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.     

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     

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.     

Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg.

The F420-reducing hydrogenase and the non-F420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively stained F420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, parallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided into smaller protein masses. Electron microscopy of negatively stained samples taken from intermediate steps of the purification process revealed the presence of enzyme particles bound to inside-out membrane vesicles. Linker particles of 10 to 20 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold labelling confirmed that the F420-reducing hydrogenase is a membrane-bound enzyme. Electron microscopy of the negatively stained purified non-F420-reducing hydrogenase revealed that the enzyme is composed of three subunits exhibiting different diameters (5, 4, and 2 to 3 nm). According to immunogold labelling experiments, approximately 70% of the non-F420-reducing hydrogenase protein molecules were located at the cell periphery; the remaining 30% were cytoplasmic. No linker particles were observed for this enzyme.     

The magnetic properties of the nickel cofactor F430 in the enzyme methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum.

Preparations of gamma-aminobutyrate (GABA)/benzodiazepine receptor from pig cerebral cortex are composed of three major bands of polypeptides (51, 55 and 57 kDa) which are purified in a ratio of approx. 2:1:1 respectively. Treatment of purified receptor preparations with cyclic AMP-dependent protein kinase resulted in major incorporation of 32P into the 55 kDa band only. The maximum incorporation achieved was 0.6 mol of 32P/mol of 55 kDa polypeptide. The phosphorylated receptor subunit (beta-subunit) displays the same apparent Mr as a band labelled irreversibly with the GABA receptor agonist [3H]muscimol. The two nonphosphorylated subunit polypeptides (51 and 57 kDa) are each labelled irreversibly with [3H]flunitrazepam and are recognized by anti-peptide antibodies specific for alpha-subunits.     

Tetrahydromethanopterin-dependent serine transhydroxymethylase from Methanobacterium thermoautotrophicum

Serine transhydroxymethylase of Methanobacterium thermoautotrophicum has been purified to within 95% of homogeneity. Activity was strictly dependent on tetrahydromethanopterin, tetrahydrofolate being unable to serve as the acceptor C₁ units from l-serine. The native protein has a molecular weight of about 102,000 daltons. The enzyme shows maximal activity at 60°C, has a pH optimum of 8.1, and required pyridoxal-5-phosphate and Mg²⁺ for optimal activity.     

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     

F390 synthetase and F390 hydrolase from Methanobacterium thermoautotrophicum (strain delta H).

Factor F390 is the 8-OH adenylated form of the deazaflavin coenzyme F420, which is a central electron carrier in methanogenic bacteria. The enzymes catalysing the formation of F390 from ATP and F420 (F390 synthetase) and its hydrolysis into AMP and F420 (F390 hydrolase) were isolated and partially purified from Methanobacterium thermoautotrophicum. Both enzymes were oxygen-stable. The F390 synthetase tended to coelute with coenzyme F420 reducing hydrogenase during all purification steps. The 30-fold purified enzyme was still contaminated with the hydrogenase. The F390 hydrolase was purified 135-fold to a specific activity of 8.6 mumol/min/mg protein. The colourless enzyme consisted of one polypeptide of approximately 27,000 kd.     

Coenzyme F420 dependence of the methylenetetrahydromethanopterin dehydrogenase of Methanobacterium thermoautotrophicum

To identify the electron acceptor of the methylenetetrahydromethanopterin dehydrogenase of Methanobacterium thermoautotrophicum, we have purified the enzyme to homogeneity. The purified enzyme is absolutely dependent on coenzyme F420 (a 7,8-didemethyl-8-hydroxy-5-deazariboflavin derivative) for activity. Several alternative electron acceptors are ineffectual in the reaction. Changes in the absorption spectra of reaction mixtures indicate that 1.1 mol of coenzyme F420 is reduced per mol of substrate oxidized. The reaction is reversible and the equilibrium favors oxidation of methylenetetrahydromethanopterin.     

Expression,purification, and characterization of the oligomeric states of rubredoxin oxidoreductase from Methanobacterium thermoautotrophicum

The rubredoxin oxidoreductase from Methanobacterium thermoautotrophicum was successfully overexpressed in Escherichia coli with 6x His-Tag and purified by using Ni-NTA technology. It was characterized by SDS and native polyacrylamide gel electrophoresis (PAGE), as well as size-exclusion chromatography. The protein was pure, judged by SDS-PAGE, but three or more oligomeric species were observed by native PAGE and size-exclusion chromatography. The smallest rubredoxin oxidoreductase species is the dimer. The multiple species are stable and remain in their respective oligomeric states, judged by the chromatographic and electrophoretic results. A model is proposed in order to explain the structural basis for these results.     

An extended-X-ray-absorption-fine-structure (e.x.a.f.s.) study of coenzyme F430 from Methanobacterium thermoautotrophicum.

Coenzyme F430 is a nickel porphinoid found in all methanogenic bacteria. Extended-X-ray-absorption-fine-structure (e.x.a.f.s.) spectra have been recorded above the nickel K-edge of coenzyme F430 and two model compounds, (5,10,15,20-tetramethylporphinato) nickel(II) and (5,10,15,20-tetramethylchlorinato)-nickel(II). The results show that the four nickel-nitrogen distances in F430 are split, with two nitrogen atoms at 0.192 nm and two at 0.210 nm.     

Ni(II) and Ni(I) forms of pentaalkylamide derivatives of cofactor F430 of Methanobacterium thermoautotrophicum.

