Information on EC 4.2.1.104 - cyanase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
4.2.1.104
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RECOMMENDED NAME
GeneOntology No.
cyanase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
carbamate + H+ = NH3 + CO2
show the reaction diagram
spontaneous
-
-
-
cyanate + HCO3- + 2 H+ = NH3 + 2 CO2
show the reaction diagram
cyanate + HCO3- + H+ = carbamate + CO2
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carbon-nitrogen lyase reaction
-
-
-
-
elimination of CO2
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
cyanate degradation
-
-
NIL
-
-
Nitrogen metabolism
-
-
cyanate degradation
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SYSTEMATIC NAME
IUBMB Comments
carbamate hydro-lyase
This enzyme, which is found in bacteria and plants, is used to decompose cyanate, which can be used as the sole source of nitrogen [6,7]. Reaction (1) can be considered as the reverse of 'carbamate = cyanate + H2O', where this is assisted by reaction with bicarbonate and carbon dioxide (see mechanism above) [2], and hence is classified in sub-subclass 4.2.1. Bicarbonate functions as a recycling substrate [2].
CAS REGISTRY NUMBER
COMMENTARY hide
37289-24-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
K12
-
-
Manually annotated by BRENDA team
gene cynS
UniProt
Manually annotated by BRENDA team
gene cynS
UniProt
Manually annotated by BRENDA team
strain CECT5344, expression in Escherichia coli DH5 alpha
TrEMBL
Manually annotated by BRENDA team
strain 21; strain 21, isolated from environments under anthropogenic contaminations, selected by resistance to cyanide
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-
Manually annotated by BRENDA team
strain 18; strain 18, associated strains, isolated from environments under anthropogenic contaminations, selected by resistance to cyanide
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-
Manually annotated by BRENDA team
strain 18; strain 18, associated strains, isolated from environments under anthropogenic contaminations, selected by resistance to cyanide
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-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
strain UTEX625
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-
Manually annotated by BRENDA team
gene cynS
UniProt
Manually annotated by BRENDA team
gene tetur28g02430
UniProt
Manually annotated by BRENDA team
halophilic sulfur-oxidizing gamma-proteobacterium
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
an ancient gene transfer occurred before the diversification within the Tetranychidae family
malfunction
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
cyanate + ?
?
show the reaction diagram
Cyanate + bicarbonate
?
show the reaction diagram
Cyanate + bicarbonate
CO2 + carbamate
show the reaction diagram
cyanate + HCO3- + 2 H+
NH3 + 2 CO2
show the reaction diagram
NCO- + HCO3-
NH4+ + CO2
show the reaction diagram
NCO- + HCO3- + 2 H+
NH3 + 2 CO2
show the reaction diagram
NCO- + HCO3- + H+
NH3 + CO2
show the reaction diagram
NCO- + HCO3- + H+
NH4+ + 2 CO2
show the reaction diagram
studies on decomposition of externally supplied cyanate, depending on the CynABDS operon, light and internal pools of HCO3- and CO2
-
-
?
NCO- + HCO3- + H+
NH4+ + CO2
show the reaction diagram
utilization of exogenous cyanate as a niche source of C and N in cyanobacteria
-
-
?
additional information
?
-
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
cyanate + ?
?
show the reaction diagram
Cyanate + bicarbonate
?
show the reaction diagram
cyanate + HCO3- + 2 H+
NH3 + 2 CO2
show the reaction diagram
NCO- + HCO3- + 2 H+
NH3 + 2 CO2
show the reaction diagram
NCO- + HCO3- + H+
NH3 + CO2
show the reaction diagram
NCO- + HCO3- + H+
NH4+ + CO2
show the reaction diagram
Q59948
utilization of exogenous cyanate as a niche source of C and N in cyanobacteria
-
-
?
additional information
?
