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Life with carbon monoxide.

This review focuses on how microbes live on CO as a sole source of carbon and energy and with CO by generating carbon monoxide as a metabolic intermediate. The use of CO is a property of organisms that use the Wood-L jungdahl pathway of autotrophic growth. The review discusses when CO metabolism originated, when and how it was discovered, and what properties of CO are ideal for microbial growth. How CO sensing by a heme-containing transcriptional regulatory protein activates the expression of CO metabolism-linked genes is described. Two metalloenzymes are the cornerstones of growth with CO: CO dehydrogenase (CODH) and acetyl-CoA synthase (ACS). CODH oxidizes CO to CO2, providing low-potential electrons for the cell, or alternatively reduces CO2 to CO. The latter reaction, when coupled to ACS, forms a machine for generating acetyl-CoA from CO2 for cell carbon synthesis. The recently solved crystal structures of CODH and ACS along with spectroscopic measurements and computational studies provide insights into novel bio-organometallic catalytic mechanisms and into the nature of a 140 A gas channel that coordinates the generation and utilization of CO. The enzymes that are coupled to CODH/ACS are also described, with a focus on a corrinoid protein, a methyltransferase, and pyruvate ferredoxin oxidoreductase.

Acetate-CoA Ligase↗

Noncobalamin vitamin B12 analogues in human red cells, liver, and brain.

Analogues of vitamin B12 which appear to be noncobalamin corrinoids appear to be present in human red cells, liver, and brain. Their sources, nature, and effects require study, particularly with reference to their positive and/or negative effects on vitamin B12 metabolism. In normal persons, they are concentrated in liver, with only small quantities in red cells and still smaller quantities in brain. Their concentrations in disease states will be of interest, particularly in persons with varying degrees of neurological damage associated with deficiencies in vitamin B12 or analogue metabolism.

Adult↗

The biosynthesis of vitamin B12.

The use of 13C-Fourier transform nuclear magnetic resonance (F.t.-n.m.r.) has led to the observation that while 8 molecules of [2-13C]ALA are incorporated into vitamin B12 in P. shermanii, [5-13C]ALA labels only seven of the carbon atoms of cyanocobalamin, i.e. one of the amino methyl groups of ALA is "lost" in the process. It has also been confirmed that seven of the methyl groups of B12 are derived from 13CH3-enriched methionine and further that the chirality of the gemdimethyl grouping at C12 labelled with [13CH3]methionine is R. A soluble enzyme mixture from the 37000 or 100000 g supernatant of disrupted cells of P. shermanii converts both 14 C-labelled ALA and [14C]uro'gen III to cobyrinic acid, the simplest corrinoid material on the pathway to vitamin B12 and the coenzyme, in presence of NADPH, Co2+, Mg2+, S-adenosyl-methionine and glutathione. Multiply-labelled uro'gens (13C, 14C and 3H) have been used to show that incorporation takes place without randomization. A sequence for corrin synthesis from uro'gen III is presented.

Aminolevulinic Acid↗

Autotrophic synthesis of activated acetic acid from CO2 in Methanobacterium thermoautotrophicum. Synthesis from tetrahydromethanopterin-bound C1 units and carbon monoxide.

The synthesis of acetyl-CoA from CO2, H2, and various C1 compounds was studied in vitro with extracts and with protein fractions of Methanobacterium thermoautotrophicum. Acetyl-CoA synthesis from CO2 and H2 by extracts required CO2 reduction to CH4 to proceed. Both processes were highly stimulated by formaldehyde which served as the carbon precursor of both CH4 and the CH3 group of acetate. Carbon monoxide in combination with formaldehyde dramatically stimulated the acetyl-CoA synthesis up to 150-fold. In this system, which did not require CO2 reduction to the formaldehyde and CO level, acetyl-CoA synthesis was no longer dependent on CH4 formation. The soluble (100,000 X g supernatant) cell protein was resolved into a protein fraction [45-60% (NH4)2SO4-fraction] which catalyzed acetyl-CoA synthesis at a specific rate of 15 nmol X min-1 X (equivalent of mg cell protein)-1 (60 degrees C). This oxygen-sensitive enzyme reaction required dithioerythritol for activity and was strictly dependent on coenzyme A, CO, and N5,N10-methylene tetrahydromethanopterin, N5-methyl tetrahydromethanopterin or formaldehyde plus tetrahydromethanopterin. The incorporation of formaldehyde is explained by the spontaneous formation of methylene tetrahydromethanopterin. The product of the reaction, acetyl-CoA, was quantitatively derived from CO (carboxyl of acetate) and a C1 derivative of tetrahydromethanopterin (methyl of acetate). The C1 derivative of tetrahydromethanopterin could not be replaced by a C1 derivative of tetrahydrofolate or by methyl-coenzyme M; ATP was not required. The active protein fraction contained CO dehydrogenase and at least on corrinoid protein. These results provide strong biochemical arguments for the proposed mechanism of autotrophic acetyl-CoA synthesis in Methanobacterium.

