Showing papers on "Methanosarcina barkeri published in 2001"

A paramagnetic species with unique EPR characteristics in the active site of heterodisulfide reductase from methanogenic archaea

Abstract: Heterodisulfide reductase (Hdr) from methanogenic archaea is an iron–sulfur protein that catalyses the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (H-S-CoM) and coenzyme B (H-S-CoB). In EPR spectroscopic studies with the enzyme from Methanothermobacter marburgensis, we have identified a unique paramagnetic species that is formed upon reaction of the oxidized enzyme with H-S-CoM in the absence of H-S-CoB. This paramagnetic species can be reduced in a one-electron step with a midpoint-potential of −185 mV but not further oxidized. A broadening of the EPR signal in the 57Fe-enriched enzyme indicates that it is at least partially iron based. The g values (gxyz= 2.013, 1.991 and 1.938) and the midpoint potential argue against a conventional [2Fe−2S]+, [3Fe−4S]+, [4Fe−4S]+ or [4Fe−4S]3+ cluster. This species reacts with H-S-CoB to form an EPR silent form. Hence, we propose that only a half reaction is catalysed in the presence of H-S-CoM and that a reaction intermediate is trapped. This reaction intermediate is thought to be a [4Fe−4S]3+ cluster that is coordinated by one of the cysteines of a nearby active-site disulfide or by the sulfur of H-S-CoM. A paramagnetic species with similar EPR properties was also identified in Hdr from Methanosarcina barkeri.

Microbial growth by a net heat up-take: a calorimetric and thermodynamic study on acetotrophic methanogenesis by Methanosarcina barkeri.

Abstract: To answer the intriguing question whether or not endothermic microbial growth exists, and in particular, to verify Heijnen and van Dijken's prediction (1992), acetotrophic methanogen, Methanosarcina barkeri, has been cultivated in a highly sensitive bench-scale calorimeter (an improved Bio-RC1 reaction calorimeter) in a pH auxostat fashion. A growth yield of 0.043 C-mol C-mol(-1) has been obtained and a cell density as high as 3 g L(-1) was attained. Heat uptake during growth has indeed been quantitatively measured with calorimetry, resulting in a heat yield of +145 kJ C-mol(-1). Thermodynamics of the growth of acetotrophic methanogens was analyzed in detail. The changes in Gibbs energy, enthalpy, and entropy during growth of M. barkeri were compared with some typical aerobic and anaerobic growth processes of different microorganisms on various substrates. In the growth of M. barkeri on acetate, the retarding effect of the positive enthalpy change on the driving force of growth is overcompensated by the large positive entropy change, resulting from converting one organic molecule (acetic acid) to two gaseous products, CH(4) and CO(2). Both the enthalpy and the entropy increases are due partially to the transition of these two products into the gaseous phase. The thermodynamic role of this phase transition for the growth process is analyzed. Microbial growth characterized by enthalpy increase and correspondingly by a large increase in entropy may be called enthalpy-retarded growth.

Characterization of a heme-dependent catalase from Methanobrevibacter arboriphilus.

Abstract: Recently it was reported that methanogens of the genus Methanobrevibacter exhibit catalase activity. This was surprising, since Methanobrevibacter species belong to the order Methanobacteriales, which are known not to contain cytochromes and to lack the ability to synthesize heme. We report here that Methanobrevibacter arboriphilus strains AZ and DH1 contained catalase activity only when the growth medium was supplemented with hemin. The heme catalase was purified and characterized, and the encoding gene was cloned. The amino acid sequence of the catalase from the methanogens is most similar to that of Methanosarcina barkeri.

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.

Abstract: Evidence is presented that, in Methanosarcina barkeri oxaloacetate synthesis, an essential and major CO2 fixation reaction is catalyzed by an apparent α4β4-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 α-ketoglutarate, and severely inhibited by ATP, ADP, and excess Mg2+. 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)ProX5Phe(Tyr) to be fully conserved in biotin-dependent enzymes; it might function as the hinge for their “swinging arms.”

Methanococcus jannaschii generates L-proline by cyclization of L-ornithine.