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-oxoglutarate
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-
3-nitropropionate
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-
acetate
-
competitive to bicarbonate
ammonium
negative regulation even in the presence of cyanate. The negative effect exerted by ammonium on cyanate assimilation seems to take place at the level of gene expression, since the addition of ammonium to cells growing in cyanate had no effect on either cyanase activity or cyanate consumption
chloride
Cyanate
formate
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-
fumarate
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-
Glutarate
-
-
Hydroxymalonate
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malate
-
D- and L-isomer
Maleate
-
-
malonate
methyl methanethiosulfonate
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-
methylmalonate
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-
N-ethylmaleimide
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-
N3-
-
competitive to cyanate
Na-azide
oxalate
oxaloacetate
p-hydroxymercuribenzoate
-
sodium chloride
-
succinate
-
-
Sulfoacetate
-
-
Tetranitromethane
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Thiocyanate
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kinetics of cyanase activity at pH 7.5 and 37°C
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
bicarbonate
Cyanate
cyanide
positive regulation
NtcA
-
Urea
strong inducer of cyanase activity
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.048 - 29
Cyanate
additional information
additional information
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
15
chloride
Thiohalophilus thiocyanatoxydans
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-
6
Thiocyanate
Thiohalophilus thiocyanatoxydans
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-
additional information
additional information
Thiohalophilus thiocyanatoxydans
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kinetics of cyanase activity at pH 7.5 and 37°C, presence of 3 mM NaHCO3, cyanate concentrations between 0.2 and 2 mM
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.01
-
in absence of bicarbonate
0.038
purified recombinant mutant E94L, pH 7.7, 27°C
0.04
purified recombinant mutant S117A, pH 7.7, 27°C
0.047
-
complemented deficient Escherichia coli cells
0.22
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in the presence of 3 mM bicarbonate and 2 mM cyanate
0.303
purified recombinant enzyme, pH 4.8, 27°C
0.43
purified recombinant enzyme, pH 7.4, 55°C
0.633
purified recombinant enzyme, pH 5.7, 27°C
0.75
-
; activity determined every 15 to 30 min in the presence of 2 mM KCNO and 2 mM HCO3- by formation of NH4+, absent activity if bacteria are grown with NH4 indicates inducible synthesis of cyanase
0.8
purified recombinant enzyme, pH 7.4, 4°C
1.26
-
; pH optimum 8.1, activity determined every 15 to 30 min in the presence of 2 mM KCNO and 2 mM HCO3- by formation of NH4+, pH values of 7.0, 8.0 and 9.1, absent activity if bacteria are grown with NH4+ indicates inducible synthesis of cyanase
2.286
purified recombinant wild-type enzyme, pH 7.7, 27°C
5.56
purified recombinant enzyme, pH 7.4, 26°C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.4 - 8.8
using potassium phosphate buffer (50 mM) the enzyme retains more than 50% activity over a wide pH range after incubation at 27°C for 10 min
4.8 - 8.8
active within that range
6.7 - 8.8
over 75% of maximal activity at pH 6.7-8.8, no activity at pH 5.7 and pH 4.8
additional information
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 55
activity range
19 - 34
27 - 80
quite stable even at high temperatures
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Serratia proteamaculans (strain 568)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14000
-
10 * 14000, estimated by gel filtration, SDS-PAGE, similar to apparent masses of cyanase of Escherichia coli
14661
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x * 14661, minimum molecular weight calculated from amino acid composition
15200
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x * 15200, SDS-PAGE
16350
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8 or 10 * 16350 (4 or 5 disulfide linked dimers), amino acid sequence determination,
16362
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x * 16362, amino acid sequence calculation
18600
-
calculated from amino acid sequence
19000
SDS-PAGE, monomer calculated: 18100 + His-tag: 600
21900
10 * 21900, about, sequence calculation, homology modelling of monomers, coimmunoprecipitation, and gel filtration
22000
10 * 22000, about, sequence calculation, homology modelling of monomers, coimmunoprecipitation, and gel filtration
50000
x * 50000, recombinant MBP-CynS fusion enzyme, SDS-PAGE
70000
-
gel filtration
150000
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E. coli, sucrose density gradient centrifugation, gel filtration
170000
-
crystal structure
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
decamer
homodecamer
monomer
