Max-Planck-Institut fur terrestrische Mikrobiologie, Karl-von-Frisch-Strase, D-35043 Marburg, and Laboratorium fur Mikrobiologie, Fachbereich Biologie, Philipps-Universitat, Karl-von-Frisch-Strase, D-35032 Marburg, Germany
In 1933, Stephenson & Stickland (1933a) published that they had isolated from river mud, by the single cell technique, a methanogenic organism capable of growth in an inorganic medium with formate as the sole carbon source.

Biochemistry of methanogenesis: a tribute to Marjory Stephenson:1998 Marjory Stephenson Prize Lecture

Methanoarchaea, the largest and most phylogenetically diverse group in the Archaea domain, have evolved energy-yielding pathways marked by one-carbon biochemistry featuring novel cofactors and enzymes. All of the pathways have in common the two-electron reduction of methyl-coenzyme M to methane catalyzed by methyl-coenzyme M reductase but deviate in the source of the methyl group transferred to coenzyme M. Most of the methane produced in nature derives from acetate in a pathway where the activated substrate is cleaved by CO dehydrogenase/acetyl-CoA synthase and the methyl group is transferred to coenzyme M via methyltetrahydromethanopterin or methyltetrahydrosarcinapterin. Electrons for reductive demethylation of the methyl-coenzyme M originate from oxidation of the carbonyl group of acetate to carbon dioxide by the synthase. In the other major pathway, formate or H2 is oxidized to provide electrons for reduction of carbon dioxide to the methyl level and reduction of methyl-coenzyme to methane. Methane is also produced from the methyl groups of methanol and methylamines. In these pathways specialized methyltransferases transfer the methyl groups to coenzyme M. Electrons for reduction of the methyl-coenzyme M are supplied by oxidation of the methyl groups to carbon dioxide by a reversal of the carbon dioxide reduction pathway. Recent progress on the enzymology of one-carbon reactions in these pathways has raised the level of understanding with regard to the physiology and molecular biology of methanogenesis. These advances have also provided a foundation for future studies on the structure/function of these novel enzymes and exploitation of the recently completed sequences for the genomes from the methanoarchaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii.

Enzymology of one-carbon metabolism in methanogenic pathways

Methanogenic archaea have an unusual type of metabolism because they use H2 + CO2, formate, methylated C1 compounds, or acetate as energy and carbon sources for growth. The methanogens produce methane as the major end product of their metabolism in a unique energy-generating process. The organisms received much attention because they catalyze the terminal step in the anaerobic breakdown of organic matter under sulfate-limiting conditions and are essential for both the recycling of carbon compounds and the maintenance of the global carbon flux on Earth. Furthermore, methane is an important greenhouse gas that directly contributes to climate changes and global warming. Hence, the understanding of the biochemical processes leading to methane formation are of major interest. This review focuses on the metabolic pathways of methanogenesis that are rather unique and involve a number of unusual enzymes and coenzymes. It will be shown how the previously mentioned substrates are converted to CH4 via the CO2-reducing, methylotrophic, or aceticlastic pathway. All catabolic processes finally lead to the formation of a mixed disulfide from coenzyme M and coenzyme B that functions as an electron acceptor of certain anaerobic respiratory chains. Molecular hydrogen, reduced coenzyme F420, or reduced ferredoxin are used as electron donors. The redox reactions as catalyzed by the membrane-bound electron transport chains are coupled to proton translocation across the cytoplasmic membrane. The resulting electrochemical proton gradient is the driving force for ATP synthesis as catalyzed by an A1A0-type ATP synthase. Other energy-transducing enzymes involved in methanogenesis are the membrane-integral methyltransferase and the formylmethanofuran dehydrogenase complex. The former enzyme is a unique, reversible sodium ion pump that couples methyl-group transfer with the transport of Na+ across the membrane. The formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation of methanofuran. Furthermore, the review addresses questions related to the biochemical and genetic characteristics of the energy-transducing enzymes and to the mechanisms of ion translocation.

The unique biochemistry of methanogenesis.

