Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

Respiration in Organic Acid-Forming Molds

Cytochrome c, coenzyme Q and lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1, 1, 1, 27) in Rhizopus oryzae were studied in order to investigate the connection between the mechanism of lactate formation and terminal respiration.

Cytochrome c was extracted easily and in good yield by the addition of cetyltrimethyl ammonium bromide to mycelial suspensions. It was purified by calcium phosphate gel and Amberlite IRC-50 resin chromatography.

Coenzyme Q was extracted with ethanol, purified by chromatography on silicic acid, and, following crystallization from a mixture of ethanol and methanol, was identified as coenzyme Q₀.

Lactic dehydrogenase was partially purified and some of its properties were investigated.

Rhizopus oryzae at an early growth stage in shake culture produced almost no lactate. At this stage, the mycelia were rich in cytochrome c and FAD. On the contrary, those of later growth stages fermented a larger amount of the glucose to lactate and the contents of cytochrome c and FAD were lower than in the young mycelia.

Surface cultures produced lactate at a rate very nearly equivalent to the rate of glucose consumption. Addition of zinc to the medium resulted in decreased lactate production, but no increase was observed in the mycelial content of either cytochrome c or FAD in this case. On the other hand, increased quantities of FMN were found in mycelia from shake or surface cultures when zinc was added.     

Enzymic properties of a mutant of Escherichia coli K 12 lacking nitrate reductase

Summary A pleiotropic mutant of Escherichia coli K ₁₂ lacking reduced NAD: nitrate oxidoreductase, soluble formate dehydrogenase and membrane-bound formate:ferricytochrome b₁ oxidoreductase is described. Levels of several other enzymes and cytochromes have been measured and found to differ little from those normally present in the wild type with the exceptions of cytochrome c₅₂₂, reduced NAD:cytochrome c oxidoreductase and reduced NAD:nitrite oxidoreductase which are very high. Although the affected gene maps in a different position from that reported for chl A by other workers it seems likely that the two loci are identical.     

Soluble electron-transport activities in fresh and aged turnip tissue

Summary A soluble (supernatant) fraction from turnips catalyses the reduction of both FeCN and DCPIP but usually not cytochrome c in the presence of either NADH or NADPH. Slicing and aging turnip tissue induces an increase in these activities as well as the development of an NADH-cytochrome c reductase activity.(NH₄)₂SO₄ and Sephadex fractionation indicated that at least three enzymes were involved: an NADH-cytochrome-c reductase, an NADH-FeCN reductase, and an NAD(P)H-DCPIP and FeCN reductase. While the latter reductase had an acid pH optimum, indicating vacuolar origin, the NADH-cytochrome-c and FeCN reductases both had neutral pH optima, indicating cytoplasmic origin. Characterization of the NADH-specific reductases indicated that NADH-FeCN reductase may be a soluble form of the microsomal membrane NADH dehydrogenase but that NADH-cytochrome-c reductase may be normally soluble and possibly involved in cyanide-sensitive NADH oxidation.The induced development of all three reductases was inhibited by 6-methylpurine, ethionine and cycloheximide, indicating dependence on both RNA and protein synthesis. The inhibition by cycloheximide could be reversed but this reversion required a 20-h washing-out period to be complete.Abbreviations DCPIP 2,6-dichlorophenol indophenol - FeCN ferricyanide - NO QNO 2-n-nonylhydroxyquinoline-N-oxide - pCMB p-chloromercuribenzoate - SF soluble fraction     

Sulfolobus tokodaii ST2133 is characterized as a thioredoxin reductase-like ferredoxin:NADP+ oxidoreductase

The putative gene (st2133) for ferredoxin:NADP⁺ oxidoreductase (FNR) from Sulfolobus tokodaii, a thermoacidophilic crenarchaeon, was heterologously expressed. About 90 % of the purified product was a homodimer containing 0.46 mol FAD/mol subunit, and showing NADPH:DCPIP oxidoreductase activity, V _max being 1.38 and 21.8 U/mg (70 °C) in the absence and presence of 1 mM FMN. NADPH was a much better electron donor than NADH with various electron acceptors, such as oxygen, hydrogen peroxide, DCPIP, cytochrome c, and dithiobisnitrobenzoate. Most of the reactions were activated by 15- to 140-fold on addition of FMN, while FAD was 5–10 times less effective. Ferredoxin (Fd) from S. tokodaii served as an electron carrier in both Fd-dependent NADPH formation and NADPH-dependent Fd reduction. ST2133 belongs to the thioredoxin reductase-like protein family, which is slightly distantly related to FNR family proteins from bacteria, plants and man. This is the first report on FNR from a crenarchaeon, providing a clue to the recycling of Fd during archaeal metabolism.     

