Nickel,a component of factor F 430 from Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum, growing on medium supplemented with 2 mol ⁶³NiCl₂/l, was found to take up 1.2 mol ⁶³Ni per g cells (dry weight). More than 70% of the radioisotope was incorporated into a compound, which dissociated from the protein fraction after heat treatment, was soluble in 70% acetone, and could be purified by chromatography on QAE-Sephadex A-25, Sephadex G-25, and DEAE cellulose. The purified ⁶³Ni labelled compound had an absorption spectrum and properties identical to those of factor F ₄₃₀ and is therefore considered to be identical with factor F ₄₃₀.Factor F ₄₃₀, a compound of molecular weight higher than 1000 with an absorbance maximum at 430 nm, has recently been purified from Methanobacterium thermoautotrophicum (Gunsalus and Wolfe, 1978). The structure and function of this compound are not yet known.     

Sirohydrochlorin,a precursor of factor F430 biosynthesis in Methanobacterium thermoautotrophicum

Factor F₄₃₀ is a nickel-containing coenzyme of methanogenic bacteria with porphinoid structure which is derived from uroporphyrinogen III. It is shown that sirohydrochlorin is metabolized by cell free extracts of Methanobacterium thermoautotrophicum to factor F₄₃₀ demonstrating that this compound, or a reduced form of it, is an intermediate in the biosynthesis of F₄₃₀, and not only of vitamin B₁₂ and siroheme.     

Incorporation of 8 succinate per mol nickel into factors F430 by Methanobacterium thermoautotrophicum

Factors F₄₃₀ from methanogenic bacteria have recently been shown to contain nickel and it has been speculated that they may have a nickel tetrapyrrole structure. This assumption was tested by determining whether succinate is incorporated by growing Methanobacterium thermoautotrophicum into three factors F₄₃₀. Succinate is assimilated by Methanobacterium thermoautotrophicum into the amino acids glutamate, arginine and proline and into tetrapyrroles rather than other cell components. It was found that per mol nickel 8–9 mol of succinate were incorporated into the three factors F₄₃₀ which is the amount predicted for a tetrapyrrole structure. Since the three factors F₄₃₀ only contained significant amounts of glutamate rather than arginine or proline, the incorporation data suggest that factors F₄₃₀ are nickel tetrapyrrole compounds. Spectral properties of the three factors F₄₃₀, apparent molecular weights, and the absence of phosphor in these compounds are also described.     

Separation and quantification of cofactors from methanogenic bacteria by high-performance liquid chromatography: optimum and routine analyses

Four assays were developed, employing high-performance liquid chromatography, which gave optimal detection and separation of derivatives of 7-methylpterin, coenzyme F₄₂₀, factor F₄₃₀ or vitamin B₁₂. In addition an assay was developed in which thirteen of these cofactors can be separated and quantified simultaneously and which can be used in routine analysis of methanogenic populations in anaerobic digesters. The application of the different assays is demonstrated by analyses of extracts of pure cultures of Methanobacterium thermoautotrophicum and Methanosarcina barkeri and of sludge from a methanogenic fluidized bed reactor. Mixtures of authentic methanogenic cofactors were used in reference analyses and a relative peak area method was employed to identity the various cofactors in the extracts.     

Reversible conversion of coenzyme F420 to the 8-OH-AMP and 8-OH-GMP esters,F390-A and F390-G,on oxygen exposure and reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum

Intracellular levels of F₃₉₀ (AMP and GMP adducts of the 5-deazaflavin cofactor F₄₂₀) in Methanobacterium thermoautotrophicum were analysed after gasing fermenter cultures with several consecutive cycles of substrate gas and gas mixtures containing 5% oxygen. No F₃₉₀ was detected in growing cells, hydrogen starved cells and CO₂ starved cells prior to O₂ contamination. Also, no F₃₉₀ was found in hydrogen depleted cells after O₂ treatment. Exposure of exponentially growing cells and CO₂ starved cells to oxygen lead to the formation of F₃₉₀ species; the increase in the detected amount of F₃₉₀ was coupled to a decrease of the F₄₂₀ level. As soon as anaerobiosis was reestablished F₃₉₀ cofactors were degraded and growth proceeded. Independent of the physiological condition of Methanobacterium thermoautotrophicum methanopterin was formed upon O₂ exposure. After normal growth conditions were restored the level of detected methanopterin decreased again.     

