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 .
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
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
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
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
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
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
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
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
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
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
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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].
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
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
no activity with ethylene, methylene blue, or anthraquinone disulfonate, the purified enzyme has no coenzyme A-acetylating aldehyde dehydrogenase activity
no activity with ethylene, methylene blue, or anthraquinone disulfonate, the purified enzyme has no coenzyme A-acetylating aldehyde dehydrogenase activity
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
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
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
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
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
in isoprenoid biosynthesis, the protein (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase performs a 2H+/2e- reduction and deoxygenation of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate, yielding isopentenyl diphosphate and dimethylallyl diphosphate. In addition to its role as a 2H+/2e- reductase in isoprenoid biosynthesis, IspH can catalyse a second class of reactions: the addition of water to acetylene groups to produce aldehyde and ketone products
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
site-directed mutagenesis of the residue involved in electron transfer between the two cofactors, the exchange of Lys48 against alanine does not affect catalysis
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
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
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)
4.2.1.112 acetylene hydratase
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