Information on EC 3.5.5.8 - thiocyanate hydrolase

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The expected taxonomic range for this enzyme is: Proteobacteria

EC NUMBER
COMMENTARY hide
3.5.5.8
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RECOMMENDED NAME
GeneOntology No.
thiocyanate hydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
thiocyanate + 2 H2O = carbonyl sulfide + NH3 + HO-
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of thiocyanate
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
thiocyanate degradation II
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SYSTEMATIC NAME
IUBMB Comments
thiocyanate aminohydrolase
The enzyme from Thiobacillus thioparus catalyses the first step in the degradation of thiocyanate.
CAS REGISTRY NUMBER
COMMENTARY hide
142539-65-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
alphaproteobacterium sp.
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Manually annotated by BRENDA team
alphaproteobacterium sp. THI201
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
methacrylonitrile + H2O
?
show the reaction diagram
thiocyanate + H2O
carbonyl sulfide + NH3 + HO-
show the reaction diagram
thiocyanate + H2O
carbonyl sulfide + NH3 + OH-
show the reaction diagram
additional information
?
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does not use thiosulfate as a substrate
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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
thiocyanate + H2O
carbonyl sulfide + NH3 + HO-
show the reaction diagram
thiocyanate + H2O
carbonyl sulfide + NH3 + OH-
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
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contains 0.41 atoms Fe2+ per heterotrimer
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetate
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inhibitory at 20 mM
azide
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inhibitory at 20 mM
Borate
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inhibitory at 20 mM
Br-
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inhibitory at 20 mM
Cl-
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inhibitory at 20 mM
Cyanate
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significant inhibition
nitrate
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inhibitory at 20 mM
nitrite
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inhibitory at 20 mM
sulfite
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inhibitory at 20 mM
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Thiocyanate
alphaproteobacterium sp.
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maximum activity at 20-40 mM thiocyanate
additional information
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expression of thiocyanate hydrolase is promoted by its activator protein P15K
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
11 - 160
Methacrylonitrile
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 57
Methacrylonitrile
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.03
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crude extract, in 0.1 M potassium phosphate buffer at 37C and pH 7.5
3.3
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after 110fold purification, in 0.1 M potassium phosphate buffer at 37C and pH 7.5
32
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purified native enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 6.5
alphaproteobacterium sp.
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7.5 - 8
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 70
alphaproteobacterium sp.
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 70
alphaproteobacterium sp.
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30 - 40
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enzyme shows high activity within this range
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
126000
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gel filtration
130000
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gel filtration
140000
142000
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native PAGE
240000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dodecamer
heterododecamer
heterohexamer
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2 * 29000 + 2 * 19000 + 2 * 17000, gel filtration
hexamer
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2 * 19000 + 2 * 23000 + 2 * 32000, deduced from amino acid sequence, SDS-PAGE
trimer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, using 0.1 M potassium phosphate (pH 7.2) containing 2.0 M ammonium sulfate for purified SCNase or 0.1 M potassium phosphate (pH 6.9) containing 1.5 M Na/K tartrate for spontaneously activated SCNase and inactivated SCNase, respectively; hanging drop vapor diffusion method, using 0.1 M potassium phosphate (pH 7.2) containing 2.0 M ammonium sulfate for purified SCNase or 0.1 M potassium phosphate (pH 6.9) containing 1.5 M Na/K tartrate for spontaneously activated SCNase and inactivated SCNase, respectively; hanging drop vapor diffusion method, using 0.1 M potassium phosphate (pH 7.2) containing 2.0 M ammonium sulfate for purified SCNase or 0.1 M potassium phosphate (pH 6.9) containing 1.5 M Na/K tartrate for spontaneously activated SCNase and inactivated SCNase, respectively
hanging drop vapour diffusion method with 0.1 M potassium phosphate (pH 7.6) containing 1.5 M Na/K L-(+) tartrate or 0.1 M potassium phosphate (pH 7.2) containing 2 M ammonium sulfate for apo- or native SCNase, respectively
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mutant R136W, hanging drop vapor diffusion method, using 0.1 M potassium phosphate (pH 7.2) and 1.4 M sodium potassium tartrate
O66187 and O66186 and O66188
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
alphaproteobacterium sp.
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incubating recombinant enzyme on ice for 3 h reduces the enzyme activity to 2% of the original activity and subsequent heating at 60C for 15 min returns the activity to the original level
30 - 80
alphaproteobacterium sp.
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the activity of the recombinant enzyme is completely retained after heat treatment at temperatures from 30 to 70C, but is decreased by heating at 80C for 5 min to 80%, to 60% at 90C, and to 50% at 100C
60
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treatment of the enzyme at 60C for 5 min decreases activity below 10% of the original
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
when recombinant SCNase, containing gammaCys131-SO2 and gammaCys133-SO(H) modifications in the cobalt center, is stored at 20C under aerobic conditions for 4 months, no enzymatic activity is detected
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712237, 712237
when recombinant SCNase, containing gammaCys131-SO2- and gammaCys133-SO(H) modifications in the cobalt center, is stored at 20C under aerobic conditions for 4 months, no enzymatic activity is detected
712237
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, recombinant enzyme, 1 week, complete loss of activity (72% of the original activity is recovered by the heat treatment of 60C for 15 min)
alphaproteobacterium sp.
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, DEAE-Sephacyl, hydroxylapatite
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DEAE-Sepharose column chromatography, Mono Q GI column chromatography, and Superdex 200 gel filtration
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hydroxyapatite column chromatography (native enzyme) and glutathione Sepharose column chromatography (recombinant enzyme). The activity of the enzyme purified at room temperature is 1.4times that of the enzyme purified at 4C
alphaproteobacterium sp.
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native enzyme from strain THI115, and recombinant enzyme from Echerichia coli strain BL21(DE3) by three steps of butyl-resin chromatography, hydroxyapatite and ion exchange chromatography, or by 4 steps of butyl-resin chromatography and gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis
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expressed in Escherichia coli BL21(DE3) cells; expressed in Escherichia coli BL21(DE3) cells; expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21-AI(DE3) cells
O66187 and O66186 and O66188
expressed in Escherichia coli Rosetta-gami B and BL21(DE3) cells
alphaproteobacterium sp.
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expression in Escherichia coli
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expression of the alpha, beta, and gamma subunits in Escherichia coli, the subunits assemble to an apo-heterododecamer (alphabetagamma)4 like the wild-type enzyme exhibiting no catalytic activity due to lack of bound Co2+ irrespective of the cobalt concentration in the medium, coepression of enzyme with activator protein P15K leads to a functional Co2+-bound enzyme which exhibits 78% of native enzyme activity, optimization of conditions, overview
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R136W
O66187 and O66186 and O66188
the mutant of the gamma subunit shows no thiocyanate hydrolysis activity but does catalyze the hydration of nitriles
R90F
O66187 and O66186 and O66188
the mutant of the beta subunit shows no thiocyanate hydrolysis activity but does catalyze the hydration of nitriles
R136W
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the mutant of the gamma subunit shows no thiocyanate hydrolysis activity but does catalyze the hydration of nitriles
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R90F
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the mutant of the beta subunit shows no thiocyanate hydrolysis activity but does catalyze the hydration of nitriles
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