Interconversion of F430 derivatives of methanogenic bacteria

F₄₃₀ is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F₄₃₀ is thermolabile and in the presence of acetonitrile or C10 _in4 ^sup- two epimerization products are obtained upon heating; in the absence of these compounds F₄₃₀ is oxidized to 12, 13-didehydro-F₄₃₀. The latter is stereoselectively reduced under H₂ atmosphere to F₄₃₀ by cell-free extracts of M. barkeri or M. thermoautotrophicum. H₂ may be replaced by the reduced methanogenic electron carrier coenzyme F₄₂₀.Abbreviations CH₃S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F₄₃₀ Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F₄₃₀ and 12,13-di-epi-F₄₃₀ the 12, 13- and 12, 13-derivatives of F₄₃₀ - 12, 13-didehydro-F₄₃₀ F₄₃₀ oxidized at C-12 and C-13 - coenzyme F₄₂₀ 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F₄₂₀H₂ reduced coenzyme F₄₂₀ - MV⁺ methylviologen semiquinone - HPLC high-performance liquid chromatography     

Nickel dependence of factor F430 content in Methanobacterium thermoautotrophicum

Factor F₄₃₀ is a yellow compound of unknown structure present in methanogenic bacteria. It has recently been shown to contain nickel. In this communication the influence of the nickel concentration in the growth medium on the factor F₄₃₀ content of Methanobacterium thermoautotrophicum and on the nickel content of factor F₄₃₀ was studied. It was found: (1) The content of factor F₄₃₀ in the cells was strongly dependent on the nickel concentration of the growth medium. Cells grown on media with 2.5 M NiCl₂ contained 28 times as much factor F₄₃₀ per g as those grown on media with 0.075 M NiCl₂; (2) factor F₄₃₀ was synthesized in nickel deprived cells only upon the addition of nickel Nickel uptake paralleled factor F₄₃₀ synthesis; (3) independent of the nickel concentration in the growth medium, the extinction coefficient at 430 nm of factor F₄₃₀ per mol nickel was always near 22,500 cm^-1 (mol Ni)^-1. These findings indicate that nickel is an essential component of factor F₄₃₀.Dedicated to Professor Otto Kandler on the occasion of his 60th birthday     

Photoreactivation in Methanobacterium thermoautotrophicum

The sensitivity of three methanogenic bacteria towards ultraviolet irradiation was similar to the UV-sensitivity of Escherichia coli. The lethal effects of UV-irradiation in Methanobacterium thermoautotrophicum Marburg and in Methanobacterium thermoautotrophicum H but not in Methanococcus vannielii were reversed by exposure to visible light. In cell suspensions of Methanobacterium thermoautotrophicum that had been irradiated to 0.1% survival, 90% of the UV-caused damage was photorepairable. The in vivo action spectrum for photoreactivation suggests that in this organism a deazaflavin, probably F₄₂₀, functions as the chromophore of the photoreactivating enzyme.     

Function of H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum in coenzyme F420 reduction with H2

Summary In most methanogenic archaea, two hydrogenase systems that can catalyze the reduction of coenzyme F₄₂₀ (F₄₂₀) with H₂ are present: (1) the F₄₂₀-reducing hydrogenase, which is a nickel iron-sulfur flavoprotein composed of three different subunits, and (2) the N ⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase system, which is composed of H₂-forming methylenetetrahydromethanopterin dehydrogenase and F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase, both metal-free proteins without an apparent prosthetic group. We report here that in nickel-limited chemostat cultures of Methanobacterium thermoautotrophicum, the specific activity of the F₄₂₀-reducing Ni/Fe-hydrogenase was essentially zero, whereas that of the H₂-forming methylenetetrahydromethanopterin dehydrogenase was six times higher, and that of the F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase was four times higher than in cells grown under non-nickel-limited conditions. This evidence supports the hypothesis that when M. thermoautotrophicum grows under conditions of nickel limitation, the reduction of F₄₂₀ with H₂ is catalyzed by the metal-free methylenetetrahydromethanopterin dehydrogenase system. Received: 9 September 1997 / Accepted: 30 October 1997     

