Coenzyme F420 dependent N5, N10-methylenetetrahydromethanopterin dehydrogenase in methanol grown Methanosarcina barkeri

Enßle, M.; Zirngibl, C.; Linder, Dietmar; Thauer, Rudolf K.

doi:10.1007/bf00244966

Cited by 31 publications

(18 citation statements)

References 39 publications

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“…The curve‐fitting data were in agreement with those from Lineweaver–Burk plots (Supporting Information http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12475/suppinfo). The V max was slightly lower than that of the organisms listed in Table , but its K m value was similar or even lower than those given for the various organisms in Table (Enßle et al ., ; Klein et al ., ; Schwörer et al ., ). When methylenetetrahydrofolate (functionally and structurally similar to methylene‐H 4 MPT) was used instead of methylene‐H 4 MPT, the specific activity was less than 0.01 U mg −1 .…”

Section: Resultsmentioning

confidence: 99%

A reversed genetic approach reveals the coenzyme specificity and other catalytic properties of three enzymes putatively involved in anaerobic oxidation of methane with sulfate

Kojima

Moll

Kahnt

et al. 2014

Environmental Microbiology

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Consortia of anaerobic methanotrophic (ANME) archaea and delta-proteobacteria anaerobically oxidize methane coupled to sulfate reduction to sulfide. The metagenome of ANME-1 archaea contains genes homologous to genes otherwise only found in methanogenic archaea, and transcription of some of these genes in ANME-1 cells has been shown. We now have heterologously expressed three of these genes in Escherichia coli, namely those homologous to genes for formylmethanofuran : tetrahydromethanopterin formyltransferase, methenyltetrahydromethanopterin cyclohydrolase (Mch) and coenzyme F420 -dependent methylenetetrahydromethanopterin dehydrogenase (Mtd), and have characterized the overproduced enzymes with respect to their coenzyme specificity and other catalytic properties. The three enzymes from ANME-1 were found to catalyse the same reactions and with similar specific activities using identical coenzymes as the respective enzymes in methanogenic archaea, the apparent Km for their substrates being in the same concentration range. The results support the proposal that anaerobic oxidation of methane to CO₂in ANME involves the same enzymes and coenzymes as CO₂reduction to methane in methanogenic archaea. Interestingly, the activity of Mch and the stability of Mtd from ANME-1 were found to be dependent on the presence of 0.5-1.0 M potassium phosphate, which suggested that ANME-1 archaea contain high concentrations of lyotropic salts, presumably as compatible solutes.

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Section: Resultsmentioning

confidence: 99%

A reversed genetic approach reveals the coenzyme specificity and other catalytic properties of three enzymes putatively involved in anaerobic oxidation of methane with sulfate

Kojima

Moll

Kahnt

et al. 2014

Environmental Microbiology

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“…Reactions (2-4) are reversible (Thauer, 1998). The enzymes involved display high turnover numbers (k cat ) and each represents as much as 0?5-1 % of the total cellular protein (Enßle et al, 1991;Ma & Thauer, 1990;Schwörer & Thauer, 1991;te Brömmelstroet et al, 1990te Brömmelstroet et al, , 1991a. Thus, the catalytic capacities of the hydrogenase, dehydrogenase and reductase substantially exceed the specific rate of methane formation.…”

Section: Introductionmentioning

confidence: 99%

“…However, methanol-grown cells contain high levels of F 420 -reducing hydrogenase (Michel et al, 1995), whilst F 420 serves as the electron carrier in two reactions of the methyl group-oxidation pathway, notably N 5 -methyl-H 4 MPT and N 5 ,N 10 -methylene-H 4 MPT oxidation (reversed reactions 3 and 4) (Enßle et al, 1991;Schwörer & Thauer, 1991;te Brömmelstroet et al, 1991a;Thauer, 1998). During growth on methanol, the compound serves as both the energy and carbon source.…”

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confidence: 99%

Hydrogen concentrations in methane-forming cells probed by the ratios of reduced and oxidized coenzyme F420

2005

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Coenzyme F 420 is the central low-redox-potential electron carrier in methanogenic metabolism. The coenzyme is reduced under hydrogen by the action of F 420 -dependent hydrogenase. The standard free-energy change at pH 7 of F 420 reduction was determined to be "15 kJ mol "1 , irrespective of the temperature (25-65 6C). Experiments performed with methane-forming cell suspensions of Methanothermobacter thermautotrophicus incubated under various conditions demonstrated that the ratios of reduced and oxidized F 420 were in thermodynamic equilibrium with the gas-phase hydrogen partial pressures. During growth in a fed-batch fermenter, ratios changed in connection with the decrease in dissolved hydrogen. For most of the time, the changes were as expected for thermodynamic equilibrium between the oxidation state of F 420 inside the cells and extracellular hydrogen. Also, methanol-metabolizing, but not acetate-converting, cells of Methanosarcina barkeri maintained the ratios of reduced and oxidized coenzyme F 420 in thermodynamic equilibrium with external hydrogen. The results of the study demonstrate that F 420 is a useful probe to assess in situ hydrogen concentrations in H 2 -metabolizing methanogens.

