A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F420H2 dehydrogenase from M. mazei Gö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F420H2 was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. TheKm values for 2-hydroxyphenazine and phenazine were 35 and 250 μM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F420-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport of M. mazei Gö1.
The proton translocating electron transport systems (F 420 H 2 :heterodisulfide oxidoreductase and H 2 :heterodisulfide oxidoreductase) of Methanosarcina mazei Go È1 were inhibited by diphenyleneiodonium chloride (DPI) indicated by IC 50 values of 20 nmol DPI´mg ±1 protein and 45 nmol DPI´mg ±1 protein, respectively. These effects are due to a complex interaction of DPI with key enzymes of the electron transport chains. It was found that 2-hydroxyphenazine-dependent reactions as catalyzed by F 420 -nonreducing hydrogenase, F 420 H 2 dehydrogenase and heterodisulfide reductase were inhibited. Interestingly, the H 2 -dependent methylviologen reduction and the heterodisulfide reduction by reduced methylviologen as catalyzed by the hydrogenase and the heterodisulfide reductase present in washed membranes were unaffected by DPI, respectively. Analysis of the redox behavior of membrane-bound cytochromes indicated that DPI inhibited CoB-S-S-CoM-dependent oxidation of reduced cytochromes and H 2 -dependent cytochrome reduction. Membrane-bound and purified F 420 H 2 dehydrogenase were inhibited by DPI irrespectively whether methylviologen + metronidazole or 2-hydroxyphenazine were used as electron acceptors. Detailed examination of 2-hydroxy-phenazine-dependent F 420 H 2 -oxidation revealed that DPI is a competitive inhibitor of the enzyme, indicated by the K m value for 2-hydroxyphenazine, which increased from 35 mm to 100 mm in the presence of DPI. As DPI and phenazines are structurally similar with respect to their planar configuration we assume that the inhibitor is able to bind to positions where interaction between phenazines and components of the electron transport systems take place. Thus, electron transfer from reduced 2-hydroxyphenazine to cytochrome b 2 as part of the heterodisulfide reductase and from H 2 to cytochrome b 1 as subunit of the membranebound hydrogenase is affected in the presence of DPI. In case of the F 420 H 2 dehydrogenase electron transport from FAD or from FeS centers to 2-hydroxyphenazine is inhibited.Keywords: cytochrome, diphenyleneiodonium; heterodisulfide reductase; methanogenic Archaea; Methanosarcina; NADH dehydrogenase; phenazine.The process of methanogenesis as performed by methanogenic archaea is coupled to energy conservation by electron transport phosphorylation [1]. In Methanosarcina mazei Go È1 two membrane-bound electron transport systems have been discovered. The F 420 H 2 :heterodisulfide oxidoreductase is involved in methanol degradation and consists of F 420 H 2 dehydrogenase [2,3] and heterodisulfide reductase [4]. Electron transfer between the enzymes is probably mediated by methanophenazine, a membrane integral electron carrier which was recently isolated from Ms. mazei Go È1 [5]. The F 420 H 2 dehydrogenase catalyzes the oxidation of F 420 H 2 , which is formed during oxidation of one of four methanol molecules. The remaining methyl moities are transferred to 2-mercaptoethanesulfonate (CoM-SH). In the final step of methanogenesis the methyl-S-CoM reductase catalyzes t...
Washed membranes prepared from H2+CO2- or formate-grown cells of Methanococcus voltae catalyzed the oxidation of coenzyme F420H2 and the reduction of the heterodisulfide (CoB-S-S-CoM) of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate, which is the terminal electron acceptor of the methanogenic pathway. The reaction followed a 1:1 stoichiometry according to the equation: F420H2 + COB-S-S-CoM --> F420 + CoM-SH + CoB-SH. These findings indicate that the reaction depends on a membrane-bound F420H2-oxidizing enzyme and on the heterodisulfide reductase, which remains partly membrane-bound after cell lysis. To elucidate the nature of the F420H2-oxidizing protein, washed membranes were solubilized with detergent, and the enzyme was purified by sucrose density centrifugation, anion-exchange chromatography, and gel filtration. Several lines of evidence indicate that F420H2 oxidation is catalyzed by a membrane-associated F420-reducing hydrogenase. The purified protein catalyzed the H2-dependent reduction of methyl viologen and F420. The apparent molecular mass and the subunit composition (43, 37, and 27 kDa) are almost identical to those of the F420-reducing hydrogenase that has already been purified from Mc. voltae. Moreover, the N-terminus of the 37-kDa subunit is identical to the amino acid sequence deduced from the fruG gene of the operon encoding the selenium-containing F420-reducing hydrogenase from Mc. voltae. A distinct F420H2 dehydrogenase, which is present in methylotrophic methanogens, was not found in this organism.
F RPH H P -dependent CoB-S-S-CoM reduction as catalyzed by the F RPH H P :heterodisulfide oxidoreductase from Methanosarcina strains was observed in a defined system containing purified F RPH H P dehydrogenase from Methanosarcina mazei Go ë1, 2-hydroxyphenazine and purified heterodisulfide reductase from Methanosarcina thermophila. The process could be divided into two partial reactions: (1) reducing equivalents from F RPH H P were transferred to 2-hydroxyphenazine by the F RPH H P dehydrogenase with a V m x value of 12 U/mg protein; (2) reduced 2-hydroxyphenazine acted as electron donor for CoB-S-S-CoM reduction as catalyzed by the heterodisulfide reductase. The specific activity was 14^16 U/mg protein at 37³C and 60^70 U/mg protein at 60³C. The partial reactions could be combined in the presence of both enzymes. Under these conditions reduced 2-hydroxyphenazine was rapidly oxidized by the heterodisulfide reductase thereby producing the electron acceptor for the F RPH H P dehydrogenase. Above a concentration of 50 W WM of 2-hydroxyphenazine, the specific activity of the latter enzyme reached the V m x value. When other phenazines or quinone derivatives were used as electron carriers, the activity of F RPH H P -dependent CoB-S-S-CoM reduction was much lower than the rate obtained with 2-hydroxyphenazine. Thus, this water-soluble analogue of methanophenazine best mimics the natural electron acceptor methanophenazine in aqueous systems.z 1998 Federation of European Biochemical Societies.
Resistente Stärke ist der Teil der Stärke, der im Verdauungstrakt gesunder Personen durch Wirtsenzyme nicht abgebaut wird. Nach oraler Aufnahme mit der Nahrung gelangen RS-Präparate in den Dickdarm und werden dort von der intestinalen Mikroflora zu kurzkettigen Fettsäuren (SCFA) fermentiert. SCFAs nehmen für den Erhalt eines gesunden Dickdarmepithels eine herausragende Rolle ein. Ihnen wird eine protektive Rolle bei entzündlichen Darmerkrankungen und Darmkrebs zugeschrieben. Ziel dieses Beitrages ist die Beschreibung der Technologie zur Herstellung einer neuen resistenten Stärke (Neo-Amylose) sowie die Charakterisierung des Produktes.Das Edukt für die Herstellung von Neo-Amylose ist Saccharose. Diese wird in einer biokatalytischen Polymerisation mit Hilfe des Enzyms Amylosucchrase (E.C. 2.4.1.4) aus Neisseria polysaccharea umgesetzt. Das Enzym katalysiert dabei die Spaltung von Saccharose in Glucose und Fructose sowie die Bindung des Glucose-Monomers an das nicht-reduzierende Ende eines alpha-1,4-D-Glucans:
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