In vivo measurement of F420 fluorescence in cultures of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum could be followed by measuring the intensity of fluorescence directly in the culture vessel, avoiding conventional time-consuming extraction procedures of fluorescent coenzymes. The influence of light scattering by the bacteria was investigated. It could be shown, that light scattering had only little effect on the measurement of coenzyme F₄₂₀ fluorescence. However, culture fluorescence did not correlate to methanogenic activity, due to superposition of bacterial fluorescence by fluorescence from cell-free coenzyme which accumulates in the culture medium. By use of time-resolved laser spectroscopy, different fluorescence lifetimes were obtained for intracellular (1.0 ns) and extracellular (2.5 ns) components, respectively. A combination of this technique with photobleaching measurements for direct determination of F₄₂₀ content of bacteria in a culture is proposed.     

Large-Scale Production of Coenzyme F420-5,6 by Using Mycobacterium smegmatis

Production of coenzyme F₄₂₀ and its biosynthetic precursor FO was examined with a variety of aerobic actinomycetes to identify an improved source for these materials. Based on fermentation costs, safety, and ease of growth, Mycobacterium smegmatis was the best source for F₄₂₀-5,6. M. smegmatis produced 1 to 3 μmol of intracellular F₄₂₀ per liter of culture, which was more than the 0.85 to 1.0 μmol of F₄₂₀-2 per liter usually obtained with Methanobacterium thermoautotrophicum and ~10-fold higher than what was previously reported for the best aerobic actinomycetes. An improved chromatography system using rapidly flowing quaternary aminoethyl ion-exchange material and Florisil was used to more quickly and easily purify F₄₂₀ than with previous methods.     

A method for estimating phosphate in the presence of phytate and its application to the determination of phytase

Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80°C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF₄₃₀, the prosthetic group of methylcoenzyme M reductase, F₅₆₀, an oxidation product of this compound, coenzyme F₄₂₀, F₃₄₂, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF₄₃₀, coenzyme F₄₂₀, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied.     

Biosynthesis of coenzyme F420 and methanopterin in Methanobacterium thermoautotrophicum

Growing cultures of Methanobacterium thermoautotrophicum were supplemented with [U-¹⁴C]adenosine or [1-¹⁴C]adenosine. 7,8-Didemethyl-8-hydroxy-5-deazariboflavin (factor F₀) and 7-methylpterin were isolated from the culture medium. Hydrolysis of cellular RNA yielded purine and pyrimidine nucleotides. The ribose side chain of proffered adenosine is efficiently incorporated into cellular adenosine and guanosine nucleotide pools but not into pyrimidine nucleotides. Thus, M. thermoautotrophicum can utilize exogenous adenosine by direct phosphorylation without hydrolysis of the glycosidic bond, and AMP can be efficiently converted to GMP. Factor F₀ and 7-methylpterin had approximately the same specific activities as the purine nucleotides. It follows that the ribityl side chain of factor F₀ is derived from the ribose side chain of a nucleotide precursor by reduction. The pyrazine ring of methanopterin is formed by ring expansion involving the ribose side chain of the precursor, GTP.Abbreviations Factor F₀ 8-hydroxy-6,7-didemethyl-5-deazariboflavin - APRT adenine phosphoribosyltransferase - GPRT guanine phosphoribosyltransferase - PRPP phosphoribosylpyrophosphate - HPLC high performance liquid chromatography     

