Information on EC 2.3.1.169 - CO-methylating acetyl-CoA synthase

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The expected taxonomic range for this enzyme is: Bacteria, Archaea

EC NUMBER
COMMENTARY hide
2.3.1.169
-
RECOMMENDED NAME
GeneOntology No.
CO-methylating acetyl-CoA synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
acetyl-CoA + a [Co(I) corrinoid Fe-S protein] = CO + CoA + a [methyl-Co(III) corrinoid Fe-S protein]
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
demethylation
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Carbon fixation pathways in prokaryotes
-
-
methanogenesis from acetate
-
-
Microbial metabolism in diverse environments
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-
NIL
-
-
reductive acetyl coenzyme A pathway
-
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reductive acetyl coenzyme A pathway I (homoacetogenic bacteria)
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reductive acetyl coenzyme A pathway II (autotrophic methanogens)
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SYSTEMATIC NAME
IUBMB Comments
acetyl-CoA:corrinoid protein O-acetyltransferase
Contains nickel, copper and iron-sulfur clusters. Involved, together with EC 1.2.7.4, carbon-monoxide dehydrogenase (ferredoxin), in the synthesis of acetyl-CoA from CO2 and H2.
CAS REGISTRY NUMBER
COMMENTARY hide
176591-19-8
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64972-88-9
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
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expression of the Cdh1- and Cdh2-encoding genes is regulated differentially in response to growth phase and to changing substrate conditions. CdhA3 clearly affects expression of cdh1, suggesting that it functions in signal perception and transduction rather than in catabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + corrinoid protein
CoA + CO + methylcorrinoid protein
show the reaction diagram
CH3-(corrinoid/iron-sulfur protein) + CO + HS-CoA
CH3-CO-S-CoA + corrinoid/iron-sulfur protein
show the reaction diagram
CH3-CO-S-CoA + H+ + tetrahydromethanopterin
CH3-tetrahydromethanopterin + CO + HS-CoA
show the reaction diagram
-
-
?
CH3-CO-S-CoA + tetrahydrosarcinapterin + H2O
CH3-tetrahydrosarcinapterin + CO2 + H+ + electron
show the reaction diagram
CH3-tetrahydrofolate + CO + HS-CoA
CH3-CO-S-CoA + tetrahydrofolate
show the reaction diagram
CH3-tetrahydrosarcinapterin + CO + HS-CoA
CH3-CO-S-CoA + tetrahydrosarcinapterin
show the reaction diagram
-
-
-
?
CH3I + CO + HS-CoA
CH3-CO-S-CoA + HI
show the reaction diagram
CO + H2O
CO2 + H+ + electron
show the reaction diagram
CO + methyl-X + HS-CoA
CH3-CO-S-CoA + HX
show the reaction diagram
CO2 + H+ + electron
CO + H2O
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + corrinoid protein
CoA + CO + methylcorrinoid protein
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ferredoxin
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Fd-II, which harbors two [4Fe-4S] clusters and is an electron acceptor for CODH, serves as a redox activator of ACS. Catalytic one-electron redox-active species in the CO/acetyl-CoA exchange reaction. Incubation of ACS with Fd-II and CO leads to the formation of the NiFeC species. FdII is purified from Moorella thermoacetica, overview
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[4Fe-4S]-center
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protein contains in average 3.7 Fe atoms and 1.6 Ni atoms per monomer molecule, which is consistent with the presence of a [NipNid] [Fe4S4] center. Preparation shows a broad absorption at the 420 nm region
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co3+
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part of the methylcorrinoid protein
copper
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the acetyl-CoA synthase active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Distorted Cu(I)-S3 site in the fully active enzyme in solution. Average Cu-S bond length of 2.25 A and a metal neighbor at 2.65 A, consistent with the Cu-Ni distance observed in the crystal structure. Cu-SCoA intermediate in the mechanism of acetyl-CoA synthesis. Essential and functional role for copper in the enzyme
Cu
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the Ni in cluster A can be replaced by Cu yielding an inactive form of the acetyl-CoA synthase
Cu+
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capture of Ni2+, Cu+ and Zn2+ by thiolate sulfurs of an N2S2Ni complex
Cu2+
the enzyme has a metallocofactor containing iron, sulfur, copper, and nickel, the cofactor responsible for the assembly of acetyl-CoA contains a [Fe4S4] cubane bridged to a copper-nickel binuclear site
Nickel
Zn
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the Ni in cluster A can be replaced by Zn yielding an inactive form of the acetyl-CoA synthase
Zn2+
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capture of Ni2+, Cu+ and Zn2+ by thiolate sulfurs of an N2S2Ni complex
