Methylation of Carbon Monoxide Dehydrogenase from<i>Clostridium thermoaceticum</i>and Mechanism of Acetyl Coenzyme A Synthesis

Barondeau, D.P.; Lindahl, Paul A.

doi:10.1021/ja963597k

Cited by 106 publications

(161 citation statements)

References 63 publications

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“…However, we found that the EPR signal intensity was unchanged by the presence of a strong reductant (1 mM Ti 3ϩ -citrate). This indicates that disappearance of the NiFeC signal upon reaction with an appropriate methyl donor or during catalytic turnover (8,18,23) takes place by the reversal of reaction d, with the shift in equilibrium becoming favorable only in the presence of substrates.…”

Section: Discussionmentioning

confidence: 99%

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

Gencic

Grahame

2003

Journal of Biological Chemistry

113

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The methanogenic Archaea utilize a unique metabolic pathway for degradation of acetate under anaerobic conditions, and cleavage of acetate thereby accounts for a major proportion of the methane formed in the environment. The central reaction in this pathway is carried out by an unusual multienzyme complex, designated acetyl-CoA decarbonylase/synthase (ACDS), 1 which contains five different polypeptide subunits and accounts for as much as 25% of the soluble protein in species such as Methanosarcina thermophila and Methanosarcina barkeri growing on acetate. The ACDS complex catalyzes cleavage of the acetyl C-C bond using the substrates acetylCoA and tetrahydrosarcinapterin (H 4 SPt), a tetrahydrofolate analog which serves as methyl acceptor, and yields the products CoA, N 5 -methyltetrahydrosarcinapterin, CO 2 , and two reducing equivalents, as given in Reaction 1 (1).This overall reaction is made up of a series of partial reactions catalyzed by different protein subcomponents of the ACDS complex as shown in Scheme 1 (2). Acetyl-CoA binds to the ␤ subunit, and under low redox potential conditions, as required for activity, transfers the acetyl group to a nucleophilic center on the enzyme forming an acetyl-enzyme species and releasing CoA (Scheme 1, acetyl transfer) (2, 3). The acetyl intermediate then undergoes C-C bond cleavage by a reaction that is presumed to involve metal-based decarbonylation and/or methyl group migration (Scheme 1, cleavage). The nascent methyl group is then transferred to a corrinoid cofactor present on the ␥␦ subcomponent, which catalyzes subsequent methyl transfer to the substrate H 4 SPt (Scheme 1, methyl transfer) (2). The carbonyl group is oxidized to CO 2 by a process involving the ␣⑀ CO dehydrogenase subcomponent, with regeneration of the reduced form of the ␤ subunit. Previous studies on the ␤ subunit have focused on a C-terminally truncated form of the protein purified from the native ACDS complex following partial proteolytic digestion (2-4).The genes encoding the five ACDS subunits are arranged together in an operon along with one additional open reading frame in all species of Methanosarcina and in certain other methanogens as well (5-7, 9).2 The operon structure is shown in Scheme 2, with the designated genes and corresponding subunit molecular masses indicated for M. thermophila TM-1. The additional open reading frame encodes an accessory protein thought to be involved in nickel insertion, and nickel is present in both the large CO dehydrogenase subunit ␣ (CdhA) and in the ␤ subunit (CdhC) containing the active site for

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Section: Discussionmentioning

confidence: 99%

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

Gencic

Grahame

2003

Journal of Biological Chemistry

113

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“…This species was initially assumed to be the A-cluster itself, but Barondeau and Lindahl found that reductants unable to reduce A ox can effect methylation (50). Other known redox sites on the enzyme were also eliminated, suggesting that ACS Ct contains an unidentified n = 2 redox site which they called the D-site (not related to the D-cluster).…”

Section: -4 Electron Reduction (40)mentioning

confidence: 99%

“…Other mechanisms employ the A red -CO state as a catalytic intermediate (1), but there is evidence that this state inhibits catalysis (50,54). Further studies are required to resolve this.…”

Section: +mentioning

confidence: 99%

The Ni-Containing Carbon Monoxide Dehydrogenase Family: Light at the End of the Tunnel?