MTH187 from Methanobacterium thermoautotrophicum has three HEAT-like Repeats

With the completion of genome sequencing projects, there are a large number of proteins for which we have little or no functional information. Since protein function is closely related to three-dimensional conformation, structural proteomics is one avenue where the role of proteins with unknown function can be investigated. In the present structural project, the structure of MTH187 has been determined by solution-state NMR spectroscopy. This protein of 12.4 kDa is one of the 424 non-membrane proteins that were cloned and purified for the structural proteomic project of Methanobacterium thermoautotrophicum [Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C.H. and Edwards, A.M. (2000) Prog. Biophys. Mol. Biol., 73, 339–345]. Methanobacterium thermoautotrophicum is a thermophilic archaeon that grows optimally at 65 °C. A particular characteristic of this microorganism is its ability to generate methane from carbon dioxide and hydrogen [Smith, D.R., Doucette-Stamm, L.A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., Reeve, J. N. et al. (1997) J. Bacteriol., 179, 7135–7155].Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Structure data have been deposited at PDB (1TE4) and NMR data at BMRB (5629).Olivier Julien and Isabelle Gignac - Both authors contributed equally to this work.     

Isolation and Characterization of Methanobacterium thermoautotrophicum ΔH Mutants Unable To Grow under Hydrogen-Deprived Conditions

By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of Methanobacterium thermoautotrophicum that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F₄₂₀-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F₃₉₀, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen.     

Structural characterization and physiological function of component B from Methanosarcina thermophila

The electron donor (component B) to the methyl coenzyme M methylreductase system from Methanosarcina thermophila was isolated as the 7-methyl derivative and characterized. Gas chromatography-mass spectrometry and ¹H NMR analyses identified this derivative as 7-methylthioheptanoylthreonine phosphate (CH₃-S-HTP), indicating that the original component B had the same structure (HS-HTP) as previously determined for component B from Methanobacterium thermoautotrophicum. The heterodisulfide of HS-HTP and coenzyme M (HS-CoM, 2-mercaptoethanesulfonate) was enzymatically reduced in cell extracts using electrons supplied by either H₂ or CO, confirming that HS-HTP was a functional molecule in M. thermophila.     

Non-enzymatic ammonia formation from glutamine under growth conditions for Methanobacterium thermoautotrophicum

Glutamine was found to be converted to NH₃ and pyroglutamic acid (5-oxo-2-pyrrolidine carboxylic acid; 5-oxoproline) under conditions of growth for Methanobacterium thermoautotrophicum at rates sufficient to satisfy the organism's nitrogen requirements.     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.     

Spectroscopic investigation of the nickel-containing porphinoid cofactor F<Subscript>430</Subscript>. Comparison of the free cofactor in the +1, +2 and +3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent,red and ox forms

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F₄₃₀, a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F₄₃₀ in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F₄₃₀, variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F₄₃₀M) in the +1, +2 and +3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle orbitals that are unique to Ni(I) forms of F₄₃₀. Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d–d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F₄₃₀ macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0549-9Abbreviations F₄₃₀ cofactor 430 - F₄₃₀M penta-methylated form of cofactor 430 - Ni(I)F₄₃₀M F₄₃₀M with the nickel atom in the +1 oxidation state - Ni(II)F₄₃₀M F₄₃₀M with the nickel atom in the +2 oxidation state - Ni(III)F₄₃₀M F₄₃₀M with the nickel atom in the +3 oxidation state - MCR methyl-coenzyme M reductase - MCRox1 MCR exhibiting the MCR-ox1 EPR signal - MCRox1-silent EPR silent form of MCR obtained from the MCRox1 form - MCRred1 MCR exhibiting the EPR signals red1c and/or red1m - MCRred1c MCRred1 in the presence of coenzyme M - MCRred1m MCRred1 in the presence of methyl-coenzyme M - MCRred2 MCR exhibiting both the red1 and red2 EPR signals - MCRred1-silent EPR silent form of MCR obtained from the MCRred1 form - MCRsilent EPR silent form of MCR     

Tetrahydromethanopterin,a coenzyme involved in autotrophic acetyl coenzyme A synthesis from 2 CO2 in Methanobacterium

Methanobacterium thermoautotrophicum, a methane forming archaebacterium, grows autotrophically by synthesizing activated acetic acid from 2 CO₂. It is demonstrated in vitro that the methyl group of acetate is derived from methenyl tetrahydromethanopterin, which is known to be a one-carbon carrying coenzyme in CO₂ reduction to methane. The direct acetate precursors are suggested to be methyl tetrahydromethanopterin (“activated methanol”) and “activated carbon monoxide”.