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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1 mM, complete inhibition. 1,10-Phenanthroline additionally has inhibitory effects on growth of Peptoclostridium difficile
2,2-dipyridyl
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1 mM, complete inhibition. 2,2-Dipyridyl additionally has inhibitory effects on growth of Peptoclostridium difficile
5,5'dithiobis-(2-nitrobenzoic acid)
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inhibits the acetyl-CoA/CO exchange reaction
CN-
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inhibitor on the CoA/acetyl-CoA exchange, 98% inhibition at 1.2 mM
CO2
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inhibitor on the CoA/acetyl-CoA exchange
CoA
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at concentration above 10 mM, 50% inhibition of acetyl-CoA synthesis from methyl iodide at 15 mM
dephospho-CoA
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inhibitor on the CoA/acetyl-CoA exchange, 75% inhibition at 0.44 mM
desulfo-CoA
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inhibitor on the CoA/acetyl-CoA exchange, 30% mM at 2.1 mM
Dithionite
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inhibits reverse methyl group transfer, when it is preincubated with methylated enzyme but not when it is preincubated with Co+-iron-sulfur protein
Fe2+
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in cofactor ferredoxin(II), which harbors two [4Fe-4S] clusters
Mersalyl acid
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inhibits the acetyl-CoA/CO exchange reaction
methyl iodide
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inhibits the acetyl-CoA/CO exchange reaction
N2O
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inhibitor on the CoA/acetyl-CoA exchange
Sodium dithionite
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inhibits the acetyl-CoA/CO exchange reaction
Ti3+-citrate
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inhibits reverse methyl group transfer, when it is preincubated with methylated enzyme but not when it is preincubated with Co+-iron-sulfur protein
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additional information
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no inhibition of the exchange reaction by methyl- and phenylglyoxal, and butanedione
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CO
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two effects: stimulation and inhibition on CoA/acetylCoA exchange
Ferredoxin
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.28
acetyl-CoA
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exchange activity: C14 from the carboxyl group of acetyl-CoA with C12 from CO
14.7
CH3I
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pH 7.3, 22°C
0.087 - 0.36
CO
4.3
CoA
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pH 7.3, 22°C, acetyl-CoA synthesis from methyl iodide
0.066 - 0.53
methylcorrinoid protein
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00012 - 0.142
acetyl-CoA
additional information
additional information
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.66
HS-CoA
Peptoclostridium difficile
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at 1 atm CO, pH not specified in the publication, temperature not specified in the publication
9541
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.4 - 1.5
CO
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.12
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pH 6.8, acetyl-CoA synthesis, in absence of ferredoxin II
0.41
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pH 6.8, acetyl-CoA synthesis, in presence of 1 mM ferrous ammonium sulfate
0.49
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pH 6.8, acetyl-CoA synthesis, in presence of ferredoxin II
0.74
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pH 6.8, acetyl-CoA synthesis, in presence of 4 mM ATP
0.8
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pH 6.8, acetyl-CoA synthesis, in absence of ATP and Fe2+
28
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40°C, CoA/acetyl-CoA exchange
additional information
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70 mol of CO exchanged per min/mol of enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.4
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CO/acetyl-CoA exchange reaction
5.8
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Tris-maleate buffer, the rate of acetyl-CoA synthesis increases with decreasing pH, at pH values below 5.8 the rate of acetyl-CoA synthesis decreases slightly
6
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exchange activity: C14 from the carboxyl group of acetyl-CoA with C12 from CO
6.2
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acetyl-CoA exchange assay at
6.5
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CO exchange assay at
6.7
