1. The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD+, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H2 removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H2 scavenging and acetate producing microorganisms. 相似文献
6.
Chevalier Frdric Herbinire-Gaboreau Juline Charif Delphine Mitta Guillaume Gavory Frdric Wincker Patrick Grve Pierre Braquart-Varnier Christine Bouchon Didier 《BMC microbiology》2012,12(1):1-18
Background
Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.Results
Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.Conclusions
Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production. 相似文献7.
Femke D. Marvin-Sikkema Teresa M. Pedro Gomes Jean-Philippe Grivet Jan C. Gottschal Rudolf A. Prins 《Archives of microbiology》1993,160(5):388-396
In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes malic enzyme, NAD(P)H: ferredoxin oxidoreductase, pyruvate: ferredoxin oxidoreductase, hydrogenase, acetate: succinate CoA transferase and succinate thiokinase leading to the formation of H2, CO2, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O2-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role-especially in zoospores — as H2-evolving, ATP-generating and O2-scavenging organelles.Abbrevations DTT
Dithiotreitol
- PEP
Phosphoenol pyruvate 相似文献
8.
Rhodospirillum rubrum is able to produce H2 during fermentation anaerobically in the dark in two ways, namely through formate hydrogen lyase and through the nitrogenase. After chemotrophic preculture aerobically in the dark formate hydrogen lyase was synthesized after a lag phase, whilst after phototrophic preculture a slight activity was present from the beginning of the anaerobic dark culture. During fermentation metabolism its activity increased noticeably. Hydrogen production through the nitrogenase occurred if the nitrogenase had been activated during phototrophic preculture. It ceased during fermentation metabolism after about 3 1/2 h anaerobic dark culture. The CO insensitive H2 production by the nitrogenase could be partially inhibited by N2. Potential activity of this system, however, remained and could be increased under conditions of nitrogenase induction. It seems therefore possible that synthesis of nitrogenase under N-deficiency can occur during fermentation metabolism in the same way as the formation of the photosynthetic apparatus in order to prepare for subsequent phototrophic metabolism.Abbreviations CAP
chloramphenicol
- DSM
Deutsche Sammlung von Mikroorganismen, Göttingen
- FHL
formate hydrogen lyase
- O.D
optical density
- PFL
pyruvate formate lyase 相似文献
9.
The fermentative metabolism of Rhodospirillum rubrum (strain Ha, F1, S1) was studied after transfering the cells from aerobic to anaerobic dark culture conditions. Pyruvate was metabolized mainly to acetate and formate, and to a lesser extent to CO2 and propionate, by all strains. Therefore, pyruvate formate lyase would appear to be the characteristic key enzyme of the dark anaerobic fermentation metabolism in R. rubrum. Strain F1 and S1 metabolized the formate further to H2 and CO2. It is concluded that this cleavage was catalysed by a formate hydrogen lyase system. Strain Ha was unable to metabolize formate. The cleavage of formate and the synthesis of poly--hydroxy-butyric acid were increased by a low pH value (6.5). Fermentation equations and schemes of the pyruvate metabolism are discussed. 相似文献
10.
Pyruvate fermentation inRhodospirillum rubrum (strains F1, S1, and Ha) was investigated using cells precultured on different substrates anaerobically in the light and than transferred to anaerobic dark conditions. Pyruvate formate lyase was always the key enzyme in pyruvate fermentation but its activity was lower than in cells which have been precultured aerobically in darkness. The preculture substrate also had a clear influence on the pyruvate formate lyase activity. Strains F1 and S1 metabolized the produced formate further to H2 and CO2. A slight production of CO2 from pyruvate, without additional H2-production, could also be detected. It was concluded from this that under anaerobic dark conditions a pyruvate dehydrogenase was also functioning. On inhibition of pyruvate formate lyase the main part of pyruvate breakdown was taken over by pyruvate dehydrogenase.When enzyme synthesis was inhibited by chloramphenicol, propionate production in contrast to formate production was not affected. Protein synthesis was not significant during anaerobic dark culture. Bacteriochlorophyll. however, showed, after a lag phase, a clear rise.Abbreviations Bchl
Bacteriochlorophyll
- CoA
Coenzyme A
- DSM
Deutsche Sammlung von Mikroorganismen (Göttingen)
- OD
optical density
- PHBA
poly--hydroxybutyric acid
- R
Rhodospirillum 相似文献
11.