Some properties of formate dehydrogenase,accumulation and incorporation of 185W-tungsten into proteins of Clostridium formicoaceticum

Formate dehydrogenase of Clostridium formicoaceticum used only methyl and benzyl viologen, but not NAD as electron acceptor. The S_0.5 values were 0.9×10^-4 M for formate and 5.8×10^-3 M for methyl viologen. Using potassium phosphate buffer a pH-optimum of 7.9 was observed. The initial velocity of the formate dehydrogenase activity reached a maximum at 70°C, whereas the activity was stable only up to 50°C. The level of formate dehydrogenase in C. formicoaceticum was increased to its maximum when 10^-6 M selenite and 10^-7 M tungstate were added to a synthetic medium. Addition of molybdate instead of tungstate did not increase the level of formate dehydrogenase. ¹⁸⁵W-tungsten was concentrated about 100-fold by C. formicoaceticum; molybdate had no major effect on the uptake of tungsten. ¹⁸⁵W-tungsten was found almost exclusively in the soluble fluid and was predominantly recovered after chromatography in a protein of about 88000 molecular weight. Occasionally a labelled protein of low molecular weight was observed. Again molybdate added even in high molar excess did not influence the labelling pattern. No radioactivity peak could be obtained at the elution peak of formate dehydrogenase activity. The extreme instability of formate dehydrogenase prevented further purification.Abbreviations FDH formate dehydrogenase - DTE dithioerythritol - HEPES hydroxyethylpiperazine N-2-ethane sulconic acid - TEA triethylamine - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulfate - TTC triphenyltetrazolium     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

On the acceptor specificity of glycollate oxidase of Nicotiana tabacum

Summary The effect of compounds on the activity of ammonium sulphate preparations of glycollate oxidase from Nicotiana tabacum cv. John Williams' Broadleaf and the aurea mutant Su/su is reported. Coupling to DCPIP as terminal oxidant under anaerobic conditions gave greater rates of glycollate oxidation than when measured as O₂ uptake in the presence of cyanide. The enzyme also linked to DCPIP in the presence of O₂, showing that it is a facultative aerobic dehydrogenase. Catalytic amounts of PMS stimulated enzyme-dependent oxygen uptake and DCPIP reduction under aerobic and anaerobic conditions. This further suggests that an intermediate carrier, or alternate acceptor, depending on concentration, exists before O₂ in vivo. Naturally occurring quinoid compounds may fulfill such a role, as evidenced by the enhancement of aerobic DCPIP reduction upon addition of catalytic amounts of caffeic and chlorogenic acid. The observation that PMS, caffeic and chlorogenic acid, biopterin, 6-hydroxy-2-amino-4-hydroxypteridine and a quinone extract of N. tabacum quenched the inhibitory effect of blue light on tobacco glycollate oxidase, is in accordance with the possible function of such compounds in glycollate oxidation.Abbreviation DCPIP 2,6-dichlorophenolindophenol - FMN flavin mononucleotide - PMS phenazine methosulphate     

Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in Rhodospirillum rubrum

Zusammenfassung Es wird die 25–30 fache Anreicherung einer löslichen NADH-Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.) aus R. rubrum durch Gelfiltration an Sephadex G 200 und Chromatographie an DEAE-Cellulose beschrieben. Das Enzym ist bei DEAE-Cellulosechromatographie nur in Gegenwart von FMN oder NADH stabil. Menadion, Ferricyanid, DCPIP, p-Benzochinon und Cytochrom c sind als Elektronenacceptoren wirksam. Cytochrom c ist ein schlechter Acceptor. Das pH-Optimum der Reaktion liegt bei 8,4. Km für NADH ist 3,0 · 10^-5 m. NADPH wird nur mit etwa 3–5% des Wertes von NADH umgesetzt. Die prosthetische Gruppe des Enzyms ist FMN, Km für FMN ist 0,3 · 10^-7 m. Das Enzymprotein wird bei Verdünnung in 0,05 m Puffer inaktiviert; FMN und FAD sowie NADH und NADPH haben einen stabilisierenden Effekt. Durch höhere Pufferkonzentrationen wird das Enzym zunehmend inaktiviert. Die Inaktivierung besteht in einer Labilisierung oder Abspaltung der prosthetischen Gruppe vom Enzymmolekül. Verschiedene Metalle inaktivieren das Enzym ebenfalls.