In most organisms, tetrahydrofolate (H(4)folate) is the carrier of C(1) fragments between formyl and methyl oxidation levels. The C(1) fragments are utilized in several essential biosynthetic processes. In addition, C(1) flux through H(4)folate is utilized for energy metabolism in some groups of anaerobic bacteria. In methanogens and several other Archaea, tetrahydromethanopterin (H(4)MPT) carries C(1) fragments between formyl and methyl oxidation levels. At first sight H(4)MPT appears to resemble H(4)folate at the sites where C(1) fragments are carried. However, the two carriers are functionally distinct, as discussed in the present review. In energy metabolism, H(4)MPT permits redox-flux features that are distinct from the pathway on H(4)folate. In the reductive direction, ATP is consumed in the entry of carbon from CO(2) into the H(4)folate pathway, but not in entry into the H(4)MPT pathway. In the oxidative direction, methyl groups are much more readily oxidized on H(4)MPT than on H(4)folate. Moreover, the redox reactions on H(4)MPT are coupled to more negative reductants than the pyridine nucleotides which are generally used in the H(4)folate pathway. Thermodynamics of the reactions of C(1) reduction via the two carriers differ accordingly. A major underlying cause of the thermodynamic differences is in the chemical properties of the arylamine nitrogen N(10) on the two carriers. In H(4)folate, N(10) is subject to electron withdrawal by the carbonyl group of p-aminobenzoate, but in H(4)MPT an electron-donating methylene group occurs in the corresponding position. It is also proposed that the two structural methyl groups of H(4)MPT tune the carrier's thermodynamic properties through an entropic contribution. H(4)MPT appears to be unsuited to some of the biosynthetic functions of H(4)folate, in particular the transfer of activated formyl groups, as in purine biosynthesis. Evidence bearing upon whether H(4)MPT participates in thymidylate synthesis is discussed. Findings on the biosynthesis and phylogenetic distribution of the two carriers and their evolutionary implications are briefly reviewed. Evidence suggests that the biosynthetic pathways to the two carriers are largely distinct, suggesting the possibility of (ancient) separate origins rather than divergent evolution. It has recently been discovered that some eubacteria which gain energy by oxidation of C(1) compounds contain an H(4)MPT-related carrier, which they are thought to use in energy metabolism, as well as H(4)folate, which they are thought to use for biosynthetic reactions.

Tetrahydrofolate and tetrahydromethanopterin compared: functionally distinct carriers in C1 metabolism

Methane is a product of the energy-yielding pathways of the largest and most phylogenetically diverse group in the Archaea. These organisms have evolved three pathways that entail a novel and remarkable biochemistry. All of the pathways have in common a reduction of the methyl group of methyl-coenzyme M (CH3-S-CoM) to CH4. Seminal studies on the CO2-reduction pathway have revealed new cofactors and enzymes that catalyze the reduction of CO2 to the methyl level (CH3-S-CoM) with electrons from H2 or formate. Most of the methane produced in nature originates from the methyl group of acetate. CO dehydrogenase is a key enzyme catalyzing the decarbonylation of acetyl-CoA; the resulting methyl group is transferred to CH3-S-CoM, followed by reduction to methane using electrons derived from oxidation of the carbonyl group to CO2 by the CO dehydrogenase. Some organisms transfer the methyl group of methanol and methylamines to CH3-S-CoM; electrons for reduction of CH3-S-CoM to CH4 are provided by the oxidation of methyl groups to CO2.

Biochemistry of methanogenesis.

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

The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri was purified 313-fold to a specific activity of 470 mumol min-1 mg-1 at 37 degrees C and pH 7.8. At this stage, the enzyme was pure as judged from polyacrylamide gel electrophoresis. The monofunctional enzyme was oxygen stable, but the presence of a detergent proved to be essential for its stability. Like the cyclohydrolase purified from Methanobacterium thermoautotrophicum (A. A. Dimarco, M. I. Donnelly, and R. S. Wolfe, J. Bacteriol. 168:1372-1377, 1986), the protein showed an apparent Mr of 82,000, and it is composed of two identical subunits as was concluded from nondenaturating and denaturating polyacrylamide gel electrophoresis. The enzymes from M. thermoautotrophicum and M. barkeri markedly differ with respect to the hydrolysis product of 5,10-methenyltetrahydromethanopterin: 5-formyl- and 10-formyltetrahydromethanopterin, respectively. The apparent Km for 5,10-methenyltetrahydromethanopterin was 0.57 mM at 37 degrees C and pH 7.8.

C. M. H. Hensgens

Papers

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

Purification and properties of 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri.