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Information on EC 4.2.1.112 - acetylene hydratase and Organism(s) Syntrophotalea acetylenica and UniProt Accession Q71EW5
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4.2.1.112 acetylene hydrataseIUBMB Comments
This is a non-redox-active enzyme that contains two molybdopterin guanine dinucleotide (MGD) cofactors, a tungsten centre and a cubane type [4Fe-4S] cluster .The tungsten centre binds a water molecule that is activated by an adjacent aspartate residue, enabling it to attack acetylene bound in a distinct hydrophobic pocket . Ethylene cannot act as a substrate .
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Word Map
- 4.2.1.112
- pelobacter
- acetylenicus
-
tungstoenzyme
- wiv
-
nonredox
-
bioinspired
-
w-containing
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e-4-hydroxy-3-methyl-but-2-enyl
-
qm-only
- pterin
The taxonomic range for the selected organisms is: Syntrophotalea acetylenica
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
acetylene hydratase, tungsten-dependent acetylene hydratase, (e)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
additional information
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the enzyme structurally belongs to the dimethyl sulfoxide reductase family of enzymes
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
acetaldehyde = acetylene + H2O
acetylene hydratase harbors two pyranopterins bound to tungsten, and a [4Fe-4S] cluster. Tungsten is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. The enzyme activity requires a strong reductant suggesting (IV) as the active oxidation state. Two different types of reaction pathways have been proposed, the reaction does not involve a net electron transfer. The nature of the oxygen ligand of the W center in the enzyme is crucial to formulate a reaction mechanism. Residue Asp13 is catalytically important because it activates the oxygen atom for the addition to the C-C triple bond. Reaction mechanism analysis, overview
acetaldehyde = acetylene + H2O
acetylene hydratase harbors two pyranopterins bound to tungsten, and a [4Fe-4S] cluster. Tungsten is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. The enzyme activity requires a strong reductant suggesting (IV) as the active oxidation state. Two different types of reaction pathways have been proposed, the reaction does not involve a net electron transfer. The nature of the oxygen ligand of the W center in the enzyme is crucial to formulate a reaction mechanism. Residue Asp13 is catalytically important because it activates the oxygen atom for the addition to the C-C triple bond. Representation of the five-step catalytic cycle, with Asp13 acting as a key player in the mechanism, and W binding and activating C2H2, and providing electrostatic stabilization to the transition states and intermediates
acetaldehyde = acetylene + H2O
active site channel structure and detailed catalytic mechanism
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acetaldehyde = acetylene + H2O
reaction mechanism involving molybdenum-cofactor and tungsten/iron-sulfur cluster, structure-function modeling, overview
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acetaldehyde = acetylene + H2O
active-site access, active-site architecture, and reaction mechanism, overview
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acetaldehyde = acetylene + H2O
active-site modeling and reaction mechanism involving direct coordination of the substrate to the tungsten ion, followed by a nucleophilic attack by a water molecule concerted with a proton transfer to a second-shell aspartate, which then reprotonates the substrate. A tungsten-bound hydroxide plays the key role performed by Asp13 in the enzyme, rate-limiting proton transfer step from Asp13 residue to the C2 center of the vinyl anion
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acetaldehyde = acetylene + H2O
active-site modeling and reaction mechanism, detailed overview. The mechanism starts with a ligand exchange step, in which the acetylene substrate displaces the tungsten-bound water molecule and binds to the metal in an eta2 fashion. Then the nucleophilic attack takes place nearly perpendicularly to the W-C-C plane, concerted nucleophilic attack-proton transfer transition state and the resulting vinyl anion intermediate, W=C=CH2 vinylidene intermediate, overview. To complete the reaction, the vinyl alcohol now needs only to tautomerize to acetaldehyde. At this point, the vinyl alcohol can be released and the interconversion can take place outside the active site. Alternative mechanisms, overview
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
acetaldehyde hydro-lyase (acetylene-forming)
This is a non-redox-active enzyme that contains two molybdopterin guanine dinucleotide (MGD) cofactors, a tungsten centre and a cubane type [4Fe-4S] cluster [2].The tungsten centre binds a water molecule that is activated by an adjacent aspartate residue, enabling it to attack acetylene bound in a distinct hydrophobic pocket [2]. Ethylene cannot act as a substrate [1].
CAS REGISTRY NUMBER
COMMENTARY
75788-81-7
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
acetylene + H2O
acetaldehyde
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-
-
-
?
acetylene + H2O
acetaldehyde
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-
the subsequent disproportionation of acetaldehyde yields acetate and ethanol
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?
acetylene + H2O
acetaldehyde
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the hydration reaction is unique for a tungsten and molybdenum containing enzyme, which are usually redox enzymes
-
-
r
acetylene + H2O
acetaldehyde
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the active site residues Asp13, Lys48, and Ile142 are involved in catalysis. Asp13 close to W(IV) coordinates two molybdopterin-guanosine-dinucleotide ligands, Lys48 couples the [4Fe-4S] cluster to the W site, and Ile142 is part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene
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-
r
additional information
?
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the enzyme is highly specific for acetylene
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-
?
additional information
?
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the enzyme is highly specific for acetylene
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-
?
additional information
?
-
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no activity with ethylene, methylene blue, or anthraquinone disulfonate, the purified enzyme has no coenzyme A-acetylating aldehyde dehydrogenase activity
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-
?
additional information
?
-
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no activity with propyne, ethylene, and acetonitrile, calculated barriers for hydration of the compounds compared to acetylene, overview
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
acetylene + H2O
acetaldehyde
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-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
[4Fe-4S] cluster
low potential ferredoxin-type [4Fe-4S] cluster, the [4Fe-4S] has a midpoint potential around -410 mV
molybdenum cofactor
-
molybdopterin cofactor, 0.94 mol per mol of enzyme in purified recombinant enzyme
additional information
cofactors bis-WPT guanine dinucleotide and [4Fe-4S] cluster are buried deep inside a four-domain fold, as typically observed for enzymes of the DMSOR family
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tungsto-bis(pyranopterin guanine dinucleotide)
harbors two pyranopterins bound to tungsten, the enzyme contains 1.3-1.4 mol pyranopterin-guaninedinucleotide cofactor (MGD)
tungsto-bis(pyranopterin guanine dinucleotide)
the enzyme harbors two pyranopterins bound to tungsten, structure overview
molybdopterin guanine dinucleotide
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1.3 mol of cofactor per mol of enzyme
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
in [4Fe-4S] cluster, the enzyme contains 3.7-3.9 mol Fe/mol enzyme
Molybdenum
a Mo-dependent active form of AH (Mo-AH) can also be obtained from Pelobacter acetylenicus
Tungsten
bound with two pyranopterins, the enzyme contains 0.4-0.5 mol W/mol enzyme
Fe2+
Iron
Iron-sulfur cluster
-
dependent on, the enzyme is a tungsten/iron-sulfur protein, 4.8 mol of iron per mol of enzyme and 3.9 mol od acid-labile sulfur per mol of enzyme
Molybdenum
Tungsten
additional information
Fe2+
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dependent on, the enzyme is a tungsten/iron-sulfur protein with [3Fe-4S], 2.0 gAV, low potential [4Fe-4S] cluster which is highly sensitive to oxidation, 4.8 mol of iron per mol of enzyme, 3.9 mol of acid-labile sulfur per mol of enzyme
Fe2+
-
dependent on, the enzyme is a tungsten/iron-sulfur protein with [4Fe-4S], N-terminal binding cluster Cys-Xaa-Cys-Xaa-Cys
Iron
-
non-redox-active tungsten/[4Fe-4S] enzyme, a cubane-type [4Fe:4S] cluster, structure analysis, overview
Iron
-
acetylene hydratase is a tungsten, iron-sulfur enzyme, contains 4.8 mol of iron per mol of enzyme
Molybdenum
-
1.3 mol of molybdopterin-guanine dinucleotide per mol of enzyme
Tungsten
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a non-redox-active tungsten/[4Fe-4S] enzyme, contains two molybdopterin guanine dinucleotide cofactors, MGD, designated P and Q, structure analysis, bis-molybdopterin guanine dinucleotide-ligated tungsten atom, overview, the tungsten center binds a water molecule that is activated by an adjacent aspartate residue, enabling to attack acetylene bound in a distinct, hydrophobic pocket, this mechanism requires a strong shift of pKa of the aspartate, caused by nearby low-potential [4Fe:4S] cluster, overview
Tungsten
-
dependent on, the enzyme is a tungsten/iron-sulfur protein
Tungsten
-
dependent on, the enzyme is a tungsten/iron-sulfur protein, 0.4 mol of tungsten per mol of enzyme
Tungsten
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dependent on, the enzyme is a tungsten/iron-sulfur protein, 0.5 mol of tungsten per mol of enzyme
Tungsten
-
acetylene hydratase is a tungsten, iron-sulfur enzyme, active W(IV) state structure, contains 0.4 mol of tungsten per mol of enzymeoverview
additional information
the dependence of AH activity on the applied redox potential gives a midpoint potential of -340 mV. Mo-dependent enzyme is approximately 10fold less active than the native W-dependent enzyme. Attempts to insert vanadium into the enzyme's active site fail
additional information
the dependence of AH activity on the applied redox potential gives a midpoint potential of -340 mV. Mo-dependent enzyme is approximately 10fold less active than the native W-dependent enzyme. W is generally preferred over Mo in low-potential redox catalysis
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
HgCl2
-
reduces enzyme activity by 40% at 0.01 mM, 80% at 0.1 mM, and 98% at 0.2 mM
additional information
oxidation of AH with one equivalent [Fe(CN)6]3-
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additional information
oxidation of AH with one equivalent [Fe(CN)6]3-
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additional information
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no inhibition by citrate, ascorbate, tiron, ferrocene, and EDTA, no inhibition by ethylene at 8 mM
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ti(III)citrate
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a strong reductant is required for activity
additional information
-
the enzyme is inducible by acetylene feeding
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
the enzyme activity depends on the redox status, overview
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additional information
additional information
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the enzyme activity depends on the redox status, overview
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additional information
additional information
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the enzyme activity depends on the redox status, overview
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
activity of different purifications, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
additional information
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the organism is grown anaerobically in tungstate-supplemented fresh water medium
additional information
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the organism is grown in carbonate-buffered, sulfide-reduced, and tungstate-supplemented fresh water medium, optimally at pH 6.8-7.0
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
the enzyme activity is localized exclusively in the soluble fraction of the cell extract
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
additional information
evolution
the enzyme belongs to the tungsten containing enzymes, and is a member of the dimethyl sulfoxide reductase (DMSOR) family of enzymes. The W center coordinated by the two MGDs with the [4Fe-4S] cluster in close proximity, is unique for an enzyme of the DMSOR family
evolution
the enzyme belongs to the tungsten containing enzymes, it is a unique W, Fe-S enzyme and a member of the dimethyl sulfoxide reductase (DMSOR) family of enzymes. The W, Fe-S-dependent enzyme might have arosen recently as a means for microbes to take advantage of local anthropogenic sources of acetylene or it represents the relic of some ancestral biochemical process
additional information
Mo-dependent enzyme is approximately 10fold less active than the native W-dependent enzyme. Active site cavity structure of Pelobacter acetylenicus acetylene hydratase, overview. A C2H2 molecule docked computationally at the AH active site gives an excellent fit in the pocket of the hydrophobic ring with its carbon atoms positioned directly above the oxygen ligand and the carboxylic acid group of active site residue Asp13
additional information
Mo-dependent enzyme is approximately 10fold less active than the native W-dependent enzyme. Active site cavity structure of Pelobacter acetylenicus acetylene hydratase, overview. A C2H2 molecule docked computationally at the AH active site gives an excellent fit in the pocket of the hydrophobic ring with its carbon atoms positioned directly above the oxygen ligand and the carboxylic acid group of Asp13
additional information
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study of the chemoselectivity of tungsten-dependent acetylene hydratase, overview
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
73000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
additional information
additional information
domain I (residues 4-60) harbors the [4Fe-4S] cluster, ligated by the four cysteine residues Cys9, Cys12, Cys16 and Cys46. Domains II (residues 65-136 and 393-542) and III (residues 137-327) have an alphabetaalpha-fold with homologies to the NAD-binding fold of dehydrogenases
additional information
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four domain structure with active site access pathway, active site architecture, overview
additional information
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analysis of the active W(IV) state three-dimensional structure, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
high resolution crystal structure determination of the W-dependent enzyme crystallized under the exclusion of dioxygen (N2/H2 (94%/6% v/v)) at 1.26 A resolution, PDB ID 2E7Z
purified enzyme, the mother liquor contains plus 15% (v/v) 2-methyl-2,5-pentanediol, X-ray diffraction structure determination and analysis at 1.26-1.95 A resolution, modeling
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purified native enzyme, sitting drop vapour diffusion method in a 95%N2/5%H2 atmosphere, 10 mg/ml protein in 5 mM HEPES-NaOH, pH 7.5, and 3 mM dithionite or Ti(III)citrate, mixing with an equal volume of 0.002 ml of precipitant solution equilibrated against 0.3 ml of reservoir, 20°C, 3 weeks, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement
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purified recombinant NarG-fusion acetylene hydratase, sitting and hanging drop vapor diffusion methods, small crystals after 3 to 4 weeks, 6.5-10 mg/ml protein in solution containing 5 mM HEPES-NaOH, pH 7.5, and 7.5 mM Na2S2O4, cryoprotection by 20% v/v 2-methyl-2,4-pentanediol, X-ray diffraction structure determination and analysis
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D13A
site-directed mutagenesis of catalytically important Asp13, a direct neighbor of the [4Fe-4S] coordinating Cys12, forms a close hydrogen bond of 2.41 A to the oxygen ligand of the W ion, the mutant shows significant loss of activity compared to wild-type
D13E
site-directed mutagenesis of catalytically important Asp13, a direct neighbor of the [4Fe-4S] coordinating Cys12, forms a close hydrogen bond of 2.41 A to the oxygen ligand of the W ion, the mutant shows unaltered activity compared to wild-type
I142A
site-directed mutagenesis, Ile142 is part of the hydrophobic ring that is proposed to form the substrate binding cavity at the end of the access tunnel towards the active site, its exchange against alanine results in a strong loss of activity
K48A
site-directed mutagenesis of the residue involved in electron transfer between the two cofactors, the exchange of Lys48 against alanine does not affect catalysis
I142A
-
site-directed mutagenesis, the mutant shows a marked loss of activity compared to the wild-type enzyme
K48A
-
site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme
additional information
-
construction of active-site variants, and of a fusion protein of the N-terminal chaperone binding site of the Escherichia coli nitrate reductase NarG to the AH gene improving the yield and activity of AH and its variants significantly, overview
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme activity is stable even after prolonged storage of the cell extract or of the purified protein under air
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
native enzyme 29.7fold by ammonium sulfate fractionation, ion exchange chromatography, and gel filtration to homogeneity
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native enzyme 7.6fold by ammonium sulfate fractionation, ion exchange chromatography, and gel filtration to homogeneity
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under air at room temperature, native enzyme 240-fold by ammonium sulfate fractionation, anion exchange chromatography, gel filtration, and a second anion exchange chromatography step to homogeneity
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloning of AH gene in Escherichia coli strain JM109, expression of wild-type enzyme, active-site variants of the enzyme, and of the nitrate reductase N-terminal chaperone binding site NarG-fusion enzyme in Escherichia coli strain BL21(DE3)
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Einsle, O.; Niessen, H.; Abt, D.J.; Seiffert, G.; Schink, B.; Huber, R.; Messerschmidt, A.; Kroneck, P.M.H.
Crystallization and preliminary x-ray analysis of the tungsten-dependent acetylene hydratase from Pelobacter acetylenicus
Acta Crystallogr. Sect. F
F61
299-301
2005
Syntrophotalea acetylenica, Syntrophotalea acetylenica WoAcy1 / DSM 3246
Meckenstock, R.U.; Krieger, R.; Ensign, S.; Kroneck, P.M.H.; Schink, B.
Acetylene hydratase of Pelobacter acetylenicus. Molecular and spectroscopic properties of the tungsten iron-sulfur enzyme
Eur. J. Biochem.
264
176-182
1999
Syntrophotalea acetylenica
Yadav, J.; Das, S.K.; Sarkar, S.
A functional mimic of the new class of tungstoenzyme acetylene hydratase
J. Am. Chem. Soc.
119
4315-4316
1997
Syntrophotalea acetylenica
-
Rosner, B.M.; Schink, B.
Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein
J. Bacteriol.
177
5767-5772
1995
Syntrophotalea acetylenica, Syntrophotalea acetylenica WoAcy1 / DSM 3246
Seiffert, G.B.; Ullmann, G.M.; Messerschmidt, A.; Schink, B.; Kroneck, P.M.H.; Einsle, O.
Structure of the non-redox-active tungsten/[4Fe:4S] enzyme acetylene hydratase
Proc. Natl. Acad. Sci. USA
104
3073-3077
2007
Syntrophotalea acetylenica
Vincent, M.A.; Hillier, I.H.; Periyasamy, G.; Burton, N.A.
A DFT study of the possible role of vinylidene and carbene intermediates in the mechanism of the enzyme acetylene hydratase
Dalton Trans.
39
3816-3822
2010
Syntrophotalea acetylenica
Antony, S.; Bayse, C.
Theoretical studies of models of the active site of the tungstoenzyme acetylene hydratase
Organometallics
28
4938-4944
2009
Syntrophotalea acetylenica
-
Liao, R.; Himo, F.
Theoretical study of the chemoselectivity of tungsten-dependent acetylene hydratase
ACS Catal.
1
937-944
2011
Syntrophotalea acetylenica
-
Tenbrink, F.; Schink, B.; Kroneck, P.M.
Exploring the active site of the tungsten, iron-sulfur enzyme acetylene hydratase
J. Bacteriol.
193
1229-1236
2011
Syntrophotalea acetylenica, Syntrophotalea acetylenica WoAcy1 / DSM 3246
Liao, R.Z.; Yu, J.G.; Himo, F.
Mechanism of tungsten-dependent acetylene hydratase from quantum chemical calculations
Proc. Natl. Acad. Sci. USA
107
22523-22527
2010
Syntrophotalea acetylenica
Kroneck, P.M.H.
Acetylene hydratase a non-redox enzyme with tungsten and iron-sulfur centers at the active site
J. Biol. Inorg. Chem.
21
29-38
2016
Gordonia alkanivorans, Norcadia rhodochorous, Rhodobacter capsulatus, Rhodococcus opacus, Rhodococcus ruber, Rhodococcus sp. A1, Syntrophotalea acetylenica (Q71EW5)
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