James G. Ferry

Frontiers, Opportunities, and Challenges in Biochemical and Chemical Catalysis of CO2 Fixation

Two major energy-related problems confront the world in the next 50 years. First, increased worldwide competition for gradually depleting fossil fuel reserves (derived from past photosynthesis) will lead to higher costs, both monetarily and politically. Second, atmospheric CO_2 levels are at their highest recorded level since records began. Further increases are predicted to produce large and uncontrollable impacts on the world climate. These projected impacts extend beyond climate to ocean acidification, because the ocean is a major sink for atmospheric CO2.1 Providing a future energy supply that is secure and CO_2-neutral will require switching to nonfossil energy sources such as wind, solar, nuclear, and geothermal energy and developing methods for transforming the energy produced by these new sources into forms that can be stored, transported, and used upon demand.

A left-hand beta-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila.

A carbonic anhydrase from the thermophilic archaeon Methanosarcina thermophila that exhibits no significant sequence similarity to known carbonic anhydrases has recently been characterized. Here we present the structure of this enzyme, which adopts a left‐handed parallel beta‐helix fold. This fold is of particular interest since it contains only left‐handed crossover connections between the parallel beta‐strands, which so far have been observed very infrequently. The active form of the enzyme is a trimer with three zinc‐containing active sites, each located at the interface between two monomers. While the arrangement of active site groups differs between this enzyme and the carbonic anhydrases from higher vertebrates, there are structural similarities in the zinc coordination environment, suggestive of convergent evolution dictated by the chemical requirements for catalysis of the same reaction. Based on sequence similarities, the structure of this enzyme is the prototype of a new class of carbonic anhydrases with representatives in all three phylogenetic domains of life.

Role of bacterial siderophores in dissolution of hornblende

Hornblende, a common mineral in granitic soils, may act as a source for a variety of metals needed by bacterial species for enzyme function (e.g., Fe, Zn, Mn, Cu, Co, Mo, V, Ni). A species of the bacterial genus Streptomyces was cultured from an Adirondack soil and isolated because of its ability to grow robustly in low Fe medium with hornblende present. Studies with unbuffered culture medium, to discover whether Streptomyces sp. cultures affected solution pH, showed a decrease of 2.0 pH units in 21 d, then an increase of 3.0 pH units at 56 d. Cells that adhered to the hornblende surface at 56 days were difficult to remove, presumably because of mycelial growth deep into pits and cracks. Decreases and increases in pH may have been due to production of organic acids and ammonia respectively. Increases in pH could also have been related to release of components during death of organisms. In a buffered medium, Streptomyces sp. increased the initial Fe release rate from hornblende approximately fivefold over that of an abiotic control. A catechol derivative, produced by the Streptomyces sp. and characterized by chromatography and mass spectrometry, is presumed to cause this Fe release enhancement. Hornblende dissolution was also analyzed in the presence of a commercially available hydroxamate siderophore, desferrioxamine mesylate (DFAM). DFAM is the methane sulfonate form of one of many siderophores known to be a product of streptomycetes. The rate of Fe release obtained when incubating the hornblende with 24 μm of DFAM was similar to the rate observed in the presence of the Streptomyces sp. isolate. Higher concentrations of DFAM increased the dissolution rate nonlinearly, described by the rate equation R = (7.6 × 10−13)C0.47, where R is the release rate of Fe (mol/m2s), and C is the concentration (mol/l) of DFAM. The DFAM also increased release of Al and Si from hornblende into solution; however, these release rates were not increased by addition of the Streptomyces sp. alone. Preferential release of Al and Si in the presence of DFAM, but not in the presence of bacteria alone, may be related to the difference in selectivity of catechol vs. hydroxamate siderophores. Addition of Streptomyces sp. in the presence of DFAM at three concentrations consistently enhanced Fe release approximately two to threefold the rate with siderophore alone. Recycling of siderophore molecules or enhanced production of one siderophore by microorganisms in the presence of other siderophores makes it difficult to predict a priori release rates when both siderophore and bacteria are present, as would be the case in natural soils.

Biochemistry of Methanogenesis

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.

Anaerobic degradation of benzoate to methane by a microbial consortium.

A stabilized consortium of microbes which anaerobically degraded benzoate and produced CH4 was established by inoculation of a benzoate-mineral salts medium with sewage sludge; the consortium was routinely subcultured anaerobically in this medium for 3 years. Acetate, formate, H2 and CO2 were identified as intermediates in the overall conversion of benzoate to CH4 by the culture. Radioactivity was equally divided between the CH4 and CO2 from the degradation of uniformly ring-labeled [14C]benzoate. The methyl group of acetate was stoichiometrically converted to CH4. Acetate, cyclohexanecarboxylate, 2-hydroxycyclohexanecarboxylate, o-hydroxybenzoic acid and pimelic acid were converted to CH4 without a lag suggesting that benzoate was degraded by a reductive pathway. Addition of o-chlorobenzoate inhibited benzoate degradation but not acetate degradation or methane formation. Two methanogenic organisms were isolated from the mixed culture, neither organism was able to degrade benzoate, showing that the methanogenic bacteria served as terminal organisms of a metabolic food chain composed of several organisms. Removal of intermediates by the methanogenic bacteria provided thermodynamically favorable conditions for benzoate degradation.

