5-Formyl-5,6,7,8-tetrahydromethanopterin is the intermediate in the process of methanogenesis in Methanosarcina barkeri.

Formylmethanofuran:tetrahydromethanopterin (H4MPT) formyltransferase and 5,10-methenyl-H4MPT cyclohydrolase purified from Methanosarcina barkeri catalyze a formyl group transfer and the hydrolysis of the methenyl function, respectively. The results from UV spectroscopy and HPLC analyses, and comparison with results obtained with the enzymes isolated from Methanobacterium thermoautotrophicum showed 5-formyl-H4MPT to be the product of the formyltransferase and cyclohydrolase reactions in M. barkeri. The findings disagree with an earlier report in which 10-formyl-H4MPT was identified as the product of the cyclohydrolase in the latter organism. In addition, it was observed that 10-formyl-H4MPT, which is non-enzymically formed from 5,10-methenyl-H4MPT at alkaline pH, becomes rapidly converted into the 5-formyl derivative. The latter finding explains why the nature of the formyl species previously had been improperly assigned.     

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.     

Purification and properties of 5,10-methylenetetrahydromethanopterin reductase, a coenzyme F420-dependent enzyme, from Methanobacterium thermoautotrophicum strain delta H

5,10-Methylenetetrahydromethanopterin reductase was purified 22-fold to apparent homogeneity from the methanogenic bacterium Methanobacterium thermoautotrophicum. The enzyme catalyzes the reduction of 5,10-methylene- to 5-methyltetrahydromethanopterin. The electron carrier coenzyme F420 is specifically used as the cosubstrate. The reductase reaction may proceed in both directions, methylene reduction is, however, thermodynamically favored. In addition, the velocity of the reaction in this direction exceeds the reverse reaction by a factor of 26. The reductase is composed of a single subunit with an estimated Mr = 35,000. The active enzyme does not contain a flavin prosthetic group or iron-sulfur clusters, in contrast to 5,10-methylenetetrahydrofolate reductases purified from eukaryotic and eubacterial sources, which catalyze an analogous reaction as the methanogenic reductase.     

PQQ-dependent methanol dehydrogenases: rare-earth elements make a difference

Methanol dehydrogenase (MDH) catalyzes the first step in methanol use by methylotrophic bacteria and the second step in methane conversion by methanotrophs. Gram-negative bacteria possess an MDH with pyrroloquinoline quinone (PQQ) as its catalytic center. This MDH belongs to the broad class of eight-bladed β propeller quinoproteins, which comprise a range of other alcohol and aldehyde dehydrogenases. A well-investigated MDH is the heterotetrameric MxaFI-MDH, which is composed of two large catalytic subunits (MxaF) and two small subunits (MxaI). MxaFI-MDHs bind calcium as a cofactor that assists PQQ in catalysis. Genomic analyses indicated the existence of another MDH distantly related to the MxaFI-MDHs. Recently, several of these so-called XoxF-MDHs have been isolated. XoxF-MDHs described thus far are homodimeric proteins lacking the small subunit and possess a rare-earth element (REE) instead of calcium. The presence of such REE may confer XoxF-MDHs a superior catalytic efficiency. Moreover, XoxF-MDHs are able to oxidize methanol to formate, rather than to formaldehyde as MxaFI-MDHs do. While structures of MxaFI- and XoxF-MDH are conserved, also regarding the binding of PQQ, the accommodation of a REE requires the presence of a specific aspartate residue near the catalytic site. XoxF-MDHs containing such REE-binding motif are abundantly present in genomes of methylotrophic and methanotrophic microorganisms and also in organisms that hitherto are not known for such lifestyle. Moreover, sequence analyses suggest that XoxF-MDHs represent only a small part of putative REE-containing quinoproteins, together covering an unexploited potential of metabolic functions.     

Cyclic 2,3-diphosphoglycerate metabolism in Methanobacterium thermoautotrophicum (strain ΔH): characterization of the synthetase reaction

The levels of cyclic 2,3-diphosphoglycerate (cDPG) in methanogenic bacteria are governed by the antagonistic activities of cDPG synthetase and cDPG hydrolase. In this paper we focus on the synthetase from Methanobacterium thermoautotrophicum. The cytoplasmic 150 kDa enzyme catalyzed cDPG synthesis from 2,3-diphosphoglycerate (apparent K_m=21 mM), Mg²⁺ (K_m=3.1 mM) and ATP (K_m=1–2 mM). In batch-fed cultures, the enzyme was constitutively present (6–6.5 nmol per min per mg protein) during the different growth phases. In continuous cultures, activity decreased in response to phosphate limitation. The synthetase reaction proceeded with maximal rate at pH 6 and at 65° C and was specifically dependent on high (>0.3M) K⁺ concentrations. The reaction conditions remarkably contrasted to those of cDPG degradation catalyzed by the previously described membrane-bound cDPG hydrolase.Abbreviations cDPG Cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-Diphosphoglycerate - 2-PG 2-Phosphoglycerate - 3-PG 3-Phosphoglycerate     

Stimulation of the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in cell-free extracts of Methanobacterium thermoautotrophicum by the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate

The conversion of formaldehyde to methylcoenzyme M in cell-free extracts of Methanobacterium thermoautotrophicum was stimulated up to 10-fold by catalytic amounts of the heterodisulfide (CoM-S-S-HTP) of coenzyme M and 7-mercaptoheptanoylthreonine phosphate. The stimulation required the additional presence of ATP, also in catalytic concentrations. ATP and CoM-S-S-HTP were mutually stimulatory on the methylcoenzyme M formation and it was concluded that the compounds were both involved in the reductive activation of the methyltetrahydromethanopterin: coenzyme M methyltransferase. Micromolar concentrations of benzyl viologen or cyanocobalamin inhibited the formaldehyde conversion; these compounds, however, strongly stimulated the reduction of CoM-S-S-HTP. The results described here closely resemble observations made on the activation and reduction of CO₂ to formylmethanofuran indicating that this step and the reductive activation of the methyltransferase are controlled by some common mechanism.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - MFR methanofuran - HS-HTP 7-mercaptoheptanoylthreonine phosphate - CoM-S-S-HTP the heterodisulfide of HS-CoM and HS-HTP - BES 2-bromoethanesulfonate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - CN-Cbl cyanocobalamin - HO-Cbl hydroxycobalamin - HBI 5-hydroxybenzimidazole - DMBI 5,6-dimethylbenzimidazole     

A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.     

Electron transfer reactions in methanogens

Abstract Methanogenic bacteria comprise a specialized group of obligately anaerobic microorganisms able to reduce a limited number of substrates to CH₄. The intermediates involved in this reduction process remain bound to a series of typical C₁-carriers. Reducing equivalents are either obtained from the oxidation of H₂ or from oxidation of carbon substrates to CO₂. Electron transfer reactions thus constitute the very essence of the process of methanogenesis.
In recent years much progress has been made in the elucidation of the special metabolic pathways and the nature of the C₁-carriers involved in methanogenic bacteria. The energy generated at the oxidoreduction reactions, notably at the methylreductase step, is conserved by ATP synthesis. The energy is used for cell carbon synthesis and, in catalytic amounts, for the reductive activation of some methanogenic enzymes. Before the condensing reaction resulting in the formation of acetyl-CoA takes place, 2 C₁-units are reduced or oxidized depending on the substrate to a carbonyl and a -CH₃ group. Formation of the latter proceeds via the methanogenic route. Intermediary cell carbon synthesis starting from acetyl-CoA involves reductive carboxylations and oxidoreductions by the participation of the enzymes of the tricarboxylic acid cycle.     

Stimulation of the methylcoenzyme M reduction by uridine-5'-diphospho-sugars in cell-free extracts of Methanobacterium thermoautotrophicum (strain delta H)

The hydrogen-dependent reduction of methylcoenzyme M catalyzed by coenzyme-depleted cell-free extracts of Methanobacterium thermoautotrophicum was stimulated by micromolar concentrations of a UDP-disaccharide present in the organism. The compound was isolated and identified as UDP-1-O-alpha-D-2-acetamido-2-deoxyglucopyranose (UDPGlcpNAc) glycosidically linked to 2-acetamido-2-deoxymannopyranosyluronic acid. Maximal stimulation was observed when both the UDP-disaccharide and mercaptoheptanoylthreonine phosphate were present in the reaction mixtures. The UDP derivative isolated was not specific in its action: other UDP-sugars tested in micromolar concentrations stimulated the methylcoenzyme M reduction to the same extent. The activated sugars presumably substitute for ATP, which is usually required in much higher concentrations to activate the methylcoenzyme M reductase system.