A series of pentaalkylamide forms of F430 and of its 12,13-diepimer have been generated and characterized. Carbodiimide-assisted N-hydroxysulfosuccinimide activation of all five peripheral carboxylates of the F430 macrocycle allows nucleophilic attack by a number of primary amines (RNH2, R- = CH3-, CH3CH2-, CF3CH2-, CH3(CH2)3-) generating the pentaalkylamide derivatives. The identity of each derivative has been verified by fast-atom bombardment mass spectrometry (FAB-MS). The solubility of these derivatives in aprotic organic solvents varies as the amine alkyl substituent (R-) is changed. Electrochemical measurements have shown that the Ni(II/I) reduction potentials in N,N-dimethylformamide (DMF) are approximately -1 V (Ag/AgCl). Reduction by sodium amalgam in THF generates the Ni(I) form of the F430 diepimer pentabutylamide. The visible and EPR spectra of this Ni(I) species are very similar to the corresponding spectra of Ni(I) F430M (Jaun, B. and Pfaltz, A. (1986) J. Chem. Soc. Chem. Commun. 1327-1329.).     

Structure of purified cytoplasmic cofactor from Methanobacterium thermoautotrophicum

The reduction of methylcoenzyme M to methane is known to require a heat stable and oxygen sensitive cofactor. Recently it has been shown that the active site of this cofactor is 7-mercaptoheptanoylthreonine phosphate. The present study shows that in the complete structure of this cofactor 7-mercaptoheptanoylthreonine phosphate is linked by pyrophosphate to two N-acetyl-glucosamine residues and an unidentified terminal group R with m/z 214. By fast-atom-bombardment mass spectrometry the intact cofactor, isolated as the mixed disulfide with 2-mercaptoethanol, was shown to have a molecular weight of 1084.5. The pyrophosphate bond is quite labile and undergoes hydrolysis or prolonged storage. This lability of the pyrophosphate bond may explain why the intact cofactor has not been isolated until now.     

Structural and functional characterization of a thioredoxin-like protein (Mt0807) from Methanobacterium thermoautotrophicum

Mt0807 is an 85-residue thiol-redox protein from the anaerobic archaebacterium Methanobacterium thermoautotrophicum. Its small size, its participation in certain redox reactions, and the presence of a "classic" glutareodoxin active-site sequence have led to the suggestion that it might be a glutaredoxin. However, studies by previous workers indicated that it exhibited neither glutaredoxin-like nor thioredoxin-like properties. To clarify the true role of this protein and its structure/functional relationship with a paralogous thioredoxin (Mt0895, 28% sequence identity) and a recently characterized orthologous protein (Mj0307, 51% sequence identity), we undertook a series of biochemical and biophysical studies. Comparative enzymatic assays and thiol titration experiments were combined with NMR structural studies and detailed 3D structure comparisons. Structurally, our results show that Mt0807 has a glutaredoxin-like fold (central four-stranded beta-sheet core surrounded by two helices on one side and a third on the other). However, more detailed comparisons with other members of the thioredoxin superfamily indicate that Mt0807 actually has several key structural and active-site characteristics more common to a thioredoxin. Furthermore, biochemical tests show that Mt0807 actually behaves as true thioredoxin. Comparisons between Mt0807 and its paralogue, Mt0895, indicate these two archaebacterial thioredoxins share very similar folds, but exhibit very different activities and likely serve somewhat different roles. On the basis of its greater relative abundance and significantly stronger redox activity, we believe that Mt0807 is the primary thioredoxin for M. thermoautotrophicum, while Mt0895 plays a minor or supportive role. We also suggest that these two molecules (Mt0807 and Mt0895) may represent a group of ancient proteins that were ancestral to both thioredoxins and glutaredoxins.     

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.     

Coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg belongs to the superfamily of adenylate-forming enzymes.

Depending on the reduction-oxidation state of the cell, some methanogenic bacteria synthesize or hydrolyze 8-hydroxyadenylylated coenzyme F420 (coenzyme F390). These two reactions are catalyzed by coenzyme F390 synthetase and hydrolase, respectively. To gain more insight into the mechanism of the former reaction, coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg was purified 89-fold from cell extract to a specific activity of 0.75 mumol.min-1.mg of protein-1. The monomeric enzyme consisted of a polypeptide with an apparent molecular mass of 41 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ftsA, the gene encoding coenzyme F390 synthetase, was cloned and sequenced. It encoded a protein of 377 amino acids with a predicted M(r) of 43,280. FtsA was found to be similar to domains found in the superfamily of peptide synthetases and adenylate-forming enzymes. FtsA was most similar to gramicidin S synthetase II (67% similarity in a 227-amino-acid region) and sigma-(L-alpha-aminoadipyl)-L-cysteine-D-valine synthetase (57% similarity in a 193-amino-acid region). Coenzyme F390 synthetase, however, holds an exceptional position in the superfamily of adenylate-forming enzymes in that it does not activate a carboxyl group of an amino or hydroxy acid but an aromatic hydroxyl group of coenzyme F420.     

Isolation of lipid activated pseudomurein precursors from Methanobacterium thermoautotrophicum

From cell extracts of the pseudomurein possessing methanogen Methanobacterium thermoautotrophicum two putative pseudomurein precursors were isolated and characterized: (1) an undecaprenyl pyrophosphate activated disaccharide pentapeptide composed of N-acetylglucosamine, N-acetyltalosaminuronic acid, alanine, glutamic acid and lysine in a molar ratio of 1:1:2:2:1 and (2) the corresponding undecaprenyl pyrophosphate activated tetrapeptide lacking one alanine residue. The isolation of these precursors show that the biosynthesis of the eubacterial murein and the methano-bacterial pseudomurein differs not only in the cytoplasmic step, as recently described, but also in the lipid stage.Abbreviations GlcNitol glucosaminitol - NAcTalNUA N-acetyltalosaminuronic acid - Udp undecaprenol - TLC thin layer chromatography