The Active Species of ‘CO<sub>2</sub>’ Utilized by Formylmethanofuran Dehydrogenase from Methanogenic Archaea

Vorholt, Julia A.; Thauer, Rudolf K.

doi:10.1111/j.1432-1033.1997.00919.x

Cited by 61 publications

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“…If the d 13 C of the dissolved CO 2 were used instead, there would have been only a small effect, i.e., resulting in 1% smaller e CO 2 ,CH 4 values. Methanogenic archaea (also homoacetogenic bacteria) are using CO 2 rather than bicarbonate as active species when reducing it to CH 4 (Vorholt and Thauer 1997). Therefore, it is not necessary to consider the d 13 C of bicarbonate.…”

Section: Discussionmentioning

confidence: 99%

Methanogenic pathway, ¹³C isotope fractionation, and archaeal community composition in the sediment of two clear‐water lakes of Amazonia

Conrad

Klose

Claus

et al. 2010

Limnology & Oceanography

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We studied the methanogenic pathway and archaeal community composition in the sediment of two clearwater lakes, Lake Batata and Lake Mussura, in Amazonia. We measured CH 4 production and d 13 C of CO 2 , CH 4 , and acetate-methyl in the presence and absence of CH 3 F, an inhibitor of acetotrophic methanogenesis. The fractionation factor of methanogenesis from CO 2 was rather high in both lake sediments, which was consistent with the low concentrations of H 2 and the small negative Gibbs free energy of hydrogenotrophic methanogenesis. The d 13 C of acetate-methyl was relatively low compared to that of organic matter and decreased further upon inhibition of acetate consumption by CH 3 F. Collectively, the data possibly suggest involvement of syntrophic acetate oxidation besides acetotrophic methanogenesis. The isotopic data were used to calculate the percent contribution of CO 2 reduction to total methanogenesis, which was rather high (approximately 53-63%). Copy numbers of bacterial and archaeal 16S ribosomal ribonucleic acid (rRNA) genes were about 10-fold higher in Lake Mussura than in Lake Batata, indicating that microbial numbers were not a limiting factor for production rates of CH 4 , which were similar in both lake sediments. The composition of the archaeal community was analyzed by cloning and sequencing of the genes coding for 16S rRNA and methyl coenzyme M reductase (mcrA), demonstrating the presence of acetotrophic Methanosaetaceae and different hydrogenotrophic methanogenic orders (Methanomicrobiales, Methanobacteriales, Methanocellales) in both lake sediments. Although methanogenic communities and pathways were principally comparable to those found in lake sediments of the midlatitudes, there were several particularities, e.g., the possible involvement of syntrophic acetate oxidation.

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Section: Discussionmentioning

confidence: 99%

Methanogenic pathway, ¹³C isotope fractionation, and archaeal community composition in the sediment of two clear‐water lakes of Amazonia

Conrad

Klose

Claus

et al. 2010

Limnology & Oceanography

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“…The cyclohydrolase activity was eluted at an NaCl concentration of 1 M. This procedure achieved 10-fold purification, yielding a cyclohydrolase preparation with a specific activity of 8.5 U : mg 21 which was devoid of formyltransferase activity (see also [5]). …”

Section: Partial Purification Of Mch From M Extorquens Am1mentioning

confidence: 99%

“…As formylH 4 MPT is not readily available, the reaction was coupled to the formation of formylH 4 MPT from methenylH 4 MPT by Mch. The H 4 MPT released during the formyltransferase reaction sponanteously reduces cytochrome c, which was detected by measuring the increase in absorbance at 550 nm (D1 ¼ 21 mM : cm 21 ). The standard assay contained 50 mM Tricine/KOH, pH 8.0, 0.6 M NaCl, 20 mM methenylH 4 MPT, 30 mM MFR, 100 mU purified Mch and 50 mM cytochrome c (horse) (Merck).…”

Section: Determination Of Enzyme Activitiesmentioning

confidence: 99%

“…The reaction was strictly dependent on all of these components ( Table 2). The activity of cytochrome c reduction by the purified Ftr complex in the coupled assay was 60 U : mg 21 . Control measurements with the partially purified Ftr from the mesophilic methanogenic archaeon Ms. barkeri produced in E. coli [9] and Mch from M. extorquens AM1 showed that cytochrome c reduction was dependent on Ftr and not on Orfs1, 2, 3.…”

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confidence: 99%

“…No flavin could be detected in preparations of the complex. In addition, molybdenum, tungsten and vanadium were all undetectable [, 0.02 mol : (mol protein) 21 ]. As mentioned above, Fmd from methanogenic archaea are molybdenum-containing or tungsten-containing iron-sulfur proteins.…”

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Characterization of the formyltransferase from Methylobacterium extorquens AM1

