Enzymology of one-carbon metabolism in methanogenic pathways.

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.

Formate dehydrogenase.

Formate is a substrate, or product, of diverse reactions catalyzed by eukaryotic organisms, eubacteria, and archaebacteria. A survey of metabolic groups reveals that formate is a common growth substrate, especially among the anaerobic eubacteria and archaebacteria. Formate also functions as an accessory reductant for the utilization of more complex substrates, and an intermediate in energy-conserving pathways. The diversity of reactions involving formate dehydrogenases is apparent in the structures of electron acceptors which include pyridine nucleotides, 5-deazaflavin, quinones, and ferredoxin. This diversity of electron acceptors is reflected in the composition of formate dehydrogenase. Studies on these enzymes have contributed to the biochemical and genetic understanding of selenium, molybdenum, tungsten, and iron in biology. The regulation of formate dehydrogenase synthesis serves as a model for understanding general principles of regulation in anaerobic organisms.

Prokaryotic carbonic anhydrases.

Carbonic anhydrases catalyze the reversible hydration of CO(2) [CO(2)+H(2)Oright harpoon over left harpoon HCO(3)(-)+H(+)]. Since the discovery of this zinc (Zn) metalloenzyme in erythrocytes over 65 years ago, carbonic anhydrase has not only been found in virtually all mammalian tissues but is also abundant in plants and green unicellular algae. The enzyme is important to many eukaryotic physiological processes such as respiration, CO(2) transport and photosynthesis. Although ubiquitous in highly evolved organisms from the Eukarya domain, the enzyme has received scant attention in prokaryotes from the Bacteria and Archaea domains and has been purified from only five species since it was first identified in Neisseria sicca in 1963. Recent work has shown that carbonic anhydrase is widespread in metabolically diverse species from both the Archaea and Bacteria domains indicating that the enzyme has a more extensive and fundamental role in prokaryotic biology than previously recognized. A remarkable feature of carbonic anhydrase is the existence of three distinct classes (designated alpha, beta and gamma) that have no significant sequence identity and were invented independently. Thus, the carbonic anhydrase classes are excellent examples of convergent evolution of catalytic function. Genes encoding enzymes from all three classes have been identified in the prokaryotes with the beta and gamma classes predominating. All of the mammalian isozymes (including the 10 human isozymes) belong to the alpha class; however, only nine alpha class carbonic anhydrase genes have thus far been found in the Bacteria domain and none in the Archaea domain. The beta class is comprised of enzymes from the chloroplasts of both monocotyledonous and dicotyledonous plants as well as enzymes from phylogenetically diverse species from the Archaea and Bacteria domains. The only gamma class carbonic anhydrase that has thus far been isolated and characterized is from the methanoarchaeon Methanosarcina thermophila. Interestingly, many prokaryotes contain carbonic anhydrase genes from more than one class; some even contain genes from all three known classes. In addition, some prokaryotes contain multiple genes encoding carbonic anhydrases from the same class. The presence of multiple carbonic anhydrase genes within a species underscores the importance of this enzyme in prokaryotic physiology; however, the role(s) of this enzyme is still largely unknown. Even though most of the information known about the function(s) of carbonic anhydrase primarily relates to its role in cyanobacterial CO(2) fixation, the prokaryotic enzyme has also been shown to function in cyanate degradation and the survival of intracellular pathogens within their host. Investigations into prokaryotic carbonic anhydrase have already led to the identification of a new class (gamma) and future research will undoubtedly reveal novel functions for carbonic anhydrase in prokaryotes.

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.

Reductive dechlorination of trichloroethylene by the CO-reduced CO dehydrogenase enzyme complex from Methanosarcina thermophila.

Trichloroethylene (TCE) was reductively dechlorinated to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene by the CO-reduced CO dehydrogenase enzyme complex from Methanosarcina thermophila; the apparent Km and Vmax values were 1.7 +/- 0.3 mM TCE and 26.2 +/- 1.7 mol TCE dechlorinated/min/mmol factor III. Factor III also catalysed the dechlorination of TCE when in the presence of titanium(III) citrate; the apparent Km and Vmax values were 1.2 +/- 0.3 mM TCE and 34.9 +/- 3.6 mol TCE dechlorinated/min/mmol factor III. The enzyme complex was resolved into the two-subunit nickel/iron-sulfur (Ni/Fe-S) component and the two-subunit factor III-containing corrinoid/iron-sulfur (Co/Fe-S) component. The Ni/Fe-S component was unable to dechlorinate TCE in the presence of CO; however, reconstitution with the Co/Fe-S component yielded the same dechlorinated products as with the CO dehydrogenase enzyme complex.

Cysteine biosynthesis in the Archaea: Methanosarcina thermophila utilizes O-acetylserine sulfhydrylase.

Two pathways for cysteine biosynthesis are known in nature; however, it is not known which, if either, the Archaea utilize. Enzyme activities in extracts of Methanosarcina thermophila grown with combinations of cysteine and sulfide as sulfur sources indicated that this archaeon utilizes the pathway found in the Bacteria domain. The genes encoding serine transacetylase and O-acetylserine sulfhydrylase (cysE and cysK) are adjacent on the chromosome of M. thermophila and possibly form an operon. When M. thermophila is grown with cysteine as the sole sulfur source, O-acetylserine sulfhydrylase activity is maximally expressed suggesting alternative roles for this enzyme apart from cysteine biosynthesis.

Identification of molybdopterin guanine dinucleotide in formate dehydrogenase from Methanobacterium formicicum.

The pterin cofactor in formate dehydrogenase isolated from Methanobacterium formicium is identified as molybdopterin guanine dinucleotide. The pterin, stabilized as the alkylated, dicarboxamidomethyl derivative, is shown to have absorption and chromatographic properties identical to those of the previously characterized authentic compound. Treatment with nucleotide pyrophosphatase produced the expected degradation products GMP and carboxyamidomethyl molybdopterin. The molybdopterin guanine dinucleotide released from the enzyme by treatment with 95% dimethyl sulfoxide is shown to be functional in the in vitro reconstitution of the cofactor-deficient nitrate reductase in extracts of the Neurospora crassa nit-1 mutant.

Enzymology of the fermentation of acetate to methane by Methanosarcina thermophila.

Biologically-produced CH4 derives from either the reduction of CO2 or the methyl group of acetate by two separate pathways present in anaerobic mierobes from the Archaea domain. Elucidation of the pathway for CO2 reduction to CH4, the first to be investigated, has yielded several novel enzymes and cofactors. Most of the CH4 produced in nature derives from the methyl group of acetate. Methanosarcina thermophila is a moderate thermophile which ferments acetate by reducing the methyl group to CH4 with electrons derived from oxidation of the carbonyl group to CO2. The pathway in M. thermophila is now understood on a biochemical and genetic level comparable to understanding of the CO2-reducing pathway. Enzymes have been purified and characterized. The genes encoding these enzymes have been cloned, sequenced, transcriptionally mapped, and their regulation defined on a molecular level. This review emphasizes recent developments concerning the enzymes which are unique to the acetate fermentation pathway in M. thermophila.

Digesta flows in sheep fed poor-quality hay supplemented with urea and carbohydrates.

Two metabolism trials were conducted with 12 yearling crossbred wethers per trial (34 and 38 kg for trials 1 and 2, respectively). The wethers, equipped with ruminal, abomasal and ileal cannulae, were randomly allotted for each trial to the following treatments: 1) hay alone or hay supplemented with 2) .9% urea, 3) 1% urea and 6.5% molasses or 4) 1% urea and 5.2% corn. Two digestive flow markers were used: Cr2O3 powder and Co-ethylenediaminetetraacetic acid (Co-EDTA). Urea and Co-EDTA were infused continuously into the rumen via cannula. Daily dry matter (DM) intake averaged 517 g. Urea supplementation improved N retention (P less than .01). Apparent digestibility of DM, acid detergent fiber (ADF) and energy was not affected by treatment. Urea and carbohydrate supplementation increased ruminal propionic acid molar proportions (P less than .05). Apparent ruminal DM digestion accounted for 41% of the total DM degraded, whereas 77.4% of the digestible ADF was degraded in the rumen. Urea supplementation increased ADF digestion in the large intestine (P less than .01). Urea and carbohydrate supplementation resulted in a stepwise increase in N flowing with the liquid phase at the abomasum. Mean retention times of the solid and liquid phases of digestive contents were similar across treatments. Overall, benefits of supplementation of poor-quality fescue hay diets by small amounts of urea and readily available carbohydrates remain questionable for sheep fed at a fixed level of intake below maintenance.

The beta and gamma classes of carbonic anhydrase.

There are currently five (alpha, beta, gamma, delta, zeta) classes of carbonic anhydrases (CA's) of which the alpha-class from mammalian sources has been studied to a much greater extent compared to the other four classes. Yet, CA's other than the alpha-class are widely distributed in Nature and play important roles in human health, the global carbon cycle, and industrial applications. In aerobic prokaryotes, beta-class CA's are implicated in maintaining internal pH and CO(2)/bicarbonate balances required for biosynthetic reactions. In anaerobic prokaryotes, beta-class CA's are implicated in the transport of CO(2) and bicarbonate across the cytoplasmic membrane that regulates pH and facilitates acquisition of substrates and product removal required for growth. In phototrophic organisms, beta-class CA's are particularly important for transport and concentration of CO(2) and bicarbonate for photosynthesis. The delta- and zeta-classes are proposed to function in marine diatoms to concentrate CO(2) for photosynthesis. Physiological roles for the gamma-class are not as well documented; however, the active site architecture and catalytic mechanism is well understood as are patterns of inhibition by sulfonamides and anions.

CO dehydrogenase.

Structurally and functionally diverse CO dehydrogenases are key components of various energy-yielding pathways in aerobic and anaerobic microbes from the Bacteria and Archaea domains. Aerobic microbes utilize Mo-Fe-flavin CO dehydrogenases to oxidize CO in respiratory pathways. Phototrophic anaerobes grow by converting CO to H2, a process initiating with a CO dehydrogenase that contains nickel and iron-sulfur centers. Acetate-producing anaerobes employ a nickel/iron-sulfur CO dehydrogenase to synthesize acetyl-CoA from a methyl group, CO, and CoA. A similar enzyme is responsible for the cleavage of acetyl-CoA by anaerobic Archaea that obtain energy by fermenting acetate to CH4 and CO2. Acetotrophic sulfate reducers from the Bacteria and Archaea also utilize CO dehydrogenase to cleave acetyl-CoA yielding methyl and carbonyl groups. These microbes obtain energy for growth via a respiratory pathway in which the methyl and carbonyl groups are oxidized to CO2, and sulfate is reduced to sulfide.

Methane from acetate.

The general features are known for the pathway by which most methane is produced in nature. All acetate-utilizing methanogenic microorganisms contain CODH which catalyzes the cleavage of acetyl-CoA; however, the pathway differs from all other acetate-utilizing anaerobes in that the methyl group is reduced to methane with electrons derived from oxidation of the carbonyl group of acetyl-CoA to CO2. The current understanding of the methanogenic fermentation of acetate provides impressions of nature's novel solutions to problems of methyl transfer, electron transport, and energy conservation. The pathway is now at a level of understanding that will permit productive investigations of these and other interesting questions in the near future.

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.

Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.

The genes for the acetate-activating enzymes, acetate kinase and phosphotransacetylase (ack and pta), from Methanosarcina thermophila TM-1 were cloned and sequenced. Both genes are present in only one copy per genome, with the pta gene adjacent to and upstream of the ack gene. Consensus archaeal promoter sequences are found upstream of the pta coding region. The pta and ack genes encode predicted polypeptides with molecular masses of 35,198 and 44,482 Da, respectively. A hydropathy plot of the deduced phosphotransacetylase sequence indicates that it is a hydrophobic polypeptides; however, no membrane-spanning domains are evident. Comparison of the amino acid sequences deduced from the M. thermophila and Escherichia coli ack genes indicate similar subunit molecular weights and 44% identity (60% similarity). The comparison also revealed the presence of several conserved arginine, cysteine, and glutamic acid residues. Arginine, cysteine, and glutamic acid residues have previously been implicated at or near the active site of the E. coli acetate kinase. The pta and ack genes were hyperexpressed in E. coli, and the overproduced enzymes were purified to homogeneity with specific activities higher than those of the enzymes previously purified from M. thermophila. The overproduced phosphotransacetylase and acetate kinase migrated at molecular masses of 37,000 and 42,000 Da, respectively. The activity of the acetate kinase is optimal at 65 degrees C and is protected from thermal inactivation by ATP. Diethylpyrocarbonate and phenylglyoxal inhibited acetate kinase activity in a manner consistent with the presence of histidine and arginine residues at or near the active site; however, the thiol-directed reagents 5,5'-dithiobis (2-nitrobenzoic acid) and N-ethylmaleimide were ineffective.

Initial steps in the anaerobic degradation of 3,4,5-trihydroxybenzoate by Eubacterium oxidoreducens: characterization of mutants and role of 1,2,3,5-tetrahydroxybenzene.

Chemical mutagenesis and antibiotic enrichment techniques were used to isolate five mutant strains of the obligate anaerobe Eubacterium oxidoreducens that were unable to grow on 3,4,5-trihydroxybenzoate (gallate). Two strains could not transform gallate and showed no detectable gallate decarboxylase activity. Two other strains transformed gallate to pyrogallol and dihydrophloroglucinol but lacked the hydrolase activity responsible for ring cleavage. A fifth strain accumulated pyrogallol, although it contained adequate levels of the enzymes proposed for the complete transformation of gallate to the ring cleavage product. The conversion of pyrogallol to phloroglucinol by cell extract of the wild-type strain was dependent on the addition of 1,2,3,5-tetrahydroxybenzene or dimethyl sulfoxide. This activity was induced by growth on gallate, while the other enzymes involved in the initial reactions of gallate catabolism were constitutively expressed during growth on crotonate. The results confirm the initial steps in the pathway previously proposed for the metabolism of gallate by E. oxidoreducens, except for the conversion of pyrogallol to phloroglucinol.

Purification and properties of methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila.

Methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila TM-1 was purified 16-fold from a cell extract to apparent homogeneity as determined by native polyacrylamide gel electrophoresis. Ninety-four percent of the methylreductase activity was recovered in the soluble fraction of cell extracts. The estimated native molecular weight of the enzyme was between 132,000 (standard deviation [SD], 1,200) and 141,000 (SD, 1,200). Denaturing polyacrylamide gel electrophoresis revealed three protein bands corresponding to molecular weights of 69,000 (SD, 1,200), 42,000 (SD, 1,200), and 33,000 (SD, 1,200) and indicated a subunit configuration of alpha 1 beta 1 gamma 1. As isolated, the enzyme was inactive but could be reductively reactivated with titanium (III) citrate or reduced ferredoxin. ATP stimulated enzyme reactivation and was postulated to be involved in a conformational change of the inactive enzyme from an unready state to a ready state that could be reductively reactivated. The temperature and pH optima for enzyme activity were 60 degrees C and between 6.5 and 7.0, respectively. The active enzyme contained 1 mol of coenzyme F430 per mol of enzyme (Mr, 144,000). The Kms for 2-(methylthio)ethane-sulfonate and 7-mercaptoheptanoylthreonine phosphate were 3.3 mM and 59 microM, respectively.

Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinase-encoding genes (pta and ack) from Methanosarcina thermophila.

Phosphotransacetylase and acetate kinase catalyze the activation of acetate to acetyl coenzyme A in the first step of methanogenesis from acetate in Methanosarcina thermophila. The genes encoding these enzymes (pta and ack) have been cloned and sequenced. They are arranged on the chromosome with pta upstream of ack (M.T. Latimer, and J. G. Ferry, J. Bacteriol. 175:6822-6829, 1993). The activities of phosphotransacetylase and acetate kinase are at least 8- to 11-fold higher in acetate-grown cells than in cells grown on methanol, monomethylamine, dimethylamine, or trimethylamine. Northern blot (RNA) analyses demonstrated that pta and ack are transcribed as an approximately 2.4-kb polycistronic message and that the regulation of enzyme synthesis occurs at the mRNA level. Primer extension analyses revealed a transcriptional start site located 27 bp upstream from the translational start of the pta gene and 24 bp downstream from a consensus archaeal boxA promoter sequence. S1 nuclease protection assays detected transcripts with four different 3' ends, each of which mapped to the beginning of four consecutive direct repeats. Northern blot analysis using an ack-specific probe detected both the 2.4-kb polycistronic transcript and a smaller 1.4-kb transcript which is the estimated size of monocistronic ack mRNA. A primer extension product was detected with an ack-specific primer; the 5' end of the product was in the intergenic region between the pta and ack genes but did not follow a consensus archaeal boxA sequence. This result, as well as detection of an additional 1.4-kb mRNA species, suggests processing of the polycistronic 2.4-kb transcript.

Characterization of the cdhD and cdhE genes encoding subunits of the corrinoid/iron-sulfur enzyme of the CO dehydrogenase complex from Methanosarcina thermophila.

The CO dehydrogenase enzyme complex from Methanosarcina thermophila contains a corrinoid/iron-sulfur enzyme composed of two subunits (delta and gamma). The cdhD and cdhE genes, which encode the delta and gamma subunits, respectively, were cloned and sequenced. The cdhD gene is upstream of and separated by 3 bp from cdhE. Both genes are preceded by apparent ribosome-binding sites. Northern (RNA) blot and primer extension analyses indicated that cdhD and cdhE are cotranscribed from a promoter located several kilobases upstream of cdhD. The putative CdhD and CdhE sequences are 37% identical to the sequences deduced from the genes encoding the beta and alpha subunits of the corrinoid/iron-sulfur enzyme from Clostridium thermoaceticum. The CdhE sequence had a four-cysteine motif with the potential to bind a 4Fe-4S cluster previously identified in the corrinoid/iron-sulfur enzyme by electron paramagnetic resonance spectroscopy. A T7 RNA polymerase/promoter system was used to produce CdhD and CdhE independently in Escherichia coli. The purified CdhD protein was reconstituted with hydroxocobalamin in the base-on configuration. The purified CdhE protein exhibited an Fe-S center and base-off cobalamin binding in which the benzimidazole base nitrogen atom was no longer a lower axial ligand to the cobalt atom.