Abstract: The established pathway for proline biosynthesis in microorganisms is shown in the upper portion of Fig. ​Fig.1.1. The reaction sequence involves (i) the phosphorylation of the δ-carboxyl of l-glutamate to form l-glutamyl-5-P, (ii) the NADH-dependent reduction of l-glutamyl-5-P to glutamic acid-γ-semialdehyde, (iii) the cyclization of glutamate-γ-semialdehyde to Δ1-pyrroline-5-carboxylic acid, and (iv) the reduction of Δ1-pyrroline-5-carboxylic acid to l-proline (2, 5, 13). As first pointed out by Selkov et al. for Methanococcus jannaschii (10), and later as a general characteristic of the genomes of most of the Archaea, genes coding for the three enzymes in this pathway are largely absent in the Archaea (4). In contrast, the genes for the biosynthesis of l-ornithine are generally present in all of the archaeal genomes (4). A simple solution to explain the absence of the proline biosynthetic genes in the genomes of some of the Archaea is that proline is derived by the cyclization of l-ornithine. This reaction could be accomplished by ornithine cyclodeaminase (EC 4.3.1.12), an enzyme which is presently considered to have a limited distribution among the bacteria (12). Ornithine cyclodeaminase was first isolated from Clostridium sporogenes, where it functions as the first step in the anaerobic catabolism of l-ornithine via proline to δ-aminovaleric acid (1). The proposed chemical steps for the mechanism of this enzyme involve the oxidative deamination of the α-amino group of ornithine to 2-oxo-5-aminopentanoic acid, which cyclizes to Δ1-pyrroline-2-carboxylic acid, which is subsequently reduced to l-proline. The ornithine cyclodeaminase has been shown to contain 1 mol equivalent of bound NAD+, which is considered to function as a recycling redox carrier in this transformation (7). Mass spectroscopic data showed that [5-15N]ornithine is converted to [15N]proline by ornithine cyclodeaminase, confirming that the initial oxidation of the ornithine is at C-2 (8). Using deuterated ornithine and [15N]ornithine, we have now demonstrated that proline in M. jannaschii is derived from ornithine by a mechanism that is analogous to that demonstrated by the ornithine cyclodeaminase. Since there is no enzyme-encoding gene with a sequence homologous to that coding for the ornithine cyclodeaminase present in the M. jannaschii genome, we propose that a currently unidentified enzyme, functioning with an analogous mechanism, is involved in proline biosynthesis in M. jannaschii. FIG. 1 Established and proposed pathways for proline biosynthesis. Preparation and analysis of cell extracts. Cell extracts of M. jannaschii, Methanosarcina thermophila strain TM-1, and Methanobacterium thermoautotrophicum strains ΔH and Marburg were prepared as previously described (16). The protein concentrations of the cell extracts used typically ranged from 7 to 26 mg/ml. Incubation with substrates. Cell extracts (50 μl) were incubated with millimolar concentrations of the substrates under argon for 2 h at 50°C. l-[2,4,4′-2H3]glutamic acid and l-[3,3′,4,4′,5′-2H6]ornithine were obtained from Cambridge Isotope Laboratories, Inc. [5-15N]ornithine was prepared from potassium [15N]-phthalimide by the following series of reactions. Potassium [15N]-phthalimide (98 atom% 15N) was reacted with dibromopropane in acetone to form 15N-labeled N-(3-bromopropyl)phthalimide (14), which was condensed with ethyl acetamidocyanoacetate in ethanol in the presence of sodium ethoxide. Acid hydrolysis of the condensation product (6 M HCl, 24 h, 110°C) and separation of the resulting products on a Dowex 50-8X (H+) column with an HCl gradient resulted in the isolation of chromatographically pure [5-15N]ornithine. Δ1-Pyrroline-5-carboxylic acid was prepared from the 2,4-dinitrophenylhydrazine derivative as previously described (6). After incubation, 0.1 M HCl in methanol (200 μl) was added, followed by centrifugation (10 min, 14,000 × g) to remove the precipitated proteins. The resulting clear liquid was evaporated to dryness with a stream of nitrogen gas, and the free amino acids contained within were converted into the methyl ester trifluoroacetyl derivatives and analyzed by gas chromatography-mass spectrometry (GC-MS) as previously described (17). Quantitation of proline and ornithine was determined from the areas of the intensities of their m/z 166 ions, or the m/z 167 or m/z 172 ions for the labeled prolines, using known mixtures of proline and ornithine for calibration. For samples not containing ornithine, the m/z 211 ion from the β-glutamate present in the M. jannaschii cell extracts (9) was used as an internal standard. The establishment of the product of the incubation as l-proline was accomplished by GC-MS of the methyl ester trifluoroacetyl derivative using a type G-TA Chiraldex column as previously described (3). As can be seen from the data presented in Table ​Table1,1, incubation of a cell extract of M. jannaschii with [2,4,4′-2H3]glutamic acid, ATP, NADH, and NADPH produced no detectable amount of labeled proline. Likewise, incubation with