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Information on EC 3.5.5.8 - thiocyanate hydrolase and Organism(s) Thiobacillus thioparus and UniProt Accession O66187

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     3 Hydrolases
         3.5 Acting on carbon-nitrogen bonds, other than peptide bonds
             3.5.5 In nitriles
                3.5.5.8 thiocyanate hydrolase
IUBMB Comments
The enzyme from Thiobacillus thioparus catalyses the first step in the degradation of thiocyanate.
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This record set is specific for:
Thiobacillus thioparus
UNIPROT: O66187
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The taxonomic range for the selected organisms is: Thiobacillus thioparus
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
hide
thiocyanate
+
2
H2O
=
carbonyl sulfide
+
NH3
+
HO-
+
2
=
+
+
Synonyms
scnase, thiocyanate hydrolase, more
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of thiocyanate
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-
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
-
SUBSTRATE
PRODUCT                       
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 + OH-
show the reaction diagram
-
-
-
?
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
NATURAL SUBSTRATE
NATURAL PRODUCT
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
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
expression of thiocyanate hydrolase is promoted by its activator protein P15K
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
11 - 160
Methacrylonitrile
11
thiocyanate
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 57
Methacrylonitrile
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32
-
purified native enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 40
-
enzyme shows high activity within this range
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
subunit alpha
UniProt
Manually annotated by BRENDA team
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
-
the organism is obligately chemilithoautotrophic sulfur-oxidizing and uses thiocyanate as sole energy source
Manually annotated by BRENDA team
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
SCNA_THITI
126
0
14488
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
126000
-
gel filtration
140000
-
native and apo-enzyme, gel filtration
142000
-
native PAGE
19000
-
2 * 19000 + 2 * 23000 + 2 * 32000, deduced from amino acid sequence, SDS-PAGE
23000
-
2 * 19000 + 2 * 23000 + 2 * 32000, deduced from amino acid sequence, SDS-PAGE
240000
-
gel filtration
32000
-
2 * 19000 + 2 * 23000 + 2 * 32000, deduced from amino acid sequence, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dodecamer
-
heterododecamer (alphabetagamma)4 of heterotrimers
heterododecamer
-
x-ray crystallography
hexamer
-
2 * 19000 + 2 * 23000 + 2 * 32000, deduced from amino acid sequence, SDS-PAGE
trimer
-
heterotrimer alphabetagamma
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
Cys113 is posttranslationally oxidized to cysteinesulfinic acid
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 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
-
mutant R136W, hanging drop vapor diffusion method, using 0.1 M potassium phosphate (pH 7.2) and 1.4 M sodium potassium tartrate
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R136W
the mutant of the gamma subunit shows no thiocyanate hydrolysis activity but does catalyze the hydration of nitriles
R90F
the mutant of the beta subunit shows no thiocyanate hydrolysis activity but does catalyze the hydration of nitriles
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
-
treatment of the enzyme at 60°C 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 20°C under aerobic conditions for 4 months, no enzymatic activity is detected
712237
when recombinant SCNase, containing gammaCys131-SO2 – and gammaCys133-SO(H) modifications in the cobalt center, is stored at 20°C under aerobic conditions for 4 months, no enzymatic activity is detected
712237
when recombinant SCNase, containing gammaCys131-SO2- and gammaCys133-SO(H) modifications in the cobalt center, is stored at 20°C under aerobic conditions for 4 months, no enzymatic activity is detected
712237
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, DEAE-Sephacyl, hydroxylapatite
-
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
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
DNA and amino acid sequence determination and analysis
-
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21-AI(DE3) cells
expression in Escherichia coli
-
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
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Shearer, J.; Kung, I.Y.; Lovell, S.; Kovacs, J.A.
A Co(III) complex in a mixed sulfur/nitrogen ligand environment: modelling the substrate- and product-bound forms of the metalloenzyme thiocyanate hydrolase
Inorg. Chem.
39
4998-4999
2000
Thiobacillus thioparus
Manually annotated by BRENDA team
Katayama, Y.; Matsushita, Y.; Kaneko, M.; Kondo, M.; Mizuno, T.; Nyunoya, H.
Cloning of genes coding for the three subunits of thiocyanate hydrolase of Thiobacillus thioparus THI 115 and their evolutionary relationships to nitrile hydratase
J. Bacteriol.
180
2583-2589
1998
Thiobacillus thioparus
Manually annotated by BRENDA team
Katayama, Y; Narahara, Y.; Inoue, Y.; Amano, F.; Kanagawa, T.; Kuraishi, H.
A thiocyanate hydrolase of Thiobacillus thioparus: a novel enzyme catalyzing the formation of carbonyl sulfide from thiocyanate
J. Biol. Chem.
267
9170-9175
1992
Thiobacillus thioparus
Manually annotated by BRENDA team
Kataoka, S.; Arakawa, T.; Hori, S.; Katayama, Y.; Hara, Y.; Matsushita, Y.; Nakayama, H.; Yohda, M.; Nyunoya, H.; Dohmae, N.; Maeda, M.; Odaka, M.
Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K
FEBS Lett.
580
4667-4672
2006
Thiobacillus thioparus, Thiobacillus thioparus THI115
Manually annotated by BRENDA team
Katayama, Y.; Hashimoto, K.; Nakayama, H.; Mino, H.; Nojiri, M.; Ono, T.; Nyunoya, H.; Yohda, M.; Takio, K.; Odaka, M.
Thiocyanate hydrolase is a cobalt-containing metalloenzyme with a cysteine-sulfinic acid ligand
J. Am. Chem. Soc.
128
728-729
2006
Thiobacillus thioparus, Thiobacillus thioparus THI115
Manually annotated by BRENDA team
Arakawa, T.; Kawano, Y.; Kataoka, S.; Katayama, Y.; Kamiya, N.; Yohda, M.; Odaka, M.
Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center
J. Mol. Biol.
366
1497-1509
2007
Thiobacillus thioparus, Thiobacillus thioparus THI115
Manually annotated by BRENDA team
Arakawa, T.; Kawano, Y.; Katayama, Y.; Nakayama, H.; Dohmae, N.; Yohda, M.; Odaka, M.
Structural basis for catalytic activation of thiocyanate hydrolase involving metal-ligated cysteine modification
J. Am. Chem. Soc.
131
14838-14843
2009
Thiobacillus thioparus (O66186), Thiobacillus thioparus (O66187), Thiobacillus thioparus (O66188)
Manually annotated by BRENDA team
Yamanaka, Y.; Arakawa, T.; Watanabe, T.; Namima, S.; Sato, M.; Hori, S.; Ohtaki, A.; Noguchi, K.; Katayama, Y.; Yohda, M.; Odaka, M.
Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase
J. Biosci. Bioeng.
116
22-27
2013
Thiobacillus thioparus (O66187 and O66186 and O66188), Thiobacillus thioparus THI115 (O66187 and O66186 and O66188), Thiobacillus thioparus THI115
Manually annotated by BRENDA team