Information on EC 4.2.1.112 - acetylene hydratase

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY
4.2.1.112
-
RECOMMENDED NAME
GeneOntology No.
acetylene hydratase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
acetaldehyde = acetylene + H2O
show the reaction diagram
-
-
-
-
acetaldehyde = acetylene + H2O
show the reaction diagram
reaction mechanism involving molybdenum-cofactor and tungsten/iron-sulfur cluster, structure-function modeling, overview
-
acetaldehyde = acetylene + H2O
show the reaction diagram
active site channel structure and detailed catalytic mechanism
-
acetaldehyde = acetylene + H2O
show the reaction diagram
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
-
acetaldehyde = acetylene + H2O
show the reaction diagram
active-site access, active-site architecture, and reaction mechanism, overview
-
acetaldehyde = acetylene + H2O
show the reaction diagram
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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acetaldehyde = acetylene + H2O
show the reaction diagram
active-site access, active-site architecture, and reaction mechanism, overview
Pelobacter acetylenicus WoAcy1
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-
PATHWAY
KEGG Link
MetaCyc Link
acetylene degradation
-
Chloroalkane and chloroalkene degradation
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Microbial metabolism in diverse environments
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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].
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
tungsten-dependent acetylene hydratase
-
-
tungsten-dependent acetylene hydratase
Pelobacter acetylenicus WoAcy1
-
-
-
additional information
-
the enzyme structurally belongs to the dimethyl sulfoxide reductase family of enzymes
CAS REGISTRY NUMBER
COMMENTARY
75788-81-7
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
isolated from soil, aerobic strain
-
-
Manually annotated by BRENDA team
Gordonia sp. aerobic
isolated from soil, aerobic strain
-
-
Manually annotated by BRENDA team
strain WoAcy1, DSM 3246
-
-
Manually annotated by BRENDA team
Pelobacter acetylenicus WoAcy1
-
-
-
Manually annotated by BRENDA team
Pelobacter acetylenicus WoAcy1
strain WoAcy1, DSM 3246
-
-
Manually annotated by BRENDA team
two aerobic isolates from soil, DSM 44186 and 44188
-
-
Manually annotated by BRENDA team
isolated from soil, aerobic strain
-
-
Manually annotated by BRENDA team
Rhodococcus ruber aerobic
isolated from soil, aerobic strain
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
study of the chemoselectivity of tungsten-dependent acetylene hydratase, overview
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetylene + H2O
acetaldehyde
show the reaction diagram
-
-
-
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
-
-
-
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
-
-
-
-
r
acetylene + H2O
acetaldehyde
show the reaction diagram
-
-
the subsequent disproportionation of acetaldehyde yields acetate and ethanol
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
-
a non-redox reaction
-
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
-
the hydration reaction is unique for a tungsten and molybdenum containing enzyme, which are usually redox enzymes
-
-
r
acetylene + H2O
acetaldehyde
show the reaction diagram
-
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
-
-
r
acetylene + H2O
acetaldehyde
show the reaction diagram
Gordonia sp. aerobic
-
-
-
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
Pelobacter acetylenicus WoAcy1
-
-, 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
-
-
r
acetylene + H2O
acetaldehyde
show the reaction diagram
Pelobacter acetylenicus WoAcy1
-
-
-, the subsequent disproportionation of acetaldehyde yields acetate and ethanol
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
Pelobacter acetylenicus WoAcy1
-
-
-
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
Rhodococcus ruber aerobic
-
-
-
-
?
additional information
?
-
-
no activity with ethylene, methylene blue, or anthraquinone disulfonate, the purified enzyme has no coenzyme A-acetylating aldehyde dehydrogenase activity
-
-
-
additional information
?
-
-
no activity with propyne, ethylene, and acetonitrile, calculated barriers for hydration of the compounds compared to acetylene, overview
-
-
-
additional information
?
-
Pelobacter acetylenicus WoAcy1
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no activity with ethylene, methylene blue, or anthraquinone disulfonate, the purified enzyme has no coenzyme A-acetylating aldehyde dehydrogenase activity
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
acetylene + H2O
acetaldehyde
show the reaction diagram
-
-
-
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
-
-
-
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
-
-
-
-
r
acetylene + H2O
acetaldehyde
show the reaction diagram
Gordonia sp. aerobic
-
-
-
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
Pelobacter acetylenicus WoAcy1
-
-
-
-
r
acetylene + H2O
acetaldehyde
show the reaction diagram
Pelobacter acetylenicus WoAcy1
-
-
-
-
?
acetylene + H2O
acetaldehyde
show the reaction diagram
Rhodococcus ruber aerobic
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
molybdenum cofactor
-
molybdopterin cofactor, 0.94 mol per mol of enzyme in purified recombinant enzyme
molybdopterin guanine dinucleotide
-
1.3 mol of cofactor per mol of enzyme
molybdopterin guanine dinucleotide
-
structure-function analysis
molybdopterin guanine dinucleotide
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Fe2+
-
dependent on, the enzyme is a tungsten/iron-sulfur protein with [4Fe-4S], N-terminal binding cluster Cys-Xaa-Cys-Xaa-Cys
Fe2+
-
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 against oxidation, 4.8 mol of iron per mol of enzyme, 3.9 mol of acid-labile sulfur per mol of enzyme
Iron
-
the enzyme is a tungsten/iron-sulfur protein
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
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
-
1.3 mol of molybdopterin-guanine dinucleotide per mol of enzyme
Molybdenum
-
dependent on, enzyme expression also requires molybdenum
Ti(III)citrate
-
a strong reductant is absolutely required for activity
Tungsten
-
dependent on, the enzyme is a tungsten/iron-sulfur protein
Tungsten
-
dependent on, the enzyme is a tungsten/iron-sulfur protein, 0.5 mol of tungsten per mol of enzyme
Tungsten
-
dependent on, tungstoenzyme
Tungsten
-
dependent on, the enzyme is a tungsten/iron-sulfur protein, 0.4 mol of tungsten per mol of enzyme
Tungsten
-
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
-
tungstoprotein
Tungsten
-
tungstoenzyme
Tungsten
-
acetylene hydratase is a tungsten, iron-sulfur enzyme, active W(IV) state structure, contains 0.4 mol of tungsten per mol of enzymeoverview
Tungsten
-
dependent on
Molybdenum
-
molybdopterin-guanine dinucleotide cofactor
additional information
-
the enzyme does not require reductive additives
additional information
-
no molybdenum detectable
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
CO
-
90% inhibition at 0.8 mM
HgCl2
-
reduces enzyme activity by 40% at 0.01 mM, 80% at 0.1 mM, and 98% at 0.2 mM
KCN
-
20% inhibition at 1 mM, 40% inhibition at 5-10 mM
nitric oxide
-
complete inhibition at 3 mM
propyne
-
competitive inhibitor
additional information
-
no inhibition by citrate, ascorbate, tiron, ferrocene, and EDTA, no inhibition by ethylene at 8 mM
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Ti(III)citrate
-
a strong reductant is required for activity
Dithionite
-
a strong reductant is required for activity
additional information
-
the enzyme is inducible by acetylene feeding
-
additional information
-
the enzyme does not require reductive additives
-
additional information
-
no activation by tungstate at 5 mM
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.014
-
Acetylene
-
pH 7.0, 30C
0.014
-
Acetylene
-
pH 7.5, 30C
additional information
-
additional information
-
the enzyme activity depends on the redox status, overview
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
10.7
-
-
purified native enzyme
13
-
-
crude cell extract of cells grown in presence of acetylene
14.2
-
-
purified recombinant enzyme, pH 7.5, 30C
26.5
-
-
purified native enzyme
69.2
-
-
purified enzyme
additional information
-
-
activity of different purifications, overview
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4.3
-
-
chromatofocusing
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
Gordonia sp. aerobic, Pelobacter acetylenicus WoAcy1, Rhodococcus ruber aerobic
-
-
-
Manually annotated by BRENDA team
Pelobacter acetylenicus WoAcy1
-
-
-
Manually annotated by BRENDA team
Pelobacter acetylenicus WoAcy1
-
-
-
Manually annotated by BRENDA team
additional information
-
the organism is grown anaerobically in tungstate-supplemented fresh water medium
Manually annotated by BRENDA team
additional information
-
the organism is grown in carbonate-buffered, sulfide-reduced, and tungstate-supplemented fresh water medium, optimally at pH 6.8-7.0
Manually annotated by BRENDA team
additional information
-
strictly anaerobic growth conditions
Manually annotated by BRENDA team
additional information
Pelobacter acetylenicus WoAcy1
-
the organism is grown anaerobically in tungstate-supplemented fresh water medium
-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
Pelobacter acetylenicus WoAcy1
-
-
-
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
60000
-
-
gel filtration
83550
-
-
MALDI mass spectrometry
85000
-
-
mass spectrometry
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
monomer
-
1 * 85000, MALDI mass spectrometry
monomer
-
1 * 73000
monomer
-
1 * 73000, SDS-PAGE
monomer
Pelobacter acetylenicus WoAcy1
-
1 * 73000, SDS-PAGE; 1 * 85000, MALDI mass spectrometry
-
additional information
-
four domain structure with active site access pathway, active site architecture, overview
additional information
-
analysis of the active W(IV) state three-dimensional structure, overview
additional information
Pelobacter acetylenicus WoAcy1
-
analysis of the active W(IV) state three-dimensional structure, overview
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
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
-
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, 20C, 3 weeks, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement
-
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
-
OXIDATION STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
the enzyme is oxygen-sensitive
-
667090, 668453
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
enzyme activity is stable even after prolonged storage of the cell extract or of the purified protein under air
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
native enzyme 29.7fold by ammonium sulfate fractionation, ion exchange chromatography, and gel filtration to homogeneity
-
native enzyme 7.6fold by ammonium sulfate fractionation, ion exchange chromatography, and gel filtration to homogeneity
-
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
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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)
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D13E
-
site-directed mutagenesis, almost inactive mutant
I142A
-
site-directed mutagenesis, the mutant shows a marked loss of activity compared to the wild-type enzyme
D13E
Pelobacter acetylenicus WoAcy1
-
site-directed mutagenesis, almost inactive mutant
-
I142A
Pelobacter acetylenicus WoAcy1
-
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
K48A
Pelobacter acetylenicus WoAcy1
-
site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme
-
additional information
Pelobacter acetylenicus WoAcy1
-
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
-