Δ1-pyrroline-5-carboxylic acid, NADH, and NADPH at the same concentrations failed to produce any detectable amount of proline (data not shown). Incubation of the cell extract with l-ornithine (9.1 mM) resulted in the production of 0.13 μmol of proline, which corresponded to the conversion of 26% of ornithine to proline. To confirm that ornithine was the sole precursor of the proline, we incubated the cell extracts with l-[3,3′,4,4′,5,5′-2H6]ornithine and measured the incorporation of six deuteriums in the generated proline, which indicated that the carbon skeleton of the proline was derived from the ornithine as an intact unit. To establish which of the nitrogens was lost in the cyclization, the experiment was repeated with [5-15N]ornithine, and the recovered proline contained 98% 15N. This result showed that the C-2 nitrogen was the one lost in the cyclization. Since the direct displacement of the C-2 amino group by the C-5 amino group is without enzymatic or chemical precedent, the most likely chemical steps for the reaction would involve the oxidation of the C-2 carbon to an imine, intramolecular cyclic addition of the C-5 nitrogen to the C-2 carbon, loss of ammonia, and reduction of the resulting imine, Δ1-pyrroline-2-carboxylic acid, as shown in lower portion of Fig. ​Fig.1.1. Alternately, the imine intermediate generated in the first oxidation could undergo hydrolysis to the keto acid before the cyclization would occur. In this case, elimination of water would produce the Δ1-pyrroline-2-carboxylic acid. Although not directly confirmed by the data, the likely choice for the coenzyme to be involved in this process would be an enzyme-bound NAD+ as occurs in ornithine cyclohydrolase. The possible involvement of coenzyme F420 must also be considered since coenzyme F420 can also effect hydride transfer reactions (15). TABLE 1 Proline formation in cell extracts of M. jannaschii Evidence supporting the involvement of either NAD+ or F420 in the reaction comes from the experiment demonstrating that the hydrogen removed during the C-2 oxidation is reincorporated at C-2 in the proline product during the reduction. Thus, the incubation of a cell extract containing 40% deuterated water with ornithine produced proline with no deuterium (Table ​(Table1,1, data for precursor 5). The conclusion from this experiment is that the hydrogen, which is removed from C-2, is not mixed with the solvent and is the same hydrogen that is incorporated in the reduction. These data are consistent with the idea that the enzyme in M. jannaschii functions with a mechanism analogous to that of ornithine cyclodeaminase from C. sporogenes. Evidence supporting the involvement of NAD+ was the observation that NADH was found to inhibit the conversion presumably by competing with the required NAD+ (data not shown). Similar data were also obtained using cell extracts of M. thermoautotrophicum strain ΔH and strain Marburg, indicating that these autotrophs generate their proline by the same mechanism as that found in M. jannaschii. Although no gene coding for ornithine cyclodeaminase can be found in the M. jannaschii genome, a gene homologous to one coding for C. sporogenes ornithine cyclodeaminase is present in the M. thermoautotrophicum ΔH (11) and Methanosarcina barkeri (http://www.jgi.doe.gov) genomes. Cell extracts of M. thermophila strain TM-1, on the other hand, were found to produce no proline from ornithine. The M. barkeri genome also has the genes for the biosynthesis of proline from glutamate via Δ1-pyrroline-5-carboxylic acid. These genomic data indicate that several different routes may be operating in the Archaea for the biosynthesis of proline. Although our data cannot rule out the possibility that additional pathways to proline function in vivo, this seems unlikely due to the efficient conversions from ornithine that were observed in vitro. In total, these observations show that an enzyme with no homology to the known ornithine cyclodeaminases is involved in proline biosynthesis in M. jannaschii. We are presently in the process of isolating the enzyme responsible for carrying out the reaction demonstrated here in order to establish the gene required for its production.

Zinc-Thiolate Intermediate in Catalysis of Methyl Group Transfer in 'Methanosarcina barkeri'

Abstract: Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkerigrowing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 10 13.7 at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn 2+ . Reconstitution with Co 2+ yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co 2+ -MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn 2+ -MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV- visible spectroscopy of Co 2+ -MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metal- thiolate formation occurs separately from H + release from the enzyme-substrate complex. Proton release to the solvent takes place from a group with apparent pKa of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a ¢G°' of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.