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Microbiology and biochemistry of the methanogenic archaeobacteria
Adv. Space Res. Vol. 9, No. 6, pp. (6)101—(6)105, 1989 Printed in Great Britain. All rights reserved.
0273—1177/89 $0.00 + .50 Copyright © 1989 COSPAR
MICROBIOLOGY AND BIOCHEMISTRY OF THE METHANOGENIC ARCHAEOBACTERIA Darren R. Abbanat, David J. Aceti, Stephen F. Baron, Katherine C. Terlesky and James G. Ferry’ Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A.
P~BSTRACT The methane producing bacteria are a diverse group of organisms that function in nature with other groups of strictly anaerobic bacteria to convert complex organic matter to methane and carbon dioxide. The methanogens belong to the archaeobacteria, a third primary kingdom distinct from all other procaryotes (eubacteria) and eucaryotes. The distinction is based on the unique structures of cell wall and membrane components present in archaeobacteria, as well as differences in the highly conserved lós rRNA sequences among the three kingdoms. In addition, the methanogens contain several novel cofactors that function as one-carbon carriers during the reduction of carbon dioxide to methane with electrons derived from the oxidation of H 2 or formate. Methanogens also convert acetate to methane by a pathway distinct from that for carbon dioxide reduction. The pathway involves activation of acetate to acetyl-SCoP~followed by decarbonylation and reduction of the methyl group to methane coupled to the oxidation of the carbonyl group to carbon dioxide. MICROBIOLOGY Methane-producing organisms (methanogens) have been isolated from a wide range of anaerobic habitats where complex organic matter is deposited, including freshwater and marine sediments, the ruinen and lower intestinal tract, and others. Methanogens are terminal organisms in microbial food chains comprised of at least three interacting metabolic groups of strictly anaerobic bacteria that together convert complex organic matter to CO2 and CR4. The complex organics are first converted to a mixture of volatile fatty acids, alcohols, carbon dioxide, and H2 by the ferinentative group. The higher fatty acids and alcohols are oxidized to acetic acid at the expense of the reduction of protons to H2, hence the name “obligate proton-reducing acetogenic bacteria”. The combined activities of these two groups produce acetic and formic acids, and H2, which are metabolized by the methanogens. The methane produced is inert in anaerobic environments and is oxidized to carbon dioxide by oxygen-requiring methylotrophic organisms only after diffusion into aerobic zones. The three orders of methanogens (Methanobacteriales, Methanococcales, and Methanomicrobiales) are characterized by extreme morphological diversity and the ability for many to proliferate in harsh environments including saturated brines and temperatures over 90°C /11. This wide diversity is even more evident in the cell wall and lipid composition. Cell walls are either pseudomurein, protein, or heteropolysaccharide. Polar membrane lipids are derivatives of isoprenyl glycerol ethers; a variety of neutral lipids constitute a significant portion of the total lipids. These unusual structures of the cell components distinguish methanogens from procaryotic organisms suggesting a deep phylogenetic division. Examination of the sequences of 16s rRNA further supports this division leading to the concept of three primary kingdoms: eucaryotes, eubacteria, and archaeobacteria /2/. In addition to the methanogens, the archaeobacteria include the extreme halophiles and the extreme thermophiles. The phenotypic characteristics of contemporary methanogens, and other archaeobacteria, has led to a hypothesis in which the ancestral organism was a strictly anaerobic thermophile that originated when the earths temperature was high (within the first billion years of formation) and the atmosphere was reducing /2/. Methanogens with growth optima around 1Corresponding author.
(6)101
D. R. Abbanat er al.
(6)102
85°Chave been isolated from deep sea hydrothermal vents that mimic these conditions /3/. In contrast, few eubacteria proliferate above 80°Cwhich is consistent with the notion that archaeobacteria are older than eubacteria. The characteristics of the universal ancestor, and evolutionary pressures that shaped the evolution of each kingdom, are important questions. BIOCHEMISTRY OF METHANOGENESIS Two pathways of methane formation are important in the dissimilation of complex organic matter: U) the reduction of carbon dioxide with H 2 or formate as the electron donors (4H2 + CO2 CH4 + 2H20, or 4HCOOH + CO2 = CH4 + 2H20 + 4CO2), and (ii) the conversion of acetate to methane and carbon dioxide tCH3COOH CO2 + CH4). The carbon dioxide reduction pathway has received considerable attention /4/. Several novel one-carbon carrying coenzytnes, unique to methanogens, participate in the process. These are methanofuran (MPR), tetrahydromethanopterin (THMP), and coenzyme M (HS-C0M). THMP is similar to pterins in eubacteria, but the structures of MFR and HS-CoM are more unique to methanogens. The initial reduction of C02, with an electron pair from H2 or HCOOH, yields formyl-MFR. The formyl group is transferred to TIDIP and then reduced with two electron pairs forming methyl-THMP. The methyl group is donated to HS-CoM forming CH3S-C0M. The final step involves a fourth cofactor (HS-HTP) which donates electrons for the reductive demethylation of CH3S-CoM forming the mixed disulfide of CoM-S-S-HTP /4/ and CH4. The mixed disulfide is reduced to the corresponding sulfhydryls with the fourth and final pair of electrons derived from either H2 or formate. ATP synthesis is coupled to electron transport by a chemiosmotic mechanism which is not fully understood. Several enzymes in the carbon dioxide reduction pathway have been characterized. The methyl reductase, catalyzing the reduction of CH3S-CoM, contains cofactor P430 a novel nickel-tetrapyrrole structurally related to porphyrin and corrin ring systems /5/. The function of F430 is unknown. Hydrogenases and formate dehydrogenases, catalyzing the oxidation of H2 and HCOOH, reduce a 5-deazaflavin (coenzyme F420) /6,7,8,9,10,11; S.F. Baron, unpublished results!. Coenzyne P420 appears to be a universal electron carrier in methanogens; although present in only a few eubacteria, its function as a low potential electron carrier is unique to methanogens. Coenzyme P420-reducing hydrogenases contain nickel and FAD, and coenzyme F420-reducing formate dehydrogenases contain tungsten or molybdenum; both enzymes contain iron-sulfur centers. The formate dehydrogenase from Methanobacterium formicicum contains FAD and a molybdopterin prosthetic group similar, but not identical, to that present in all described molybdoenzymes from eubacterial or eucaryotic sources /12/. The P420-reducing hydrogenase from M. formicicum is associated with the cytoplasmic menbrane, and participates in the formic hydrogenlyase system of this organism /S.F. Baron, unpublished results!. Carbon and electron flow during acetate conversion to methane in Methanosarcina thermophila is shown in Figure 1. Thermodynamic considerations suggest that acetate P04 #CH3*COSC0A
CoASH
ADP
ATP
1~CH
#CH3*COPOf ~
4
I
r
(PTA)
~iii~.i X-SCoA L÷ ii!~iI~
(CODH)f
I
,~
(CODH)
H20
~
~
Pd0
H2 H2
HSCoM
CODH
*
2H~ 2H~
~ ~CH3SCoM (MT)
CO2
Fdr
>._.<
_____
~Co-~CH3
3~C00 (AK)
~ #CH4 (MR) (H2HASE)
Fig. 1. Carbon and electron flow during acetate conversion to methane in Methanosarcina thermophila strain TN-i. AX, acetate kinase; PTA, phosphotransacetylase; CODH, carbon monoxide dehydrogenase; Pd, ferredoxin; H2ASE, hydrogenase; MT, methyl transferase; ME, methyl reductase. requires activation before cleavage of the carbon-carbon bond /13/. The activities of acetate kinase (ATP + CH3COO CH3COPO4 + ADP) and phosphotransacetylase (CH3COPO4 + HSCoA CH3COSCoA + PO4) are elevated in M. thernophila cells grown on acetate, compared to cells grown on alternate substrates, suggesting that the combined processes may drive the activation of acetate to CH3COSCoA in this organism /14/. The properties of the
=
Methanogenic Archaeobacteria
(6)103
acetate kinase are reported /15/. More direct evidence for involvement of acetyl-SCoA as an intermediate in the pathway was obtained with the use of a cell-free system that converted acetate to methane. The results in Table 1 show that methane produced in the cell-free system was specifically derived from the methyl group of acetate. In the experiment, the deuterated methyl group of CD 3COO was converted to deuterated methane with the deuteriums retained in the proportion predicted from previously reported whole cell experiments /16/. The results in Table 2 show that acetyl-SCoA substituted for acetate as a substrate for methanogenesis; less methane was produced from acetyl-SC0A as a result of the sensitivity of extracts to sulfhydryl groups resulting from the production of HSCoA in the reaction. It is postulated that an enzyme with carbon monoxide TABLE 1 Mass spectral analysis of methane produced from deuterated acetate in extracts of Methanosarcina thermophila.
Substrate CD3COO
C113C00
Species
Mol Z
CD3H CD2H2 CDH3 CR4 CD3H CD2H2 CDH3 CH4
83 17 0 0 0 0 0 100
Extracts were incubated with either of the above two substrates as described in Table 2. TABLE 2 Methanogenesis from acetate in a cell extracts of Methartosarcina thermophila.
Components
CR4
1 4500 (nmol) (1) eomplete (2) minus acetate 105 (i) plus acetyl CoA 502 (ii) plus CH 3S-C0M 1000 (iii) plus CH3-B12 5431 (3) minus AT? 170 (4) minus CO 41 (5) minus CoA 2834 1The complete reaction mixture (final volume 250 ul) contained cell extract (approximately 4 mg protein), 50 n~’f MES buffer pH 6.2, 25 mM MgCl 2, 48 mM acetate, 48 inN AT?, 10 units of creatine phosphokinase with 80 mM phosphocreatine (an ATP-regenerating system), 27 units glucose oxidase with 4 mM glucose (an oxygen-scrubbing system) and 100 uN coenzyine A. Incubation temperature was 45°C. The reaction mix was incubated anaerobically in stoppered vials containing a headspace of nitrogen and carbon monoxide (8:1). dehydrogenase (CODH) activity catalyzes the decarbonylation of activated acetate /17/. This hypothesis is based on a reversal of the reaction catalyzed by a CODH in the acetogenic clostridia in which acetyl-SCoA is synthesized from a methyl corrinoid, HSCoA and an enzyme-bound carbonyl derived from CO /18/. H. thermophila contains large amounts (1OZ of the cellular protein) of a CODH enzyme complex that is synthesized in acetategrown cells /191. The CODR enzyme complex contains five subunits and a Fe-Ni center with an EPR spectrum identical to the clostridial enzyme /14/. The EPR spectrum of the CODH incubated with acetyl-SC0A is perturbed which suggests that acetyl-SCoA binds to the enzyme and may be a substrate. The EPR studies also show that CO binds to the Fe-Ni center; thus, it is hypothesized that the carbonyl group of acetate is also bound to this site after decarbonylation of the activated acetate. The CODH complex also contains a corrinoid cofactor that is hypothesized to bind the methyl group of acetate after decarbonylation. Indirect evidence for the involvement of a corrinoid is presented in Table 2 where it is shown that CH3-812 also serves as a substrate for methanogenesis in the cell-free system. The methyl group is ultimately transferred to the final methyl acceptor (coenzyme H) yielding CH3S-CoM 19/ which is reductively demethylated to methane /20/; however, the intermediate methyl transfer reactions are unknown. Acetate-grown cells of M. thermophila contain a methyl transferase catalyzing the transfer of the methyl group from CH3-B12 to coenzyme M 1K. Terlesky, unpublished results!; however, evidence is not yet available directly implicating its involvement in the pathway. The electrons for
(6)104
D. R. Abbanat et a!.
the reductive step (catalyzed by CH
3S-CoM methyl reductase) are derived from oxidation of the CODH-bound carbonyl group /20/. A ferredoxin is the initial electron acceptor for the CODH /21,22/ but the electron donor to the methylreductase and intermediate electron carriers are unknown; however, evidence for the involvement of H-. as an intermediate is postulated /23,21/. M. therinophila produces and consumes H2 during growth on acetate /8/, and H2 can serve as a source of reductant for the demethylation of CH3S-CoM to methane /20/. Furthermore, a CO-oxidizing, H2-evolving enzyme system has been reconstituted with CODH, ferredoxin, and membranes /22/ purified from M. thermophila. The mechanism of ATP synthesis during acetate conversion to methane is unknown. REFERENCES 1. W.J. Jones, D.P. Nagle, and W.B. Whitman, Methanogens and the diversity of archaebacteria, Microbiol. Rev. 51, 135-177 (1987) 2. C.R. Woese, Bacterial evolution, Microbiol. Rev. 51, 221-271 (1987) 3. W.J. Jones, J.A. Leigh, F. Mayer, C.R. Woese, and R.S. Wolfe, Methanococcus jannaschii sp. nov., an extremely thermophilic methanogen from a submarine hydrothermal vent, Arch. Microbiol. 136, 254-261 (1983) 4. P.E. Rouviere, and R.S. Wolfe, Novel biochemistry of methanogenesis, J. Biol. Chem. 263, 7913-7916 (1988) 5. A. Pfaltz, B. Jaun, Fässler, A. Eschenmoser, R. Jaenchen, H.H. Gilles, G. Diekert, and R. Thauer, Zur Kenntnis des faktors P430 aus methanogenen bakterien: Struktur des porphinoiden ligandsystems, Helvet. Chim. Acta 65, 828-865 (1982) 6. J.A. Fox, D.J. Livingston, W H. Orme-Johnson, and C.T. Walsh, 8-hydroxy-5deazaflavin-reducing hydrogenase from Methanobacteriuin thernoautotrophicuxn: 1. Purification and characterization, Biochemistry USA 26, 4219-4227 (1987) 7. E. Muth, E. Morschel, and A. Klein, Purification and characterization of an 8-hyroxy-5-deazaflavin-reducing hydrogenase from the archaebacterium Methanococcus voltae, Eur. J. Biochem. 169, 571-577 (1987) 8. M.J.K. Nelson, D.P. Brown, and J.G. Ferry, FAD requirement for the reduction of coenzyme F420 by hydrogenase from Methanobacterium formicicuin, Biochem. Biophys Res. Commun. 120, 775-781 (1984) 9. N.L. Schauer, and J.G. Ferry, Composition of the coenzyme F420-dependent formate dehydrogenase from Methanobacteriuxn forsnicicum, J. Bacteriol. 165, 405411 (1986) 10. G.D. Sprott, K.M. Shaw, and T.J. Beveridge, Properties of the particulate enzyme P420-reducing hydrogenase isolated from Methanospirillum hung~j, Can. J Microbiol. 33, 896-942 (1987) 11. S. Yamazaki, A selenium-containing hydrogenase from Methanococcus vannielii, J. Biol. Chem. 257, 7926-7929 (1982) 12. H.D. May, N.L. Schauer, and J.G. Ferry, Molybdopterin cofactor from Methanobacteriuxn formicicum formate dehydrogenase, J. Bacteriol. 166, 500-504 (1986) 13. B. Eikmanns, and R.K. Thauer, Catalysis of an isotopic exchange between CO2 and the carboxyl group of acetate by Methanosarcina barkeri grown on acetate, Arch. Microbiol. 138, 365-370 (1984) 14. K.C. Terlesky, M.J. Barber, D.J. Aceti, and J.G. Ferry, EPR properties of the Ni-Fe-C center in an enzyme complex with carbon monoxide dehydrogenase activity from acetate-grown Methanosarcina thermophila. Evidence that acetyl-CoA is a physiological substrate, J. Biol. Chem. 262, 15392-15395 (1987) 15. D.J. Aceti, and J.G. Ferry, Purification and characterization of acetate kinase from Methanosarcina thermophila. Evidence for regulation of synthesis, J. Biol. Chem. in press (1988) 16. D.R. Lovley, R.H. White, and J.G. Ferry, Identification of methyl coenzyine M as an intermediate in methanogenesis from acetate in Methanosarcina spp., J. Bacteriol. 160, 521-525 (1984)
Methanogenic Archaeobactena
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17. J.A. Krzycki, R.H. Wolkin, and J.G. Zeikus, Comparison of unitrophic and mixotrophic substrate metabolism by an acetate-adapted strain of Methanosarcina barkeri, J. Bacteriol. 149, 247-254 (1982) 18. E. Pezacka, and H.G. Wood, The autotrophic pathway of acetogenic bacteria. Role of CO dehydrogenase, disulfide reductase, J. Biol. Chem. 261, 1609-1615 (1986) 19. K.C. Terlesky, M.J.K. Nelson, and J.G. Ferry, Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing a corrinoid and nickel from acetate-grown Methanosarcina thermophila, J. Bacteriol. 168, 1053-1058 (1986) 20. M.J.K. Nelson, and J.G. Ferry, Carbon monoxide-dependent methyl coenzyme M methyireductase in acetotrophic Methanosarcina spp., J. Bacteriol. 160, 526-532 (1984) 21. K.C. Terlesky, and J.G. Ferry, Purification and characterization of a ferredoxin from acetate-grown Methanosarcina therinophila, J. Biol. Chem. 263, 4080-4082 (1988) 22. K.C. Terlesky, and J.G. Ferry, Ferredoxin requirement for electron transport from the carbon monoxide dehydrogenase complex to a membrane-bound hydrogenase in acetate-grown Methanosarcina thermophila, J. Biol. Chem. 263, 4075-4079 (1988) 23. D.R. Lovley, and J.G. Ferry, Production and consumption of H
2 during growth of Methanosarcina spp. on acetate, Appl. Environ. Microbiol. 49, 247-249 (1985) ACKNOWLEDGEMENTS
This work was supported by grants 5086-200-1225 from the Gas Research Institute DMB-8i~09558 from the National Science Foundation.
JASR 9:6-H
and
0273—1177/89 $0.00 + .50 Copyright © 1989 COSPAR
MICROBIOLOGY AND BIOCHEMISTRY OF THE METHANOGENIC ARCHAEOBACTERIA Darren R. Abbanat, David J. Aceti, Stephen F. Baron, Katherine C. Terlesky and James G. Ferry’ Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A.
P~BSTRACT The methane producing bacteria are a diverse group of organisms that function in nature with other groups of strictly anaerobic bacteria to convert complex organic matter to methane and carbon dioxide. The methanogens belong to the archaeobacteria, a third primary kingdom distinct from all other procaryotes (eubacteria) and eucaryotes. The distinction is based on the unique structures of cell wall and membrane components present in archaeobacteria, as well as differences in the highly conserved lós rRNA sequences among the three kingdoms. In addition, the methanogens contain several novel cofactors that function as one-carbon carriers during the reduction of carbon dioxide to methane with electrons derived from the oxidation of H 2 or formate. Methanogens also convert acetate to methane by a pathway distinct from that for carbon dioxide reduction. The pathway involves activation of acetate to acetyl-SCoP~followed by decarbonylation and reduction of the methyl group to methane coupled to the oxidation of the carbonyl group to carbon dioxide. MICROBIOLOGY Methane-producing organisms (methanogens) have been isolated from a wide range of anaerobic habitats where complex organic matter is deposited, including freshwater and marine sediments, the ruinen and lower intestinal tract, and others. Methanogens are terminal organisms in microbial food chains comprised of at least three interacting metabolic groups of strictly anaerobic bacteria that together convert complex organic matter to CO2 and CR4. The complex organics are first converted to a mixture of volatile fatty acids, alcohols, carbon dioxide, and H2 by the ferinentative group. The higher fatty acids and alcohols are oxidized to acetic acid at the expense of the reduction of protons to H2, hence the name “obligate proton-reducing acetogenic bacteria”. The combined activities of these two groups produce acetic and formic acids, and H2, which are metabolized by the methanogens. The methane produced is inert in anaerobic environments and is oxidized to carbon dioxide by oxygen-requiring methylotrophic organisms only after diffusion into aerobic zones. The three orders of methanogens (Methanobacteriales, Methanococcales, and Methanomicrobiales) are characterized by extreme morphological diversity and the ability for many to proliferate in harsh environments including saturated brines and temperatures over 90°C /11. This wide diversity is even more evident in the cell wall and lipid composition. Cell walls are either pseudomurein, protein, or heteropolysaccharide. Polar membrane lipids are derivatives of isoprenyl glycerol ethers; a variety of neutral lipids constitute a significant portion of the total lipids. These unusual structures of the cell components distinguish methanogens from procaryotic organisms suggesting a deep phylogenetic division. Examination of the sequences of 16s rRNA further supports this division leading to the concept of three primary kingdoms: eucaryotes, eubacteria, and archaeobacteria /2/. In addition to the methanogens, the archaeobacteria include the extreme halophiles and the extreme thermophiles. The phenotypic characteristics of contemporary methanogens, and other archaeobacteria, has led to a hypothesis in which the ancestral organism was a strictly anaerobic thermophile that originated when the earths temperature was high (within the first billion years of formation) and the atmosphere was reducing /2/. Methanogens with growth optima around 1Corresponding author.
(6)101
D. R. Abbanat er al.
(6)102
85°Chave been isolated from deep sea hydrothermal vents that mimic these conditions /3/. In contrast, few eubacteria proliferate above 80°Cwhich is consistent with the notion that archaeobacteria are older than eubacteria. The characteristics of the universal ancestor, and evolutionary pressures that shaped the evolution of each kingdom, are important questions. BIOCHEMISTRY OF METHANOGENESIS Two pathways of methane formation are important in the dissimilation of complex organic matter: U) the reduction of carbon dioxide with H 2 or formate as the electron donors (4H2 + CO2 CH4 + 2H20, or 4HCOOH + CO2 = CH4 + 2H20 + 4CO2), and (ii) the conversion of acetate to methane and carbon dioxide tCH3COOH CO2 + CH4). The carbon dioxide reduction pathway has received considerable attention /4/. Several novel one-carbon carrying coenzytnes, unique to methanogens, participate in the process. These are methanofuran (MPR), tetrahydromethanopterin (THMP), and coenzyme M (HS-C0M). THMP is similar to pterins in eubacteria, but the structures of MFR and HS-CoM are more unique to methanogens. The initial reduction of C02, with an electron pair from H2 or HCOOH, yields formyl-MFR. The formyl group is transferred to TIDIP and then reduced with two electron pairs forming methyl-THMP. The methyl group is donated to HS-CoM forming CH3S-C0M. The final step involves a fourth cofactor (HS-HTP) which donates electrons for the reductive demethylation of CH3S-CoM forming the mixed disulfide of CoM-S-S-HTP /4/ and CH4. The mixed disulfide is reduced to the corresponding sulfhydryls with the fourth and final pair of electrons derived from either H2 or formate. ATP synthesis is coupled to electron transport by a chemiosmotic mechanism which is not fully understood. Several enzymes in the carbon dioxide reduction pathway have been characterized. The methyl reductase, catalyzing the reduction of CH3S-CoM, contains cofactor P430 a novel nickel-tetrapyrrole structurally related to porphyrin and corrin ring systems /5/. The function of F430 is unknown. Hydrogenases and formate dehydrogenases, catalyzing the oxidation of H2 and HCOOH, reduce a 5-deazaflavin (coenzyme F420) /6,7,8,9,10,11; S.F. Baron, unpublished results!. Coenzyne P420 appears to be a universal electron carrier in methanogens; although present in only a few eubacteria, its function as a low potential electron carrier is unique to methanogens. Coenzyme P420-reducing hydrogenases contain nickel and FAD, and coenzyme F420-reducing formate dehydrogenases contain tungsten or molybdenum; both enzymes contain iron-sulfur centers. The formate dehydrogenase from Methanobacterium formicicum contains FAD and a molybdopterin prosthetic group similar, but not identical, to that present in all described molybdoenzymes from eubacterial or eucaryotic sources /12/. The P420-reducing hydrogenase from M. formicicum is associated with the cytoplasmic menbrane, and participates in the formic hydrogenlyase system of this organism /S.F. Baron, unpublished results!. Carbon and electron flow during acetate conversion to methane in Methanosarcina thermophila is shown in Figure 1. Thermodynamic considerations suggest that acetate P04 #CH3*COSC0A
CoASH
ADP
ATP
1~CH
#CH3*COPOf ~
4
I
r
(PTA)
~iii~.i X-SCoA L÷ ii!~iI~
(CODH)f
I
,~
(CODH)
H20
~
~
Pd0
H2 H2
HSCoM
CODH
*
2H~ 2H~
~ ~CH3SCoM (MT)
CO2
Fdr
>._.<
_____
~Co-~CH3
3~C00 (AK)
~ #CH4 (MR) (H2HASE)
Fig. 1. Carbon and electron flow during acetate conversion to methane in Methanosarcina thermophila strain TN-i. AX, acetate kinase; PTA, phosphotransacetylase; CODH, carbon monoxide dehydrogenase; Pd, ferredoxin; H2ASE, hydrogenase; MT, methyl transferase; ME, methyl reductase. requires activation before cleavage of the carbon-carbon bond /13/. The activities of acetate kinase (ATP + CH3COO CH3COPO4 + ADP) and phosphotransacetylase (CH3COPO4 + HSCoA CH3COSCoA + PO4) are elevated in M. thernophila cells grown on acetate, compared to cells grown on alternate substrates, suggesting that the combined processes may drive the activation of acetate to CH3COSCoA in this organism /14/. The properties of the
=
Methanogenic Archaeobacteria
(6)103
acetate kinase are reported /15/. More direct evidence for involvement of acetyl-SCoA as an intermediate in the pathway was obtained with the use of a cell-free system that converted acetate to methane. The results in Table 1 show that methane produced in the cell-free system was specifically derived from the methyl group of acetate. In the experiment, the deuterated methyl group of CD 3COO was converted to deuterated methane with the deuteriums retained in the proportion predicted from previously reported whole cell experiments /16/. The results in Table 2 show that acetyl-SCoA substituted for acetate as a substrate for methanogenesis; less methane was produced from acetyl-SC0A as a result of the sensitivity of extracts to sulfhydryl groups resulting from the production of HSCoA in the reaction. It is postulated that an enzyme with carbon monoxide TABLE 1 Mass spectral analysis of methane produced from deuterated acetate in extracts of Methanosarcina thermophila.
Substrate CD3COO
C113C00
Species
Mol Z
CD3H CD2H2 CDH3 CR4 CD3H CD2H2 CDH3 CH4
83 17 0 0 0 0 0 100
Extracts were incubated with either of the above two substrates as described in Table 2. TABLE 2 Methanogenesis from acetate in a cell extracts of Methartosarcina thermophila.
Components
CR4
1 4500 (nmol) (1) eomplete (2) minus acetate 105 (i) plus acetyl CoA 502 (ii) plus CH 3S-C0M 1000 (iii) plus CH3-B12 5431 (3) minus AT? 170 (4) minus CO 41 (5) minus CoA 2834 1The complete reaction mixture (final volume 250 ul) contained cell extract (approximately 4 mg protein), 50 n~’f MES buffer pH 6.2, 25 mM MgCl 2, 48 mM acetate, 48 inN AT?, 10 units of creatine phosphokinase with 80 mM phosphocreatine (an ATP-regenerating system), 27 units glucose oxidase with 4 mM glucose (an oxygen-scrubbing system) and 100 uN coenzyine A. Incubation temperature was 45°C. The reaction mix was incubated anaerobically in stoppered vials containing a headspace of nitrogen and carbon monoxide (8:1). dehydrogenase (CODH) activity catalyzes the decarbonylation of activated acetate /17/. This hypothesis is based on a reversal of the reaction catalyzed by a CODH in the acetogenic clostridia in which acetyl-SCoA is synthesized from a methyl corrinoid, HSCoA and an enzyme-bound carbonyl derived from CO /18/. H. thermophila contains large amounts (1OZ of the cellular protein) of a CODH enzyme complex that is synthesized in acetategrown cells /191. The CODR enzyme complex contains five subunits and a Fe-Ni center with an EPR spectrum identical to the clostridial enzyme /14/. The EPR spectrum of the CODH incubated with acetyl-SC0A is perturbed which suggests that acetyl-SCoA binds to the enzyme and may be a substrate. The EPR studies also show that CO binds to the Fe-Ni center; thus, it is hypothesized that the carbonyl group of acetate is also bound to this site after decarbonylation of the activated acetate. The CODH complex also contains a corrinoid cofactor that is hypothesized to bind the methyl group of acetate after decarbonylation. Indirect evidence for the involvement of a corrinoid is presented in Table 2 where it is shown that CH3-812 also serves as a substrate for methanogenesis in the cell-free system. The methyl group is ultimately transferred to the final methyl acceptor (coenzyme H) yielding CH3S-CoM 19/ which is reductively demethylated to methane /20/; however, the intermediate methyl transfer reactions are unknown. Acetate-grown cells of M. thermophila contain a methyl transferase catalyzing the transfer of the methyl group from CH3-B12 to coenzyme M 1K. Terlesky, unpublished results!; however, evidence is not yet available directly implicating its involvement in the pathway. The electrons for
(6)104
D. R. Abbanat et a!.
the reductive step (catalyzed by CH
3S-CoM methyl reductase) are derived from oxidation of the CODH-bound carbonyl group /20/. A ferredoxin is the initial electron acceptor for the CODH /21,22/ but the electron donor to the methylreductase and intermediate electron carriers are unknown; however, evidence for the involvement of H-. as an intermediate is postulated /23,21/. M. therinophila produces and consumes H2 during growth on acetate /8/, and H2 can serve as a source of reductant for the demethylation of CH3S-CoM to methane /20/. Furthermore, a CO-oxidizing, H2-evolving enzyme system has been reconstituted with CODH, ferredoxin, and membranes /22/ purified from M. thermophila. The mechanism of ATP synthesis during acetate conversion to methane is unknown. REFERENCES 1. W.J. Jones, D.P. Nagle, and W.B. Whitman, Methanogens and the diversity of archaebacteria, Microbiol. Rev. 51, 135-177 (1987) 2. C.R. Woese, Bacterial evolution, Microbiol. Rev. 51, 221-271 (1987) 3. W.J. Jones, J.A. Leigh, F. Mayer, C.R. Woese, and R.S. Wolfe, Methanococcus jannaschii sp. nov., an extremely thermophilic methanogen from a submarine hydrothermal vent, Arch. Microbiol. 136, 254-261 (1983) 4. P.E. Rouviere, and R.S. Wolfe, Novel biochemistry of methanogenesis, J. Biol. Chem. 263, 7913-7916 (1988) 5. A. Pfaltz, B. Jaun, Fässler, A. Eschenmoser, R. Jaenchen, H.H. Gilles, G. Diekert, and R. Thauer, Zur Kenntnis des faktors P430 aus methanogenen bakterien: Struktur des porphinoiden ligandsystems, Helvet. Chim. Acta 65, 828-865 (1982) 6. J.A. Fox, D.J. Livingston, W H. Orme-Johnson, and C.T. Walsh, 8-hydroxy-5deazaflavin-reducing hydrogenase from Methanobacteriuin thernoautotrophicuxn: 1. Purification and characterization, Biochemistry USA 26, 4219-4227 (1987) 7. E. Muth, E. Morschel, and A. Klein, Purification and characterization of an 8-hyroxy-5-deazaflavin-reducing hydrogenase from the archaebacterium Methanococcus voltae, Eur. J. Biochem. 169, 571-577 (1987) 8. M.J.K. Nelson, D.P. Brown, and J.G. Ferry, FAD requirement for the reduction of coenzyme F420 by hydrogenase from Methanobacterium formicicuin, Biochem. Biophys Res. Commun. 120, 775-781 (1984) 9. N.L. Schauer, and J.G. Ferry, Composition of the coenzyme F420-dependent formate dehydrogenase from Methanobacteriuxn forsnicicum, J. Bacteriol. 165, 405411 (1986) 10. G.D. Sprott, K.M. Shaw, and T.J. Beveridge, Properties of the particulate enzyme P420-reducing hydrogenase isolated from Methanospirillum hung~j, Can. J Microbiol. 33, 896-942 (1987) 11. S. Yamazaki, A selenium-containing hydrogenase from Methanococcus vannielii, J. Biol. Chem. 257, 7926-7929 (1982) 12. H.D. May, N.L. Schauer, and J.G. Ferry, Molybdopterin cofactor from Methanobacteriuxn formicicum formate dehydrogenase, J. Bacteriol. 166, 500-504 (1986) 13. B. Eikmanns, and R.K. Thauer, Catalysis of an isotopic exchange between CO2 and the carboxyl group of acetate by Methanosarcina barkeri grown on acetate, Arch. Microbiol. 138, 365-370 (1984) 14. K.C. Terlesky, M.J. Barber, D.J. Aceti, and J.G. Ferry, EPR properties of the Ni-Fe-C center in an enzyme complex with carbon monoxide dehydrogenase activity from acetate-grown Methanosarcina thermophila. Evidence that acetyl-CoA is a physiological substrate, J. Biol. Chem. 262, 15392-15395 (1987) 15. D.J. Aceti, and J.G. Ferry, Purification and characterization of acetate kinase from Methanosarcina thermophila. Evidence for regulation of synthesis, J. Biol. Chem. in press (1988) 16. D.R. Lovley, R.H. White, and J.G. Ferry, Identification of methyl coenzyine M as an intermediate in methanogenesis from acetate in Methanosarcina spp., J. Bacteriol. 160, 521-525 (1984)
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17. J.A. Krzycki, R.H. Wolkin, and J.G. Zeikus, Comparison of unitrophic and mixotrophic substrate metabolism by an acetate-adapted strain of Methanosarcina barkeri, J. Bacteriol. 149, 247-254 (1982) 18. E. Pezacka, and H.G. Wood, The autotrophic pathway of acetogenic bacteria. Role of CO dehydrogenase, disulfide reductase, J. Biol. Chem. 261, 1609-1615 (1986) 19. K.C. Terlesky, M.J.K. Nelson, and J.G. Ferry, Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing a corrinoid and nickel from acetate-grown Methanosarcina thermophila, J. Bacteriol. 168, 1053-1058 (1986) 20. M.J.K. Nelson, and J.G. Ferry, Carbon monoxide-dependent methyl coenzyme M methyireductase in acetotrophic Methanosarcina spp., J. Bacteriol. 160, 526-532 (1984) 21. K.C. Terlesky, and J.G. Ferry, Purification and characterization of a ferredoxin from acetate-grown Methanosarcina therinophila, J. Biol. Chem. 263, 4080-4082 (1988) 22. K.C. Terlesky, and J.G. Ferry, Ferredoxin requirement for electron transport from the carbon monoxide dehydrogenase complex to a membrane-bound hydrogenase in acetate-grown Methanosarcina thermophila, J. Biol. Chem. 263, 4075-4079 (1988) 23. D.R. Lovley, and J.G. Ferry, Production and consumption of H
2 during growth of Methanosarcina spp. on acetate, Appl. Environ. Microbiol. 49, 247-249 (1985) ACKNOWLEDGEMENTS
This work was supported by grants 5086-200-1225 from the Gas Research Institute DMB-8i~09558 from the National Science Foundation.
JASR 9:6-H
and