Pomper

Vorholt

2001

European Journal of Biochemistry

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Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylmethanofuran -tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H 4 MPT) from N 5 -formylH 4-MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H 4 MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent K m for formylMFR ¼ 50 mM; apparent K m for H 4 MPT ¼ 30 mM). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of < 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the activesite cofactors molybdenum, molybdopterin and a [4Fe24S] cluster. Cytochrome c was found to be spontaneously reduced by H 4 MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.Keywords: C1 metabolism; methanofuran; methanogenic archaea; methylotrophic bacteria; tetrahydromethanopterin.Oxidation of formaldehyde to CO 2 in Methylobacterium extorquens AM1 was recently found to involve tetrahydromethanopterin (H 4 MPT) and a methanofuran (MFR) analog, coenzymes previously thought to be present only in methanogenic and sulfate-reducing archaea. The following enzymes have been proposed to participate in the metabolic pathway: formaldehyde-activating enzyme (Fae) [1], methyleneH 4 MPT dehydrogenases (MtdA and MtdB) [2,3], and three enzymes with high sequence identity with enzymes involved in methanogenesis: methenylH 4 MPT cyclohydrolase (Mch), formylMFR-H 4 MPT formyltransferase (Ftr), and formylMFR dehydrogenase (Fmd) [4] (Fig. 1).Of these enzymes, Fae, Mtd and Mch have been purified and characterized from M. extorquens AM1 [1 -3,5].In M. extorquens AM1, the genes for Mch, MtdB, and Fae are located in a single gene cluster [4]. This cluster also contains genes encoding polypeptides with sequence identity with Ftr and Fmd subunits (Fi...

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Biocatalytic One‐Carbon Conversion

Ragsdale

2002

Encyclopedia of Catalysis

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The review focuses on the biological machines that oxidize, reduce, and interconvert one‐carbon compounds. The focus is on the unusual cofactors and enzymes in methanogens and acetogens that are involved in anaerobic one‐carbon metabolism, which is key to the globla carbon cycle. A variety of autotrophic anaerobes can fix CO 2 into organic carbon as well as use the reduction of CO 2 as a source of energy. CO dehydrogenase, formate dehydrogenase, and formylmethanofuran dehydrogenase are metalloenzymes that reduce CO 2 to the formate oxidation level (CO, formate, or formylmethyanofuran). At the formate level, the one‐carbon compounds are converted to a cofactor‐bound form, either the formyl‐ or methenyl tetrahydromethanopterin or tetrahydrofolate derivatives, before they undergo further reduction. The next stage, reduction from the formate to the formaldehyde level, is accomplished by either methylenetetrahydrofolate or methylenetetrahydromethopterin dehydrogenase. Metalloenzymes again enter the picture at the methanol oxidation level. Afer reduction of the methylene derivatives to methyltetrahydrofolate or methyltetrahydromethanopterin by a reductase, cobalamin‐dependent methyltransferases attach the methyl group to the cobalt as enzyme‐bound methyl‐cob(III)amide. The most reduced one‐carbon compounds are at the methane level. Reduction of the methyl‐Co(III) derivatives to methane is accomplished by methyl coenzyme M reductase. Alternatively, the methyl group is reduced by acetyl‐CoA synthase to acetyl‐CoA. The oxidation of methane back to CO 2 is initiated by methane monooxygenase, while there are various microbes that can use acetyl‐CoA as an energy source.

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The Active Species of ‘CO₂’ Utilized by Formylmethanofuran Dehydrogenase from Methanogenic Archaea

Cited by 61 publications

References 57 publications

Methanogenic pathway, ¹³C isotope fractionation, and archaeal community composition in the sediment of two clear‐water lakes of Amazonia

Methanogenic pathway, ¹³C isotope fractionation, and archaeal community composition in the sediment of two clear‐water lakes of Amazonia

Characterization of the formyltransferase from Methylobacterium extorquens AM1

Biocatalytic One‐Carbon Conversion

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The Active Species of ‘CO2’ Utilized by Formylmethanofuran Dehydrogenase from Methanogenic Archaea

Cited by 61 publications

References 57 publications

Methanogenic pathway, 13C isotope fractionation, and archaeal community composition in the sediment of two clear‐water lakes of Amazonia

Methanogenic pathway, 13C isotope fractionation, and archaeal community composition in the sediment of two clear‐water lakes of Amazonia

Characterization of the formyltransferase from Methylobacterium extorquens AM1

Biocatalytic One‐Carbon Conversion

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Product

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The Active Species of ‘CO₂’ Utilized by Formylmethanofuran Dehydrogenase from Methanogenic Archaea

Methanogenic pathway, ¹³C isotope fractionation, and archaeal community composition in the sediment of two clear‐water lakes of Amazonia

Methanogenic pathway, ¹³C isotope fractionation, and archaeal community composition in the sediment of two clear‐water lakes of Amazonia