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Oxidation of reduced nicotinamide adenine dinucleotide in Rhodospirillum rubrum I. Characterization of a soluble NADH dehydrogenase

Summary A soluble NADH^* Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.] has been purified 25–30 fold from R. rubrum by gelfiltration with Sephadex G 200 and ionexchange chromatography on DEAE-Cellulose. During the second purification step the enzyme is stable only in the presence of either FMN or NADH. Electronacceptors which were found to be effective include menadione, ferricyanide, DCPIP, p-benzoquinone and cytochrome c, the latter substance being a poor acceptor. The optimum pH of the reaction is at about 8.4. Km for NADH is 3.0×10^-5 m. NADPH is oxidized at only about 3–5% the rate of NADH. The active prosthetic group of the enzyme is FMN with an appearant Km of 0.3×10^-7 m. When diluted in 0.05 m buffer the enzyme becomes rapidly inactivated. FMN, FAD and also NADH and NADPH exhibit a stabilizing protective effect. Higher concentrations of buffer cause increasing inactivation. The mechanism of the inactivation is thought to be a labilization or detachment of the prosthetic group from the enzyme molecule. Several metals also inactivate the enzyme.

     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Redox enzymes in the plant plasma membrane and their possible roles

Purified plasma membrane (PM) vesicles from higher plants contain redox proteins with low‐molecular‐mass prosthetic groups such as flavins (both FMN and FAD), hemes, metals (Cu, Fe and Mn), thiol groups and possibly naphthoquinone (vitamin K₁), all of which are likely to participate in redox processes. A few enzymes have already been identified: Monodehydroascorbate reductase (EC 1.6.5.4) is firmly bound to the cytosolic surface of the PM where it might be involved in keeping both cytosolic and, together with a b‐type cytochrome, apoplastic ascorbate reduced. A malate dehydrogenase (EC 1.1.1.37) is localized on the inner side of the PM. Several NAD(P)H‐quinone oxidoreductases have been purified from the cytocolic surface of the PM, but their function is still unknown. Different forms of nitrate reductase (EC 1.6.6.1–3) are found attached to, as well as anchored in, the PM where they may act as a nitrate sensor and/or contribute to blue‐light perception, although both functions are speculative. Ferric‐chelate‐reducing enzymes (EC 1.6.99.13) are localized and partially characterized on the inner surface of the PM but they may participate only in the reduction of ferric‐chelates in the cytosol. Very recently a ferric‐chelate‐reducing enzyme containing binding sites for FAD, NADPH and hemes has been identified and suggested to be a trans‐PM protein. This enzyme is involved in the reduction of apoplastic iron prior to uptake of Fe²⁺ and is induced by iron deficiency. The presence of an NADPH oxidase, similar to the so‐called respiratory burst oxidase in mammals, is still an open question. An auxin‐stimulated and cyanide‐insensitive NADH oxidase (possibly a protein disulphide reductase) has been characterized but its identity is still awaiting independent confirmation. Finally, the only trans‐PM redox protein which has been partially purified from plant PM so far is a high‐potential and ascorbate‐reducible b‐type cytochrome. In co‐operation with vitamin K₁ and an NAD(P)H‐quinone oxidoreductase, it may participate in trans‐PM electron transport.     