Acetyl Coenzyme A↗

The final step in methane formation. Investigations with highly purified methyl-CoM reductase (component C) from Methanobacterium thermoautotrophicum (strain Marburg).

Methyl-coenzyme M reductase (= component C) from Methanobacterium thermoautotrophicum (strain Marburg) was highly purified via anaerobic fast protein liquid chromatography on columns of Mono Q and Superose 6. The enzyme was found to catalyze the reduction of methylcoenzyme M (CH3-S-CoM) with N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP = component B) to CH4. The mixed disulfide of H-S-CoM and H-S-HTP (CoM-S-S-HTP) was the other major product formed. The specific activity was up to 75 nmol min-1 mg protein-1. In the presence of dithiothreitol and of reduced corrinoids or titanium(III) citrate the specific rate of CH3-S-CoM reduction to CH4 with H-S-HTP increased to 0.5-2 mumol min-1 mg protein-1. Under these conditions the CoM-S-S-HTP formed from CH3-S-CoM and H-S-HTP was completely reduced to H-S-CoM and H-S-HTP. Methyl-CoM reductase was specific for H-S-HTP as electron donor. Neither N-6-mercaptohexanoylthreonine phosphate (H-S-HxoTP) nor N-8-mercaptooctanoylthreonine phosphate (H-S-OcoTP) nor any other thiol compound could substitute for H-S-HTP. On the contrary, H-S-HxoTP (apparent Ki = 0.1 microM) and H-S-OcoTP (apparent Ki = 15 microM) were found to be effective inhibitors of methyl-CoM reductase, inhibition being non-competitive with CH3-S-CoM and competitive with H-S-HTP.

Disulfides↗

The energetics and sodium-ion dependence of N5-methyltetrahydromethanopterin:coenzyme M methyltransferase studied with cob(I)alamin as methyl acceptor and methylcob(III)alamin as methyl donor.

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane-associated enzyme complex that uses a methyl-transfer reaction to drive an energy-conserving sodium-ion pump. Methyl transfer occurs in two steps, first from N5-methyltetrahydromethanopterin (CH3-H4MPT) to an enzyme-bound cob(I)amide prosthetic group, and secondly from the methylated cobamide to coenzyme M (H-S-CoM). In this study, we report that methyltransferase can also use exogenous cob(I)alamin and methylcob(III)alamin as methyl acceptor and methyl donor, respectively. The enzyme catalyzes methylcob(III)alamin formation from CH3-H4MPT and cob(I)alamin (reaction a), and methyl-coenzyme M formation from methylcob(III)alamin and H-S-CoM (reaction b). Both reactions were shown to be reversible. Reaction a was catalyzed at approximately the same rate (3 U/mg) and reaction b at approximately 10% the rate (0.3 U/mg) of the physiological reaction, namely methyl transfer from CH3-H4MPT to H-S-CoM. The free energy changes (delta G0') associated with reactions a and b were both between -10 kJ/mol and -20 kJ/mol, consistent with a free energy change of approximately -30 kJ/mol determined for the physiological reaction. Reaction b but not reaction a was sodium-ion dependent. Assuming that methylation of exogenous cob(I)alamin and demethylation of exogenous methylcob(III)alamin mimic methylation and demethylation of the enzyme-bound corrinoid prosthetic group, it can be inferred that methyl transfer from the methylated cobamide prosthetic group to H-S-CoM is a site of coupling with sodium-ion translocation.

Energy Metabolism↗

O-demethylation by the homoacetogenic anaerobe Holophaga foetida studied by a new photometric methylation assay using electrochemically produced cob(I)alamin.

The previously studied complete methyl transfer sequence of tetrahydrofolate-dependent O-demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non-physiological substrates: electrochemically produced cob(I) alamin served as methyl acceptor for phenyl methyl ether demethylation, yielding methylcob(III)alamin (reaction I), and methylcob(III)alamin served as donor for tetrahydrofolate methylation, yielding 5-methyl tetrahydrofolate (reaction II). Both reactions were measured with a new and direct photometric assay of cob(I)alamin methylation (or the reverse reaction) at 540 nm, the isobestic wavelength of the cob(II)alamin/cob(I)alamin redox couple (delta epsilon 540 = 4.40 nM-1.cm-1. The rates of reactions I and II were proportional to protein concentration, unlike the complete reaction sequence. Small components of cell extract did not affect activity of reactions I and II. Isovanillate demethylation by extracts of synringate-grown cells (reaction I) required reductive activation by cob(I)alamin and was inhibited and inactivated by cob(II)alamin, indicating that the reaction mechanism was a nucleophilic attack of an enzyme-bound corrinoid in the reduced Co(I) state on the methyl carbon of the ether, rather than a radical attack. Only phenyl methyl ethers were demethylated; demethylation rates were enhanced by ortho-hydroxyl or para-carboxyl groups, but reduced by additional meta substituents. The rate of isovanillate demethylation was 81 nmol.min-1.(mg protein)-1 [0.76 mM cob(I)alamin] and apparent kinetic constants for cob(I)alamin were: Km = 1.2 mM, Vmax = 220 nmol min-1.(mg protein)-1, and Vmax/Km = 180 nmol.min-1.(mg protein) 1.mM-1 3,5-Dihydroxyanisole demethylation by extracts of 3,5-dihydroxyanisole-grown cells (also reaction I) was much slower. Reaction II did not require activation; specific activity and the specificity constant for methylcob(III)alamin were much lower.

Anisoles↗

Sodium ion translocation by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase from Methanosarcina mazei Gö1 reconstituted in ether lipid liposomes.

The N5-methyltetrahydromethanopterin (H,MPT):coenzyme M methyltransferase is a membrane associated, corrinoid-containing protein that uses the methylation of coenzyme M (HS-CoM) by methyl-tetrahydromethanopterin to drive an energy-conserving sodium ion pump. The enzyme was purified from acetate-grown Methanosarcina mazei Gö1 by a two-step solubilization with n-octyl-beta-glucoside, chromatography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL-6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 microM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei Gö1. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na-/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methyl-cobalamin could substitute for methyl-H,MPT.

Amino Acid Sequence↗

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri. Zinc dependence and thermodynamics of the methanol:cob(I)alamin methyltransferase reaction.

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl-coenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated methyltransferases 1 (MT1) and 2 (MT2). Transferase MT1, which is composed of a 50-kDa subunit, MtaB, and a 27-kDa corrinoid-harbouring subunit, MtaC, has been shown recently to catalyze the methylation of free cob(I)alamin with methanol [Sauer, K., Harms, U. & Thauer, R. K. (1997) Eur. J. Biochem. 243, 670-677]. We report here that this reaction is catalyzed by subunit MtaB overproduced in Escherichia coli. MtaB also catalyzed the formation of methanol from methylcobalamin and H2O, the hydrolysis being associated with a free-energy change deltaG(o)' of approximately +7.0 kJ/mol. MtaB was found to contain 1 mol zinc, and its activity to be zinc dependent (pK(Zn2+) = 9.3). The zinc dependence of the MT2 (MtaA)-catalyzed reaction is also described (pK(Zn2+) = 9.6).

Archaeal Proteins↗

Chemistry of methylcorrinoids related to their roles in bacterial C1 metabolism.

Corrinoids are central cofactors in bacterial metabolism, where they participate in a series of organometallic and redoxprocesses. These depend on the unique coordination chemistry and reactivity of the corrin-bound cobalt centers to which, in the complete corrins, also a nucleotide function can coordinate intramolecularly. The roles of methylcorrinoids in bacterial C1 metabolism focus around the unusual Co-C-bond.

Carbon↗

Growth temperature-dependent activity of glycerol dehydratase in Escherichia coli expressing the Citrobacter freundii dha regulon.

Using the cosmid pWE15, a genomic library of Citrobacter freundii DNA in Escherichia coli ECL707 was prepared and screened for glycerol utilization. Six out of approximately 3000 clones were positive. One clone, harboring the recombinant cosmid pRD1, expressed glycerol dehydratase in high activity when grown at 28 degrees C but not at 37 degrees C. The growth temperature had little effect on the activity of the other enzymes encoded by the dha regulon. When the glycerol-containing medium was supplemented with corrinoids, the recombinant E. coli strain produced 1,3-propanediol in high amounts at 28 degrees C.

Citrobacter freundii↗

Single-carbon chemistry of acetogenic and methanogenic bacteria.

Methanogenic and acetogenic bacteria metabolize carbon monoxide, methanol, formate, hydrogen and carbon dioxide gases and, in the case of certain methanogens, acetate, by single-carbon (C1) biochemical mechanisms. Many of these reactions occur while the C1 compounds are linked to pteridine derivatives and tetrapyrrole coenzymes, including corrinoids, which are used to generate, reduce, or carbonylate methyl groups. Several metalloenzymes, including a nickel-containing carbon monoxide dehydrogenase, are used in both catabolic and anabolic oxidoreductase reactions. We propose biochemical models for coupling carbon and electron flow to energy conservation during growth on C1 compounds based on the carbon flow pathways inherent to acetogenic and methanogenic metabolism. Biological catalysts are therefore available which are comparable to those currently in use in the Monsanto process. The potentials and limitations of developing biotechnology based on these organisms or their enzymes and coenzymes are discussed.

Acetates↗

Importance of cobalt for individual trophic groups in an anaerobic methanol-degrading consortium.

Methanol is an important anaerobic substrate in industrial wastewater treatment and the natural environment. Previous studies indicate that cobalt greatly stimulates methane formation during anaerobic treatment of methanolic wastewaters. To evaluate the effect of cobalt in a mixed culture, a sludge with low background levels of cobalt was cultivated in an upflow anaerobic sludge blanket reactor. Specific inhibitors in batch assays were then utilized to study the effect of cobalt on the growth rate and activity of different microorganisms involved in the anaerobic degradation of methanol. Only methylotrophic methanogens and acetogens were stimulated by cobalt additions, while the other trophic groups utilizing downstream intermediates, H2-CO2 or acetate, were largely unaffected. The optimal concentration of cobalt for the growth and activity of methanol-utilizing methanogens and acetogens was 0.05 mg liter-1. The higher requirement of cobalt is presumably due to the previously reported production of unique corrinoid-containing enzymes (or coenzymes) by direct utilizers of methanol. This distinctly high requirement of cobalt by methylotrophs should be considered during methanolic wastewater treatment. Methylotroph methanogens presented a 60-fold-higher affinity for methanol than acetogens. This result in combination with the fact that acetogens grow slightly faster than methanogens under optimal cobalt conditions indicates that acetogens can outcompete methanogens only when reactor methanol and cobalt concentrations are high, provided enough inorganic carbon is available.

Anaerobiosis↗

Metabolism of Dichloromethane by the Strict Anaerobe Dehalobacterium formicoaceticum.

The metabolism of dichloromethane by Dehalobacterium formicoaceticum in cell suspensions and crude cell extracts was investigated. The organism is a strictly anaerobic gram-positive bacterium that utilizes exclusively dichloromethane as a growth substrate and ferments this compound to formate and acetate in a molar ratio of 2:1. When [C]dichloromethane was degraded by cell suspensions, formate, the methyl group of acetate, and minor amounts of methanol were labeled, but there was no nuclear magnetic resonance signal corresponding to the carboxyl group of acetate. This finding and previously established carbon and electron balances suggested that dichloromethane was converted to methylene tetrahydrofolate, of which two-thirds was oxidized to formate while one-third gave rise to acetate by incorporation of CO(2) from the medium in the acetyl coenzyme A synthase reaction. When crude desalted extracts were incubated in the presence of dichloromethane, tetrahydrofolate, ATP, methyl viologen, and molecular hydrogen, dichloromethane and tetrahydrofolate were consumed, with the concomitant formation of stoichiometric amounts of methylene tetrahydrofolate. The in vitro transfer of the methylene group of dichloromethane onto tetrahydrofolate required substoichiometric amounts of ATP. The reaction was inhibited in a light-reversible fashion by 20 muM propyl iodide, thus suggesting involvement of a Co(I) corrinoid in the anoxic dehalogenation of dichloromethane. D. formicoaceticum exhibited normal growth with 0.8 mM sodium in the medium, and crude extracts contained ATPase activity that was partially inhibited by N,N'-dicyclohexylcarbodiimide and azide. During growth with dichloromethane, the organism thus may conserve energy not only by substrate-level phosphorylation but also by a chemiosmotic mechanism involving a sodium-independent F(0)F(1)-type ATP synthase.

Journal Article↗

Reductive dechlorination of tetrachloroethene to ethene by a two-component enzyme pathway.

Two membrane-bound, reductive dehalogenases that constitute a novel pathway for complete dechlorination of tetrachloroethene (perchloroethylene [PCE]) to ethene were partially purified from an anaerobic microbial enrichment culture containing Dehalococcoides ethenogenes 195. When titanium (III) citrate and methyl viologen were used as reductants, PCE-reductive dehalogenase (PCE-RDase) (51 kDa) dechlorinated PCE to trichloroethene (TCE) at a rate of 20 micromol/min/mg of protein. TCE-reductive dehalogenase (TCE-RDase) (61 kDa) dechlorinated TCE to ethene. TCE, cis-1,2-dichloroethene, and 1,1-dichloroethene were dechlorinated at similar rates, 8 to 12 micromol/min/mg of protein. Vinyl chloride and trans-1,2-dichloroethene were degraded at rates which were approximately 2 orders of magnitude lower. The light-reversible inhibition of TCE-RDase by iodopropane and the light-reversible inhibition of PCE-RDase by iodoethane suggest that both of these dehalogenases contain Co(I) corrinoid cofactors. Isolation and characterization of these novel bacterial enzymes provided further insight into the catalytic mechanisms of biological reductive dehalogenation.

Environmental Pollutants↗

Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: sequence of tceA and substrate range characterization.

The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1, 2-dibromoethane to ethene at rates of 7.5 and 30 micromol/min/mg, respectively, similar to the rates for TCE, cis-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase, tceA, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP)(2). Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Sec-independent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame, tceB, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with tceA.

Amino Acid Sequence↗

Identification of methyl halide-utilizing genes in the methyl bromide-utilizing bacterial strain IMB-1 suggests a high degree of conservation of methyl halide-specific genes in gram-negative bacteria.

Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the Proteobacteria, can grow with methyl bromide as a sole carbon and energy source. A single cmu gene cluster was identified in IMB-1 that contained six open reading frames: cmuC, cmuA, orf146, paaE, hutI, and partial metF. CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyltransferase motif. However, cmuB, identified in M. chloromethanicum and H. chloromethanicum, was not detected in IMB-1.

Alphaproteobacteria↗

Reductive dehalogenation of chlorobenzene congeners in cell extracts of Dehalococcoides sp. strain CBDB1.

Enzymatic reductive dehalogenation of tri-, tetra-, penta-, and hexachlorobenzenes was demonstrated in cell extracts with low protein concentration (0.5 to 1 micro g of protein/ml) derived from the chlorobenzene-respiring anaerobe Dehalococcoides sp. strain CBDB1. 1,2,3-trichlorobenzene dehalogenase activity was associated with the membrane fraction. Light-reversible inhibition by alkyl iodides indicated the presence of a corrinoid cofactor.

Aerobiosis↗