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“…From the linear portion of the Arrhenius plot (20 to 70°C), a value of 37.6 kJ/mol was obtained for the activation energy. A thermophilic nature is also typical for several well-characterized methanogenic enzymes from mesophilic Methanosarcina (17,21,23,26,40), indicating a possible thermophilic ancestry for this organism. At low concentrations, KCl stimulated MsbPYC, and at high concentrations, it was inhibitory; the maximum specific activity was obtained at 0.25 M KCl.…”

Section: Synthesis Of Biotin and A Biotinylated Proteinmentioning

confidence: 84%

Oxaloacetate Synthesis in the Methanarchaeon Methanosarcina barkeri : Pyruvate Carboxylase Genes and a Putative Escherichia coli -Type Bifunctional Biotin Protein Ligase Gene ( bpl/birA ) Exhibit a Unique Organization

et al. 2001

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Evidence is presented that, in Methanosarcina barkeri oxaloacetate synthesis, an essential and major CO 2 fixation reaction is catalyzed by an apparent ␣ 4 ␤ 4 -type acetyl coenzyme A-independent pyruvate carboxylase (PYC), composed of 64.2-kDa biotinylated and 52.9-kDa ATP-binding subunits. The purified enzyme was most active at 70°C, insensitive to aspartate and glutamate, mildly inhibited by ␣-ketoglutarate, and severely inhibited by ATP, ADP, and excess Mg 2؉ . It showed negative cooperativity towards bicarbonate at 70°C but not at 37°C. The organism expressed holo-PYC without an external supply of biotin and, thus, synthesized biotin. pycA, pycB, and a putative bpl gene formed a novel operon-like arrangement. Unlike other archaeal homologs, the putative biotin protein ligases (BPLs) of M. barkeri and the closely related euryarchaeon Archaeoglobus fulgidus appeared to be of the Escherichia coli-type (bifunctional, with two activities: BirA or a repressor of the biotin operon and BPL). We found the element Tyr(Phe)ProX 5 Phe(Tyr) to be fully conserved in biotindependent enzymes; it might function as the hinge for their "swinging arms."The oxaloacetate (OAA) synthesis step is an essential physiological component and a major CO 2 fixation site in a methanarchaeon (38), for it primes therein an incomplete tricarboxylic acid cycle reaction sequence that generates intermediates for the synthesis of amino acids (via OAA, ␣-ketoglutarate [␣-KG], and succinate) and tetrapyrroles (via ␣-KG) (38). Methanococcus jannaschii and Methanococcus maripaludis use a pyruvate carboxylase (PYC) for OAA synthesis (28, 37). Methanobacterium thermoautotrophicum possesses two OAA-generating enzymes: PYC and phosphoenolpyruvate carboxylase (PPC) (18,22,31). The PYC and PPC reactions take the following forms:The avenue for OAA biosynthesis in Methanosarcina species is unknown. Weimer and Zeikus (42) showed that Methanosarcina barkeri is devoid of PPC activity. We describe below PYC as an OAA-synthesizing activity in M. barkeri and present a biochemical and molecular genetic characterization of the enzyme. (Fig. 1A). Hence, the organism was capable of synthesizing biotin and of biotinylating a candidate polypeptide without any external source of biotin. The intense band at ϳ65 kDa was typical of the biotin-carrying subunit (PYCB) of an arcaheal PYC (28, 31). Thus, we purified and characterized the corresponding protein. As shown below, it possessed PYC activity and the above-mentioned avidinreacting ϳ65-kDa band indeed corresponded to the PYCB subunit of the enzyme (Fig. 1B and C). For the cells grown with a supply of biotin, the relative intensity of the ϳ65-kDa band increased. Thus, M. barkeri exhibited a less stringent version of the control on PYC synthesis seen in Methanobacterium thermoautotrophicum (31); the latter requires exogenously supplied biotin to express holo-PYC (31), although it makes biotin (33), and a similar phenomenon exists in several bacteria (reviewed in reference 31). Whether other bands in the blot (Fig. 1A) r...

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Coenzyme F420 dependent N5, N10-methylenetetrahydromethanopterin dehydrogenase in methanol grown Methanosarcina barkeri

Cited by 31 publications

References 39 publications

A reversed genetic approach reveals the coenzyme specificity and other catalytic properties of three enzymes putatively involved in anaerobic oxidation of methane with sulfate

A reversed genetic approach reveals the coenzyme specificity and other catalytic properties of three enzymes putatively involved in anaerobic oxidation of methane with sulfate

Hydrogen concentrations in methane-forming cells probed by the ratios of reduced and oxidized coenzyme F420

Oxaloacetate Synthesis in the Methanarchaeon Methanosarcina barkeri : Pyruvate Carboxylase Genes and a Putative Escherichia coli -Type Bifunctional Biotin Protein Ligase Gene ( bpl/birA ) Exhibit a Unique Organization

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