Microbial oxidation of methane and methanol: Purification and properties of a heme-containing aldehyde dehydrogenase from Methylomonas methylovora

Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methylotroph, Methylomonas methylovora are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. In the presence of an artificial electron acceptor (phenazine methosulfate), the purified enzyme catalyzes the oxidation of straight chain aldehydes (C₁-C₁₀ tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD⁺, NADP⁺, FAD, FMN, pyridoxal phosphate, and cytochrome c cannot act as electron carriers. The activity of the enzyme is inhibited by sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide and 5,5-dithiobis (2-nitrobenzoic acid)], cuprous chloride, and ferrour nitrate. The molecular weight of the enzyme as estimated by gel filtration is approximately 45000 and the subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 23000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of enzyme with sodium dithionite or aldehyde substrate resulted in the appearance of peaks at 523 nm and 552 nm. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as prosthetic group. The amino acid composition of the enzyme is also presented.Non-Standard Abbreviations PMS phenazine methosulfate - DCPIP 2,6-dichlorophenol indophenol - DEAE diethylaminoethyl     

Characterization of a new mesophilic,secondary alcohol-utilizing methanogen,Methanobacterium palustre spec. nov. from a peat bog

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H₂/CO₂ and formate in complex medium. It also grew autotrophically on H₂/CO₂. Furthermore, growth on 2-propanol/CO₂ was observed. Methane was formed from CO₂ by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO₂ apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable.On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum.An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP⁺ was the only electron carrier that was utilized. No reaction was found with NAD⁺, F₄₂₀, FMN and FAD.Abbreviations NAD⁺ nicotinamide adenine dinucleotide - NADH₂ reduced form of NAD⁺ - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH₂ reduced form of NADP⁺ - FMN flavin adenine mononucleotide - FAD flavin adenine dinucleotide - ADH alcohol dehydrogenase - F₄₂₀ 8-hydroxy-7,8-didemethyl-5-deazaflavin - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.5)     

Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q₁. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.     

Eigenschaften der NAD-spezifischen Hydrogenase aus Hydrogenomonas H 16

Zusammenfassung Aus zellfreiem Extrakt von Hydrogenomonas H 16 wurde die lösliche Hydrogenase 45 fach bis zu einer spezifischen Aktivität von 36500 E/g Protein angereichert. Das Enzym katalysiert die Reduktion von NAD mit molekularem Wasserstoff. Ein Cofaktorbedürfnis konnte nicht festgestellt werden. Der Einfluß von NADH, ATP, Bicarbonat und Magnesium auf die hydrogenasekatalysierte NAD-Reduktion war unerheblich. Das angereicherte Enzym ist flavin- und pyridinnucleotidfrei und reagiert mit NAD, nicht mit O₂, NADP, FMN, FAD oder Methylenblau. Die drei letztgenannten Wasserstoff-Acceptoren werden lediglich in Gegenwart katalytischer Mengen NAD reduziert. Die lag-Phase der Reduktion von NAD läßt sich durch Vorinkubation des Enzyms mit NADH oder Wasserstoff, nicht jedoch mit NAD eliminieren. Die Hemmung der Hydrogenasereaktion durch Sauerstoff ist gering. Reduzierende Agenzien wie Mercaptoäthanol oder Sulfid setzten die Reaktionsrate herab.Die Michaeliskonstante der löslichen Hydrogenase für molekularen Wasserstoff beträgt K _m ^H2 =1,9·10^–4 M. Die NAD-Konzentration, bei der halbmaximale Aktivität erreicht wird, beträgt [NAD]_{0,5 (V)}=1,3·10^–4 M. Das pH-Optimum wird in 0,05 M Kaliumphosphat-Puffer bei pH 8,5 und in 0,05 M Tris-HCl-Puffer bei pH 7,9 erreicht. Unter den angegebenen Bedingungen lag das Temperatur-Optimum bei 36°C. Die Aktivierungsenergie der löslichen Hydrogenase wurde als 10,4 kcal/mol ermittelt.