19000 recombinant enzyme + His-tag
octamer
oligomer
additional information
-
the monomers are composed of 2 domains, subunit arrangement, decamer is formed by 5 dimers assembled into a pentamer, model
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
selenomethionine-labeled purified recombinant enzyme, also crystals of enzyme with complexed inhibitors oxalate and chloride, 2 mM dithiothreitol, sitting drop vapour diffusion method, from 2.1 M ammonium sulfate, 50 mM Na2KPO4, pH 7.3, microseeding with large sitting drops at 18°C with wild-type crystals, 5-7 days, X-ray diffraction structure determinzation and analysis at 1.65 A resolution by multiwavelength anomalous diffraction MAD method
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.4 - 8.8
using potassium phosphate buffer (50 mM) the enzyme retains more than 50% activity over a wide pH range after incubation at 27°C for 10 min
692457
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 55
CynS is rapidly inactivated at higher temperatures and only residual activity o f 0.09 U/mg occurs at 50°C
27 - 80
quite stable even at high temperatures
30 - 70
the thermostability range represents temperatures at which the enzyme retains more than 25% of its original activity after 30 min. Higher temperatures result in the irreversible inactivation of the enzyme
100
-
1 min, complete loss of activity
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SDS
-
stable after 2 h in 5% SDS at 37°C in 0.1 M potassium phosphate buffer
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, stable for weeks
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
B; partial
-
co-purified with thiocyanate hydrolase of Thiohalophilus thiocyanoxidans, gel filtration and SDS-PAGE
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precipitation, chromatography
recombinant CynS-MBP from Escherichia coli strain HMS174 by amylose affinity chromatography
recombinant His-tagged enzyme from Escherichia coli BL21(DE3) pLys by nickel affinity chromatography and dialysis
recombinant His-tagged soluble CYN from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
expressed in Escherichia coli, cyanase gene cynS not arranged in a three-gene cluster
expression in Escherichia coli DH5 alpha
expression in Escherichia coli strain M15
gene Atcyn, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression in Escherichia coli strain BL21 (DE3) as His-tagged soluble protein
gene cynS, cynS is presumably transcribed as part of the cynABDS operon, DNA and amino acid sequence determination and analysis, phylogenetic analysis
gene cynS, DNA and amino acid sequence determination and analysis, phylogenetic analysis, overexpression in Escherichia coli strain HMS174 as maltose-binding-protein fusion protein
gene cynS, DNA and amino acid sequence determination and analysis, tightly clustered with 2 genes located upstream, which encode proteins similar to the subunits of nitrate-nitrite transporter, complementation of deficient Escherichia coli strain
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gene cynS, tightly clustered with 4 putative molybdenum cofactor biosynthesis genes located downstream
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gene Oscyn, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression in Escherichia coli strain BL21 (DE3) as His-tagged soluble protein
gene tetur28g02430, sequence comparisons and phylogenetic analysis, expression of His-tagged enzyme in Escherichia coli BL21(DE3) pLys, the Tetranychus urticae cyanase remains functionally active after horizontal gene transfer
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
AtCYN transcription is not significantly affected by KCNO treatment, but is induced by salt stress
cyanase activity is induced during growth with cyanide or cyanate, but not with ammonium or nitrate as the nitrogen source
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E94L
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme. The mutant can form trimers or a mixture of polymers but not decamers
S117A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme. The mutant can form trimers or a mixture of polymers but not decamers
E94L
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme. The mutant can form trimers or a mixture of polymers but not decamers
-
S117A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme. The mutant can form trimers or a mixture of polymers but not decamers
-
E107A
site-directed mutagenesis, soluble, no activity
E107D
site-directed mutagenesis, soluble, no activity
R104A
site-directed mutagenesis, soluble, no activity
R104L
site-directed mutagenesis, soluble, no activity
S130A
site-directed mutagenesis, soluble, no activity
S130T
site-directed mutagenesis, soluble, no activity
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
more than 85% renaturation after urea denaturation
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
environmental protection
-
potential biotechnological application in environmental detoxification
molecular biology
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