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acetyltransferase assay at
6.7 - 7.2
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assay at
6.7 - 7
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optimum for CoA/acetyl-CoA exchange
7.6
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methylation reaction assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
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acetyl-CoA exchange assay at room temperature
45
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methylation reaction assay at
70
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exchange activity: C14 from the carboxyl group of acetyl-CoA with C12 from CO
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 35
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in 10 mM Tris-maleate buffer and pH 5.8, the rate of acetyl-CoA synthesis is increased 2fold at 25°C to 35°C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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cultures are grown on methanol, acetate, or CO
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
81700
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gel filtration
1600000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
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CODH and ACS make up the two subunits of a 310 kDa alpha2beta2 heterotetrameric enzymatic complex
monomer
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1 * 82900, the enzyme exists as a monomer as well as in a 1:1 molar complex with the 73300 Da CO dehydrogenase III, SDS-PAGE
polymer
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alpha,beta,gamma,delta,epsilon, 6 * 19700 + 6 * 84500 + 6 * 63200 + 6 * 53000 + 6 * 51400, SDS-PAGE
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
49 kDa fragment containing residues 311-729 of the intact enzym. In the fragment, domains A2 and A3 have significantlymoved to each other, corresponding to a rotation around a hinge region located close to the C-terminus of the long interdomain helix; crystal structure of recombinant ACS lacking the N-terminal domain that interacts with carbon monoxide dehydrogenase shows a large reorganization of the remaining two globular domains, producing a narrow cleft of suitable size, shape, and nature to bind CoA. Sequence comparisons with homologous archaeal enzymes that naturally lack the N-terminal domain show that many amino acids lining this cleft are conserved. Besides the typical [4Fe-4S] center, the A-cluster contains only one proximal metal ion that is most likely Cu or Zn. Incorporation of a functional Ni2Fe4S4 A-cluster would require only minor structural rearrangements
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a 2.5 A resolution structure of xenon-pressurized CODH/ACS, examination of the nature of gaseous cavities within the enzyme. The cavity calculation program CAVENV accurately predicts the channels connecting the C- and A-clusters, with 17 of 19 xenon binding sites within the predicted regions. The enzyme has a channel for a small substrate, a channel plug, a flexible acetyl-CoA synthase subunit that can open to interact with a large substrate, and an interdomain cavity to putatively bind a medium-sized substrate
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sitting drop vapor diffusion at room temperature in a Coy anaerobic chamber, 0.005 ml of protein solution containing 40-60 mg/ml CODH/ACS in 50 mM Tris, pH 7.6, are mixed with 0.0075 ml of reservoir solution containing 8% polyethylene glycol MME 5000, 20% glycerol, 200 mM calcium acetate, 100 mM PIPES, pH 6.5, and 2 mM dithioerythritol, X-ray diffraction structure determination and analysis at 2.2 A resolution, multiwavelength anomalous dispersion techniques, molecular replacement
structures of the 310 kDa bifunctional CODH/acetyl-CoA synthase complex bound both with a substrate H2O/OH- molecule and with a cyanide inhibitor. Both in native crystals and identical crystals soaked in a solution containing potassium cyanide, the substrateH2O/OH- molecule exhibits binding to the unique Fe site of the C-cluster. Cyanide binding is also observed in a bent conformation to Ni of the C-cluster, adjacent the substrate H2O/OH-molecule. The bridging sulfide is not present in either structure. Findings do not support a fifth, bridging sulfide playing a catalytic role in the enzyme mechanism
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
67
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the recombinant enzyme: 10 min, 10% loss of activity, the wild-type enzyme: 10 min, no loss of activity
75
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the wild-type enzyme: 10 min, 50% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
reactions in presence of DTT, since enzyme requires strictly anaerobic conditions for stability
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the acetyl-CoA synthesis is dependent on ionic strength, the CO/acetyl-CoA exchange is independent of ionic strength