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Properties of the NAD-Specific Hydrogenase from Hydrogenomonas H 16

Summary A soluble hydrogenase from cell-free extracts of Hydrogenomonas H 16 has been purified 45-fold up to a specific activity of 36,500 units per g protein. The enzyme catalyzes the reduction of NAD with molecular hydrogen. It does not require cofactors. The NAD-reduction catalyzed by this enzyme is influenced to only a small extent by the presence of NADH, ATP, bicarbonate or magnesium ions. The enzyme is free from flavins and pyridine nucleotides, reacts only with NAD and not with oxygen, NADP, FMN, FAD or methylene blue. FMN, FAD and methylene blue are reduced only in the presence of catalytic amounts of NAD. The lag-phase of the reduction of NAD can be eliminated by preincubating the enzyme in the presence of NADH or molecular hydrogen; NAD is ineffective. The inhibition of the hydrogenase reaction by oxygen is negligible. Reducing agents such as mercaptoethanol or sulfide decreased the reaction rate.The Michaelis constant of the soluble hydrogenase for molecular hydrogen is K _m ^H2 =1.9·10^–4 M. Half maximal activity is attained at a NAD-concentration of [NAD]_{0.5 (V)}=1.3·10^–4 M. The pH-optimum is 8.5 in 0.05 M potassium phosphate buffer and 7.9 in 0.05 M Tris-HCl-buffer; reaction rates were maximal at 36°C under the conditions employed. The activation energy was calculated to be 10.4 kcal/mol.

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Abkürzungen E Enzymeinheit (mole Substrat/min) - E _x Extinktionsänderung bei der Wellenlänge x nm - Ext._x Extinktion bei der Wellenlänge x nm - KP Kaliumphosphat (KH₂PO₄-K₂HPO₄-Gemisch) - MB Methylenblau - NAD Nicotinamid-adenin-dinucleotid - NADH reduziertes - NAD; TEAE Triäthylaminoäthyl - Tris Tris(hydroxymethyl)aminomethan     

Evidence for a tungsten-stimulated aldehyde dehydrogenase activity of Desulfovibrio simplex that oxidizes aliphatic and aromatic aldehydes with flavins as coenzymes

The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts. Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor. A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 μM WO₄ ^2–. The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (K _m < 20 μM) and aromatic aldehydes (K _m < 0.32 mM) using methyl viologen as the electron acceptor. Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD⁺ or NADP⁺. ¹⁸⁵WO₄ ^2– was incorporated in vivo into D. simplex; it was found exclusively in the soluble fraction (≥ 98%). Anionic-exchange chromatography demonstrated coelution of ¹⁸⁵W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity. Received: 7 February 1997 / Accepted: 6 June 1997     

Formate dehydrogenase from the methane oxidizer Methylosinus trichosporium OB3b.

Formate dehydrogenase (NAD+ dependent) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isolated anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 cellulose and Sephacryl S-300 columns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the alpha-subunit was 53,800 +/- 2,800, and that of the beta-subunit was 102,600 +/- 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent alpha 2 beta 2 arrangement. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were detected. No other metals were found in stoichiometric amounts. When the enzyme was isolated aerobically, there was no cofactor requirement for NAD reduction; however, when isolated anaerobically, activity was 80 to 90% dependent on the addition of flavin mononucleotide (FMN) to the reaction mixture. Furthermore, the addition of formate to an active, anoxic solution of formate dehydrogenase rapidly inactivated it in the absence of an electron acceptor; this activity could be reconstituted approximately 85% by 50 nM FMN. Flavin adenine dinucleotide could not replace FMN in reconstituting enzyme activity. The Kms of formate dehydrogenase for formate, NAD, and FMN were 146, 200, and 0.02 microM, respectively. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inactivated under anoxic conditions by formate and reactivated by FMN. The evolutionary significance of this similarity is discussed.     

The stimulation of microsomal azoreduction by flavins

The reduction of the azo dye, amaranth, by rat liver microsomes is inhibited about 90% by carbon monoxide, suggesting that the reaction largely depends on cytochrome P-450. Reducing equivalents for this reaction are supplied by NADPH. This reaction is stimulated by riboflavin, FMN and FAD, as well as by methylviologen. A large fraction of the stimulated reaction is not blocked by CO, indicating that there is a pathway of electron transfer which is dependent of cytochrome P-450. Rat liver microsomes can reduce FAD, with reducing equivalents supplied by NADPH. The FADH₂ thus produced is quickly oxidized by amaranth so that two FADH₂ are oxidized for every amaranth reduced. The same stoichiometry is observed with photochemically prepared FADH₂, formed in the absence of microsomes.     