An unconventional pathway for reduction of CO2 to methane in CO-grown Methanosarcina acetivorans revealed by proteomics

Methanosarcina acetivorans produces acetate, formate, and methane when cultured with CO as the growth substrate [Rother M, Metcalf WW (2004) Proc Natl Acad Sci USA 101:16929–16934], which suggests novel features of CO metabolism. Here we present a genome-wide proteomic approach to identify and quantify proteins differentially abundant in response to growth on CO versus methanol or acetate. The results indicate that oxidation of CO to CO2 supplies electrons for reduction of CO2 to a methyl group by steps and enzymes of the pathway for CO2 reduction determined for other methane-producing species. However, proteomic and quantitative RT-PCR results suggest that reduction of the methyl group to methane involves novel methyltransferases and a coenzyme F420H2:heterodisulfide oxidoreductase system that generates a proton gradient for ATP synthesis not previously described for pathways reducing CO2 to methane. Biochemical assays support a role for the oxidoreductase, and transcriptional mapping identified an unusual operon structure encoding the oxidoreductase. The proteomic results further indicate that acetate is synthesized from the methyl group and CO by a reversal of initial steps in the pathway for conversion of acetate to methane that yields ATP by substrate level phosphorylation. The results indicate that M. acetivorans utilizes a pathway distinct from all known CO2 reduction pathways for methane formation that reflects an adaptation to the marine environment. Finally, the pathway supports the basis for a recently proposed primitive CO-dependent energy-conservation cycle that drove and directed the early evolution of life on Earth.

Methanospirillum, a New Genus of Methanogenic Bacteria, and Characterization of Methanospirillum hungatii sp.nov.

A new genus of methanogenic bacteria is described. The colonies produced by these bacteria are yellow, circular, and convex with lobate margins; an optical pattern of regular, light and dark striations throughout the colonies is a most unique and distinguishing characteristic. These striations are two cell lengths apart. Cells are gram negative and occur in filaments up to 100 μm in length. Tufts of polar flagella and a striated cell surface are revealed in electron micrographs; cell ends are blunt, not rounded. The deoxyribonucleic acid base composition of the type species is 45 mol% guanine plus cytosine. Formate or hydrogen and carbon dioxide serves as a substrate for methane formation and growth; acetate, pyruvate, methanol, ethanol, and benzoate do not. The name Methanospirillum is proposed for this new genus of spiral-shaped methanogenic bacteria. The type species, Methanospirillum hungatii sp. nov., is named in honor of R. E. Hungate. The type strain of M. hungatii is JF1 (ATCC 27890).

How to Make a Living by Exhaling Methane

Methane produced in the biosphere is derived from two major pathways. Conversion of the methyl group of acetate to CH(4) in the aceticlastic pathway accounts for at least two-thirds, and reduction of CO(2) with electrons derived from H(2), formate, or CO accounts for approximately one-third. Although both pathways have terminal steps in common, they diverge considerably in the initial steps and energy conservation mechanisms. Steps and enzymes unique to the CO(2) reduction pathway are confined to methanogens and the domain Archaea. On the other hand, steps and enzymes unique to the aceticlastic pathway are widely distributed in the domain Bacteria, the understanding of which has contributed to a broader understanding of prokaryotic biology.

The γ Class of Carbonic Anhydrases

Homologs of the gamma class of carbonic anhydrases, one of five independently evolved classes, are found in the genomic sequences of diverse species from all three domains of life. The archetype (Cam) from the Archaea domain is a homotrimer of which the crystal structure reveals monomers with a distinctive left-handed parallel beta-helix fold. Histidines from adjacent monomers ligate the three active site metals surrounded by residues in a hydrogen bond network essential for activity. Cam is most active with iron, the physiologically relevant metal. Although the active site residues bear little resemblance to the other classes, kinetic analyses indicate a two-step mechanism analogous to all carbonic anhydrases investigated. Phylogenetic analyses of Cam homologs derived from the databases show that Cam is representative of a minor subclass with the great majority belonging to a subclass (CamH) with significant differences in active site residues and apparent mechanism from Cam. A physiological function for any of the Cam and CamH homologs is unknown, although roles in transport of carbon dioxide and bicarbonate across membranes has been proposed.

Gamma carbonic anhydrases in plant mitochondria

Three genes from Arabidopsis thaliana with high sequence similarity to gamma carbonic anhydrase (γCA), a Zn containing enzyme from Methanosarcina thermophila(CAM), were identified and characterized. Evolutionary and structural analyses predict that these genes code for active forms of γCA. Phylogenetic analyses reveal that these Arabidopsis gene products cluster together with CAM and related sequences from α and γ proteobacteria, organisms proposed as the mitochondrial endosymbiont ancestor. Indeed, in vitro and in vivo experiments indicate that these gene products are transported into the mitochondria as occurs with several mitochondrial protein genes transferred, during evolution, from the endosymbiotic bacteria to the host genome. Moreover, putative CAM orthologous genes are detected in other plants and green algae and were predicted to be imported to mitochondria. Structural modeling and sequence analysis performed in more than a hundred homologous sequences show a high conservation of functionally important active site residues. Thus, the three histidine residues involved in Zn coordination (His 81, 117 and 122), Arg 59, Asp 61, Gin 75, and Asp 76 of CAM are conserved and properly arranged in the active site cavity of the models. Two other functionally important residues (Glu 62 and Glu 84 of CAM) are lacking, but alternative amino acids that might serve to their roles are postulated. Accordingly, we propose that photosynthetic eukaryotic organisms (green algae and plants) contain γCAs and that these enzymes codified by nuclear genes are imported into mitochondria to accomplish their biological function.