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme by anion exchange and hydrophobic interaction chromatography, followed by hydroxylapatite chromatography, gel filtration, and CoA affinity chromatography
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recombinant ACSCh732 and ACSChDELTAN from Escherichia coli strain NM522 by anion exchange and hydroxyapatite chromatography, followed by another and different step of anion exchange chromatography and by hydrophobic interaction chromatography to 94-95% purity forACSCh and around 98% purity for ACSChDELTAN
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recombinant ACSD beta from Escherichia coli strain NM522 by anion exchange chromatography, followed by hydrophobic interaction chromatography and another and different step of ion exchange of anion exchange chromatography to around 98% purity for ACDS beta
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recombinant C-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography under a N2 atmosphere
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression of a 49 kDa fragment containing residues 311-729 of the intact enzyme and a C-terminal His tag, in Escherichia coli
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expression of ACSD beta in Escherichia coli strain NM522
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expression of C-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3) from a pet29a(+) vector
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gene ascB, expression of full-length protein, designated ACSCh732 amino acids, and as a form lacking the 317-amino acid N-terminal domain, designated ACSChDELTAN, 415 amino acids, in Escherichia coli strain NM522
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the gene has been cloned into Escherichia coli and found to be within an 11 kb gene cluster, recombinant enzyme is inactive
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two putative operons encoding CODH/ACS, designated cdh1 (ma1016-ma1011) and cdh2 (ma3860-ma3865), both encoding bona fide CODH/ACS isoforms, as well as cdhA3 (ma4399), isogenes organisation, expression of the Cdh2-encoding genes is generally higher than that of genes encoding Cdh1, expression of cdh1, cdh2, and cdhA1 in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A219F
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mutant designed to block the tunnel through which CO and CO2 migrate. Metal clusters are properly assembled but only slowly reducible by CO. Mutant shows impaired ability of CO to migrate through the tunnel to the C-cluster and reduced catalytic activity, no cooperative CO inhibition is observed
A265M
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absence of strong cooperative inhibition of CO which characterizes wild-type enzyme
A578C
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mutant designed to block the tunnel through which CO and CO2 migrate. Metal clusters are properly assembled but only slowly reducible by CO. Mutant shows impaired ability of CO to migrate through the tunnel to the C-cluster and reduced catalytic activity, no cooperative CO inhibition is observed
F70W
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mutant designed to block the region that connects the tunnel at the betabeta interface with a water channel also located at the interface. Metal clusters are properly assembled but only slowly reducible by CO. Mutant shows impaired ability of CO to migrate through the tunnel to the C-cluster and reduced catalytic activity, no cooperative CO inhibition is observed
L215F
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mutant designed to block the tunnel through which CO and CO2 migrate. Metal clusters are properly assembled but only slowly reducible by CO. Mutant shows impaired ability of CO to migrate through the tunnel to the C-cluster and reduced catalytic activity, no cooperative CO inhibition is observed
N101Q
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mutant designed to block the region that connects the tunnel at the betabeta interface with a water channel also located at the interface. Metal clusters are properly assembled but only slowly reducible by CO. Mutant shows impaired ability of CO to migrate through the tunnel to the C-cluster and reduced catalytic activity, no cooperative CO inhibition is observed
additional information
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construction of several cdh gene knockouts, genotypic and phenotypic analysis of cdh mutants. CODH activity during aceticlastic and carboxidotrophic growth is reduced in the cdh1 mutant (strain MCD1) compared to the wild-type
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant ACS is Ni-reconstituted in the elution buffer with 6 equivalents of NiCl2 for 2 or 3 days at 27°C or at 45°C
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