Hyperthermophilic microorganisms grow at temperatures of 90 °C and above and are a recent discovery in the microbial world. They are considered to be the most ancient of all extant life forms, and have been isolated mainly from near shallow and deep sea hydrothermal vents. All but two of the nearly twenty known genera are classified asArchaea (formerly archaebacteria). Virtually all of them are strict anaerobes. The majority are obligate heterotrophs that utilize proteinaceous materials as carbon and energy sources, although a few species are also saccharolytic. Most also depend on the reduction of elemental sulfur to hydrogen sulfide (H2S) for significant growth. Peptide fermentation involves transaminases and glutamate dehydrogenase, together with several unusual ferredoxin-linked oxidoreductases not found in mesophilic organisms. Similarly, a novel pathway based on a partially non-phosphorylated Entner-Doudoroff scheme has been postulated to convert carbohydrates to acetate, H2 and CO2, although a more conventional Embden-Meyerhof pathway has also been identified in one saccharolytic species. The few hyperthermophiles known that can assimilate CO2 do so via a reductive citric acid cycle. Two So-reducing enzymes termed sulfhydrogenase and sulfide dehydrogenase have been purified from the cytoplasm of a hyperthermophile that is able to grow either with or without So. A scheme for electron flow during the oxidation of carbohydrates and peptides and the reduction of So has been proposed. However, the mechanisms by which So reduction is coupled to energy conservation in this organism and in obligate So-reducing hyperthermophiles is not known.Abbreviations ADH
alcohol dehydrogenase (ADH)
- AOR
aldehyde ferredoxin oxidoreductase
- FMOR
formate ferredoxin oxidoreductase
- FOR
formaldehyde ferredoxin oxidoreductase
- GAPDH
glyceraldehyde-3-phosphate dehydrogenase
- GDH
glutamate dehydrogenase
- GluOR
glucose ferredoxin oxidoreductase
- KGOR
2-ketoglutarate ferredoxin oxidoreductase
- IOR
indolepyruvate ferredoxin oxidoreductase
- LDH
lactate dehydrogenase
- MPT
molybopterin
- POR
pyruvate ferredoxin oxidoreductase
- PLP
pyridoxal-phosphate
- PS
polysulfide
- TPP
thiamin pyrophosphate
- So
elemental sulfur
- VOR
isovalerate ferredoxin oxidoreductase 相似文献
12.
The hyperthermophilic anaerobe Pyrococcus furiosus was found to grow on pyruvate as energy and carbon source. Growth was dependent on yeast extract (0.1%). The organism grew with doublings times of about 1 h up to cell densities of 1–2×108 cells/ml. During growth 0.6–0.8 mol acetate and 1.2–1.5 mol CO2 and 0.8 mol H2 were formed per mol of pyruvate consumed. The molar growth yield was 10–11 g cells(dry weight)/mol pyruvate. Cell suspensions catalyzed the conversion of 1 mol of pyruvate to 0.6–0.8 mol acetate, 1.2–1.5 mol CO2, 1.2 mol H2 and 0.03 mol acetoin. After fermentation of [3-14C]pyruvate the specific radioactivities of pyruvate, CO2 and acetate were equal to 1:0.01:1. Cellfree extracts contained the following enzymatic activities: pyruvate: ferredoxin (methyl viologen) oxidoreductase (0.2 U mg-1, T=60°C, with Clostridium pasteurianum ferredoxin as electron acceptor; 1.4 U mg-1 at 90°C, with methyl viologen as electron acceptor); acetyl-CoA synthetase (ADP forming) [acetyl-CoA+ADP+Piacetate+ATP+CoA] (0.34 U mg-1, T=90°C), and hydrogen: methyl viologen oxidoreductase (1.75 U mg-1). Phosphate acetyl-transferase activity, acetate kinase activity, and carbon monoxide:methyl viologen oxidoreductase activity could not be detected. These findings indicate that the archaebacterium P. furiosus ferments pyruvate to acetate, CO2 and H2 involving only three enzymes, a pyruvate:ferredoxin oxidoreductase, a hydrogenase and an acetyl-CoA synthetase (ADP forming).Non-standard abbreviations DTE
dithioerythritol
- MV
methyl viologen
- MOPS
morpholinopropane sulfonic acid
- Tricine
N-tris(hydroxymethyl)-methylglycine
Part of the work was performed at the Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karlvon-Frisch-Strasse, W-3550 Marburg/Lahn, Federal Republic of Germany 相似文献
13.
The Rnf complex is a membrane-bound ferredoxin(Fd):NAD(P)+ oxidoreductase (Fno) that couples Fd oxidation to vectorial H+/Na+ transport across the cytoplasmic membrane. Here, we produced two putative Rnf-complexes from Clostridioides difficile (Cd-Rnf) and Clostridium ljungdahlii (Cl-Rnf) for the first time in Escherichia coli. A redox-responsive low-expression system enabled Rnf assembly in the membranes of E. coli as confirmed by in vitro activity measurements. To study the physiological effects of Rnf on the metabolism of E. coli, we assembled additional Fd-dependent enzymes by plasmid-based multigene expression: (a) an Fd-linked butyrate pathway (But) from C. difficile, (b) an [FeFe]-hydrogenase (Hyd) to modulate the redox state of Fd, and (c) heterologous ferredoxins as electron carriers. The hydrogenase efficiently modulated butyrate formation by H2-mediated Fd reoxidation under nitrogen. In its functionally assembled state, Rnf severely impaired cell growth. Including Hyd in the But/Rnf background, in turn, restored normal growth. Our findings suggest that Rnf mediates reverse electron flow from NADH to Fd, which requires E. coli’s F-type ATPase to function in its reverse, ATP hydrolyzing direction. The reduced Fd is then reoxidized by endogenous Fd:NAD(P)H oxidoreductase (Fpr), which regenerates NADH and, thereby, initiates a futile cycle fueled by ATP hydrolysis. The introduction of hydrogenase interrupts this futile cycle under N2 by providing an efficient NAD(P)+-independent Fd reoxidation route, whereas under H2, Hyd outcompetes Rnf for Fd reduction. This is the first report of an Rnf complex being functionally produced and physiologically investigated in E. coli. 相似文献
14.
The hyperthermophilic anaerobe Pyrococcus furiosus was grown on maltose as energy and carbon source. During growth 1 mol maltose was fermented to 3–4 mol acetate, 6–7 mol H2 and 3–4 mol CO2. The presence of the following enzyme activities in cell extracts of maltose-grown P. furiosus indicate that the sugar is degraded to pyruvate and H2 by a modified non-phosphorylated Entner-Doudoroff-pathway (the values given in brackets are specific enzyme activities at 100 °C): Glucose: methyl viologen oxidoreductase (0.03 U/mg); 2-keto-3-deoxy-gluconate aldolase (0.03 U/mg); glyceraldehyde: benzyl viologen oxidoreductase (2.6 U/mg), glycerate kinase (2-phosphoglycerate forming) (0.48 U/mg), enolase (10.4 U/mg), pyruvate kinase (1.4 U/mg). Hexokinase, glucose-6-phosphate dehydrogenase, 2-keto-3-deoxy-6-phosphogluconate aldolase and phosphofructokinase could not be detected. Further conversion of pyruvate to acetate, CO2 and H2 involves pyruvate: ferredoxin oxidoreductase (0.4 U/mg; T=60°C with Clostridium pasteurianum ferredoxin as electron acceptor), hydrogen: methyl viologen ixodoreductase (3.4 U/mg) and ADP-dependent acetyl-CoA synthetase (1.9 U/mg). Phosphate acetyl transferase and acetate kinase could not be detected. The ADP-dependent acetyl-CoA synthetase catalyzes ATP synthesis via the mechanism of substrate level phosphorylation and apparently constitutes the only ATP conserving site during maltose catabolism in P. furiosus.This novel pathway of maltose fermentation to acetate, CO2 and H2 in the anaerobic archaeon P. furiosus may represent a phylogenetically ancient pathway of sugar fermentation.Non-standard abbreviations DTE
dithioerythritol
- MV
methyl viologen
- BV
benzyl viologen
- CHES
cyclohexylamino-ethane sulfonic acid
- ABTS
2,2-Azino-di-(3-ethylbenzthiazoliumsulfonate) 相似文献
15.
Various electron donors were found to stimulate C2H2 reduction (N2 fixation) by isolated heterocysts from Anabaena variabilis and Anabaena cylindrica. Intermediates of glycolysis and the tricarboxylic acid cycle as well as unphosphorylated sugars like glucose, fructose and erythrose were among these electron donors. The transfer of electrons from donors like H2, NADH, glyoxylate and glycollate was strictly light-dependent, whereas others like NADPH or pyruvate plus coenzyme A supported C2H2 reduction also in the dark. In all cases, the overall activity was enhanced by light. The stimulation by light was more distinct with heterocysts from A. variabilis than with heterocysts from A. cylindrica.The present communication establishes that pyruvate supports C2H2 reduction by heterocysts from either A. variabilis or A. cylindrica with rates comparable to those with other electron donors. Pyruvate could, however, support C2H2 reduction only in the presence of coenzyme A, and the concentrations of both coenzyme A and pyruvate were crucial. A pyruvate-dependent reduction of ferredoxin by extracts from heterocysts was recorded spectrophotometrically. Glyoxylate, which is an inhibitor of thiamine pyrophosphate-dependent decarboxylations, inhibited pyruvate-dependent C2H2 reduction. This result supports the conclusion that pyruvate is metabolised by pyruvate: ferredoxin oxidoreductase in heterocysts. High concentrations of pyruvate and other electron donors inhibited C2H2 reduction which suggests that nitrogenase activity in heterocysts may be controlled by the availability of electron donors.Dedicated to Professor Norbert Pfennig, Konstanz, on the occasion of his 60th birthday 相似文献
16.
Boiangiu CD Jayamani E Brügel D Herrmann G Kim J Forzi L Hedderich R Vgenopoulou I Pierik AJ Steuber J Buckel W 《Journal of molecular microbiology and biotechnology》2005,10(2-4):105-119
Anaerobic bacteria ferment glutamate via two different pathways to ammonia, carbon dioxide, acetate, butyrate and molecular hydrogen. The coenzyme B12-dependent pathway in Clostridium tetanomorphum via 3-methylaspartate involves pyruvate:ferredoxin oxidoreductase and a novel enzyme, a membrane-bound NADH:ferredoxin oxidoreductase. The flavin- and iron-sulfur-containing enzyme probably uses the energy difference between reduced ferredoxin and NADH to generate an electrochemical Na+ gradient, which drives transport processes. The other pathway via 2-hydroxyglutarate in Acidaminococcus fermentans and Fusobacterium nucleatum involves glutaconyl-CoA decarboxylase, which uses the free energy of decarboxylation to generate also an electrochemical Na+ gradient. In the latter two organisms, similar membrane-bound NADH:ferredoxin oxidoreductases have been characterized. We propose that in the hydroxyglutarate pathway these oxidoreductases work in the reverse direction, whereby the reduction of ferredoxin by NADH is driven by the Na+ gradient. The reduced ferredoxin is required for hydrogen production and the activation of radical enzymes. Further examples show that reduced ferredoxin is an agent, whose reducing energy is about 1 ATP 'richer' than that of NADH. 相似文献
17.
Andrea Veit M. Kalim Akhtar Taeko Mizutani Patrik R. Jones 《Microbial biotechnology》2008,1(5):382-394
NAD(P)H:H2 pathways are theoretically predicted to reach equilibrium at very low partial headspace H2 pressure. An evaluation of the directionality of such near‐equilibrium pathways in vivo, using a defined experimental system, is therefore important in order to determine its potential for application. Many anaerobic microorganisms have evolved NAD(P)H:H2 pathways; however, they are either not genetically tractable, and/or contain multiple H2 synthesis/consumption pathways linked with other more thermodynamically favourable substrates, such as pyruvate. We therefore constructed a synthetic ferredoxin‐dependent NAD(P)H:H2 pathway model system in Escherichia coli BL21(DE3) and experimentally evaluated the thermodynamic limitations of nucleotide pyridine‐dependent H2 synthesis under closed batch conditions. NADPH‐dependent H2 accumulation was observed with a maximum partial H2 pressure equivalent to a biochemically effective intracellular NADPH/NADP+ ratio of 13:1. The molar yield of the NADPH:H2 pathway was restricted by thermodynamic limitations as it was strongly dependent on the headspace : liquid ratio of the culture vessels. When the substrate specificity was extended to NADH, only the reverse pathway directionality, H2 consumption, was observed above a partial H2 pressure of 40 Pa. Substitution of NADH with NADPH or other intermediates, as the main electron acceptor/donor of glucose catabolism and precursor of H2, is more likely to be applicable for H2 production. 相似文献
18.
Gabriele Neuer Hermann Bothe 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,716(3):358-365
Heterocyst preparations have been obtained which actively perform nitrogen fixation (C2H2 reduction) and contain the enzymes of glycolysis and some of the tricarboxylic acid cycle. Pyruvate: ferredoxin oxidereductase has been unambiguously demonstrated in extracts from heterocysts by the formation of acetylcoenzyme A, CO2 and reduced methyl viologen (ferredoxi) from pyruvate, coenzyme A and oxidized methyl viologen (ferredoxin) as well as by the synthesis of pyruvate from CO2, acetylcoenzyme A and reduced methyl viologen. Pyruvate supports C2H2 reduction by isolated heterocysts, however, with lower activity than Na2S2O4 and H2. α-Ketoglutarate: ferredoxin oxidoreductase is absent in Anabaena cylindrica, confirming that the organism has an incomplete tricarboxylic acid cycle. 相似文献
19.
The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP
chloramphenicol
- CHI
cycloheximide
- IgG
Immunoglobulin G
- PBS
140.4 mM NaCl. 9 mM Na2HPO4, 1.3 mM NaH2PO4 (pH 74)
- SDS
sodium dodecvl sulphate
- Fd
Ferredoxin
- apoFd
Apoferredoxin
- CM-Fd
Scarboxymethylated Fd
- TCA-Fd
Fd treated with trichloroacetic acid 相似文献
20.
Boxma B Voncken F Jannink S van Alen T Akhmanova A van Weelden SW van Hellemond JJ Ricard G Huynen M Tielens AG Hackstein JH 《Molecular microbiology》2004,51(5):1389-1399
Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited. 相似文献