2002

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“…Another unusual feature of the A-cluster is that the ϩ1 state has been observed for both CODH⅐ACS (19) and ACS HT (20,21) only in complexes of the reduced enzymes with CO. There is agreement that CO (17) and the methyl group (8) bind to the Ni p site.…”

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confidence: 91%

Pulse-Chase Studies of the Synthesis of Acetyl-CoA by Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase

Seravalli

Ragsdale

2008

Journal of Biological Chemistry

103

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Carbon monoxide dehydrogenase/acetyl-CoA synthase catalyzes acetyl-CoA synthesis from CO, CoA, and a methylated corrinoid iron-sulfur protein, which acts as a methyl donor. This reaction is the last step in the Wood-Ljungdahl pathway of anaerobic carbon fixation. The binding sequence for the three substrates has been debated for over a decade. Different binding orders imply different mechanisms (i.e. paramagnetic versus diamagnetic mechanisms). Ambiguity arises because CO and CoA can each undergo isotopic exchange with acetyl-CoA, suggesting that either of these two substrates could be the last to bind to the acetyl-CoA synthase active site. Furthermore, carbonylation, CoA binding, and methyl transfer can all occur in the absence of the other two substrates. Here, we report pulsechase studies, which unambiguously establish the order in which the three substrates bind. Although a CoA pulse is substantially diluted by excess CoA in the chase, isotope recovery of a pulse of labeled CO or methyl group is unaffected by the presence of excess unlabeled CO or methyl group in the chase. These results demonstrate that CoA is the last substrate to bind and that CO and the methyl group bind randomly as the first substrate in acetyl-CoA synthesis. Up to 100% of the methyl groups and CoA and up to 60 -70% of the CO employed in the pulse phase can be trapped in the product acetyl-CoA.There are three types of CO dehydrogenase (CODH) 2 (1, 2). These include a copper molybdopterin iron-sulfur protein that is present in aerobic bacteria and an NiFeS enzyme that is present in anaerobic microbes. These enzymes allow microbes to utilize CO at the low concentrations present in the atmosphere as a carbon and electron source. A third type of CODH is highly homologous to the monofunctional NiFeS enzyme and is found tightly associated with another NiFeS enzyme called acetyl-CoA synthase (ACS), forming the CODH⅐ACS complex.CODH⅐ACS is a macromolecular machine that catalyzes the last step of the Wood-Ljungdahl pathway of anaerobic carbon dioxide fixation (Reaction 1). The 300-kDa ␣ 2 ␤ 2 heterotetramer is composed of two types of subunits. The 72-kDa ␤-subunit is CODH, which catalyzes the reversible oxidation of CO to CO 2 (Reaction 2) at the C-cluster, a novel [NiFe 4 S 4 -5 ] cluster (3, 4), whereas the 82-kDa ␣-subunit is ACS, which catalyzes the condensation of three substrates, CoA, CO, and a methyl group from the methylated corrinoid iron-sulfur protein (CH 3 -CFeSP), to produce acetyl-CoA. Methylation of the CFeSP is accomplished by a methyltransferase (MeTr), which catalyzes the transfer of a methyl group from CH 3 -H 4 folate to the Co(I) state of the CFeSP, according to Reaction 3 (5). Although MeTr contains no metals or other cofactors, the CFeSP contains a corrinoid cofactor and a [Fe 4 S 4 ] cluster, which is involved in the reactivation of the cobalt center back to the 1ϩ state whenever it has undergone oxidation (approximately once in every 1000 catalytic cycles) (6, 7). The resulting CH 3 -CFeSP becomes the methyl donor to ...

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Methylation of Carbon Monoxide Dehydrogenase fromClostridium thermoaceticumand Mechanism of Acetyl Coenzyme A Synthesis

Cited by 106 publications

References 63 publications

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

The Ni-Containing Carbon Monoxide Dehydrogenase Family: Light at the End of the Tunnel?

Pulse-Chase Studies of the Synthesis of Acetyl-CoA by Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase

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