Untersuchungen zum Cytochrom-Oxydase-System aus anaerob im Licht und aerob im Dunkeln gewachsenen Zellen von Rhodopseudomonas capsulata

Zusammenfassung Das aerobe Oxydase-System aus aerob im Dunkeln und anaerob im Licht gewachsenen Zellen von Rps. capsulata wurde untersucht. Die aus Ultraschallextrakten durch 140 000 g-Zentrifugation gewonnen Partikelfraktion katalysiert die Oxydation von NADH, Succinat und reduziertem Cytochrom c (aus Pferdeherz). Die Oxydase-Aktivitäten der Partikel aus aerob im Dunkeln gewachsenen Zellen variieren von 0,10–0,38 mole O₂/min · mg Protein und sind im Durchschnitt 10mal höher als die Oxydase-Aktivitäten der Partikel aus anaerob im Licht gewachsenen Zellen. Die Partikel enthalten Cytochrom vom b-Typ und c-Typ. Cytochrom a konnte weder in den Partikeln aus anaerob im Licht noch in den Partikeln aus aerob im Dunkeln gewachsenen Zellen nachgewiesen werden. In den Partikeln aus aerob gewachsenen Zellen ist das Verhältnis Cytochrom b:Cytochrom c größer als in den Partikeln aus anaerob im Licht gewachsenen Zellen. Die Cytochromoxydase reagiert mit Cytochrom c (aus Pferdeherz), DCPIP und TMPD. Die entsprechenden k _m -Werte betragen 5 · 10^-5 m, 1,5 · 10^-4 m und 2 · 10^-4 m. Die Cytochromoxydase hat ein breites pH-Optimum zwischen pH 8,5 und 9,5. Sättigung der Oxydase mit O₂ tritt erst bei einem Partialdruck von 15 bis 20% O₂ ein. Die Oxydase wird durch KCN und NaN₃ (50% Hemmung bei 10^-5 m), nicht aber durch CO gehemmt. Die Partikel aus aerob im Dunkeln und anaerob im Licht gewachsenen Zellen katalysieren eine mit der Succinat-Oxydation einhergehende Phosphorylierung von ADP mit P/O-Werten von maximal 0,3.

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Studies on the cytochrome oxidase system of ligh-anaerobically and dark-aerobically grown cells of Rhodopseudomonas capsulata

Summary The aerobic oxidase-system from dark-aerobically and light-anaerobically grown Rps. capsulata was investigated. The particulate fraction sedimented from ultrasonic extracts by 140,000 g-centrifugation, catalyzed the oxidation of NADH, succinate and reduced cytochrome c (horse heart). The oxidase activities of the particles from dark-aerobically grown cells were in the range of 0.10–0.38 moles O₂/min x mg protein and were usually ten times as high as the oxidase activities from light-grown cells. The particles contain cytochromes of b-type and c-type. Cytochrome of a-type could be detected neither in the particles from light-grown nor in the particles from dark-grown cells. The highest values of the relation between cytochrome b and cytochrome c were found in the particles from darkaerobically grown cells. The cytochrome oxidase reacts with cytochrome c (horse heart), DCPIP and TMPD. The k _m -values are 5×10^-5 m, 1.5×10^-4 m, or 2×10^-4 m, respectively. The cytochrome oxidase exhibits a broad pH-optimum in the range of pH 8.5–9.5. Saturation of the oxidase with O₂ is observed at a partial pressure of 15–20% O₂. The oxidase is inhibited by KCN and NaN₃ (half inhibition at 10^-5 m), but not by CO. The particles from dark-aerobically and light-anaerobically grown cells catalyze phosphorylation of ADP in the dark coupled to the oxidation of succinate with maximum P/O-values of 0.3.

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Abkürzungen ADP Adenosindiphosphat - ATP Adenosintriphosphat - BChl Bacteriochlorophyll - CCCP Carbonylcyanid-m-chlorphenylhydrazon - DCPIP 2,6-Dichlorphenolindophenol - DNP 2,4-Dinitrophenol - NAD(P) Nicotinamid-Adenin-Dinucleotid(phosphat) - NAD(P)H reduziertes NAD(P) - R. Rhodospirillum - Rps. Rhodopseudomonas - TMPD N-Tetramethyl-p-phenylendiamin     

Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus

Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)⁺ with the concurrent reduction of O₂ to H₂O₂, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and ¹⁵N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity.