Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila

Abbanat, Darren R.; Ferry, James G.

doi:10.1128/jb.172.12.7145-7150.1990

Cited by 50 publications

(29 citation statements)

References 31 publications

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“…The first biochemical evidence supporting this proposed function was obtained when it was demonstrated that the purified complex synthesizes acetyl-coA (Abbanat and Ferry, 1990). In addition to supporting the proposed function of the complex and further characterization, the results suggested different sites on the enzyme complex for acetyl-coA synthesis and CO oxidation which we further documented in our more recent studies (Lu et al, 1994).…”

Section: Comparison Of the Aminosupporting

confidence: 80%

Enzymology of the Pathway for Acetate Conversion to Methane in Methanosarcina thermophilia

Ferry¹

1999

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Section: Comparison Of the Aminosupporting

confidence: 80%

Enzymology of the Pathway for Acetate Conversion to Methane in Methanosarcina thermophilia

Ferry¹

1999

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“…Acetate kinase (3) and phosphotransacetylase (23) catalyze the activation of acetate to acetyl coenzyme A followed by decar- bonylation thought to be catalyzed by the nickel/iron-sulfur component of the CODH enzyme complex (1). The carbonyl group binds to the nickel/iron-sulfur component, and the methyl group is transferred to the corrinoid/iron-sulfur component of the CODH enzyme complex (1). The methyl acceptor for the corrinoid/iron-sulfur component is unknown; however, tetrahydromethanopterin has been shown to be an intermediate methyl carrier in M. barkeri (12).…”

Section: Methodsmentioning

confidence: 99%

“…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 alp'lyl. As isolated, the enzyme was inactive but could be reductively reactivated with titanium (III) citrate or reduced ferredoxin.…”

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confidence: 99%

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

Jablonski

Ferry

1991

J Bacteriol

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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 alp'lyl. 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°C and between 6.5 and 7.0, respectively. The active enzyme contained 1 mol of coenzyme F430 per mol of enzyme (M,r 144,000). The Kms for 2-(methylthio)ethane-sulfonate and 7-mercaptoheptanoylthreonine phosphate were 3.3 mM and 59,uM, respectively.Methanogenic bacteria obtain energy for growth by reducing carbon dioxide to methane or converting the methyl group of acetate, methanol, or methylated amines to methane. 2-(Methylthio)ethane-sulfonate (CH3-S-CoM) is a common intermediate that is reductively demethylated to CH4 by methyl coenzyme M methylreductase. The extensively studied methylreductases have a native molecular weight of approximately 300,000 and a subunit composition of a212Y2.In addition, these methylreductases contain 2 mol of a nickel-containing tetrahydrocorphin (coenzyme F430) per mol of enzyme (Mr, 300,000). The methylreductase from Methanobacterium thermoautotrophicum AH is the most extensively characterized (11, 14, 15, 26, 29-31, 33, 34, 36); the enzyme is one component of the H2-dependent methylreductase system, which has been reconstituted with protein fractions Al, A3a, and A3b, purified protein A2, and C (the methylreductase) (26,34,36). Only components A2 (34) and the methylreductase (15) have been purified to homogeneity. As isolated, the methylreductase must first be reactivated which is thought to occur by reduction of Ni(II) to Ni(I) in F430 (4). Reductive reactivation requires components A3a, A2, and ATP. H2 is oxidized by a hydrogenase in component A3b to provide electrons for the reactivation. Titanium (III) citrate can replace H2 and component A3b as an alternative source of electrons. Once reactivated, the electron donor to the methylreductase is 7-mercaptoheptanoylthreonine phosphate (HS-HTP) and the products of the reaction are CH4 and the heterodisulfide of HS-CoM and HS-HTP. The heterodisulfide is then reduced by H2 and component Al to 2-mercaptoethanesulfonate (HS-CoM) and HS-HTP.Methyl coenzyme M methylreductases from Methanosarcina spp. have ...

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“…The electron paramagnetic resonance signal is perturbed upon addition of acetyl-CoA, supporting the suggestion that acetyl-CoA is a physiological substrate for the enzyme (16). The synthesis of acetyl-CoA from CH3I, CO, and HSCoA has been reported for CODH from both C. thermoaceticum (6) and M. thermophila (1). However, the cleavage of acetyl-CoA has not been demonstrated with any purified methanogenic CO dehydrogenase.…”

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Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila

et al. 1991

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The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 ,uM, Vmax = 15 min-'. CoA is a potent inhibitor of this exchange (K1 = 25 ,uM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.Acetogens and methanogens possess novel pathways for the synthesis of acetyl-coenzyme A (acetyl-CoA) from single carbon units which allow these organisms to grow on CO2 as the sole carbon source (5). Other strains of methanogenic bacteria such as Methanosarcina species are able to grow on analogous Cl feedstocks including methanol and methylamine by using variations of this same pathway for carbon assimilation. The key enzyme in these cases is carbon monoxide dehydrogenase, a complex nickel enzyme so named for its ability to catalyze the reduction of CO2 to CO. However, the enzyme has been implicated in a number of cases as the catalyst for the carbon-carbon bond formation from suitable methyl donors and enzymatically generated CO from CO2 to give acetyl units.In the acetogenic Clostridium thermoaceticum, one molecule of CO2 is reduced to formate and formate is reduced to a methyl group by using tetrahydrofolate as a carrier; enzymatic transfer to a corrinoid iron/sulfur protein yields the methylated organocobalt species (19). Nickel-containing CO dehydrogenase (more appropriately called acetyl-CoA synthase) then acts as the site of carbon-carbon bond coupling by accepting the methyl group and catalyzing its carbonylation in the presence of CoA to give acetyl-CoA as product. The enzyme from C. thermoaceticum is the most well characterized acetogenic enzyme containing 2 Ni, 11 Fe, and 1 Zn atoms per active site; there is some discussion about the subunit composition of the functional complex as isolated: c433 [

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Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila

Cited by 50 publications

References 31 publications

Enzymology of the Pathway for Acetate Conversion to Methane in Methanosarcina thermophilia

Enzymology of the Pathway for Acetate Conversion to Methane in Methanosarcina thermophilia

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

Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila

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