Information on EC 3.5.4.23 - blasticidin-S deaminase

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

EC NUMBER
COMMENTARY hide
3.5.4.23
-
RECOMMENDED NAME
GeneOntology No.
blasticidin-S deaminase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
blasticidin S + H2O = deaminohydroxyblasticidin S + NH3
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
amidine hydrolysis
hydrolysis of C-N bond
-
-
SYSTEMATIC NAME
IUBMB Comments
blasticidin-S aminohydrolase
Catalyses the deamination of the cytosine moiety of the antibiotics blasticidin S, cytomycin and acetylblasticidin S.
CAS REGISTRY NUMBER
COMMENTARY hide
54576-55-5
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
NHL 5013
-
-
Manually annotated by BRENDA team
ATCC 9643
-
-
Manually annotated by BRENDA team
67-MI-87
-
-
Manually annotated by BRENDA team
NHL 5037
-
-
Manually annotated by BRENDA team
strain S-712 (ATCC 28865)
SwissProt
Manually annotated by BRENDA team
1004
-
-
Manually annotated by BRENDA team
1004
-
-
Manually annotated by BRENDA team
strain K55-S1
-
-
Manually annotated by BRENDA team
K55-S2
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
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
4-amino-1-(beta-D-glucuronosyl)pyrimidin-2(1H)-one + H2O
1-(beta-D-glucuronosyl)-4-hydroxypyrimidin-2(1H)-one
show the reaction diagram
acetylblasticidin S + H2O
acetyldeaminohydroxyblasticidin S
show the reaction diagram
alanylcytosinine + H2O
?
show the reaction diagram
-
-
-
-
?
blasticidin S + H2O
deaminohydroxyblasticidin S + NH3
show the reaction diagram
blasticidin S methylester + H2O
deaminohydroxyblasticidin S methylester
show the reaction diagram
-
only slightly deaminated
-
-
?
cytidine + H2O
?
show the reaction diagram
cytomycin + H2O
?
show the reaction diagram
cytosinine + H2O
?
show the reaction diagram
-
only slightly deaminated
-
-
?
demethylblasticidin S + H2O
?
show the reaction diagram
-
-
-
-
?
deoxycytidine + H2O
?
show the reaction diagram
dihydroblasticidin S + H2O
?
show the reaction diagram
-
-
-
-
?
dihydrocytomycin + H2O
?
show the reaction diagram
-
-
-
-
?
gougerotin + H2O
?
show the reaction diagram
mildiomycin + H2O
?
show the reaction diagram
phenylalanylcytosinine + H2O
?
show the reaction diagram
-
-
-
-
?
pyrimidinoblasticidin S + H2O
pyrimidinodeaminohydroxyblasticidin S
show the reaction diagram
-
-
-
-
?
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
blasticidin S + H2O
deaminohydroxyblasticidin S + NH3
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetylcytosinine
-
competitive inhibition
-
acetyldeaminohydroxyblasticidin S
-
competitive inhibition
-
caproylcytosinine
-
competitive inhibition
-
cytosinine
-
competitive inhibition
deaminohydroxyblasticidin S
-
competitive inhibition
deaminohydroxyblasticidin S methylester
-
-
deaminohydroxycytomycin S
-
competitive inhibition
-
deaminomercaptoblasticidin S
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competitive inhibition
dihydrocytomycin
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competitive inhibition
dihydrodeaminohydroxyblasticidin S
-
competitive inhibition
p-chloromercuribenzoate
-
-
p-Hydroxymercuriphenylsulfonic acid
-
leads to destabilization of the enzyme structure due to removal of required zinc
pyrimidinodeaminohydroxyblasticidin S
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competitive inhibition
Rose bengal
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0.01%, enzyme loses more than 80% of its activity, photooxidation
uracinine
-
competitive inhibition
-
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
blasticidin S
-
produces far more enzyme when grown in presence
Gougerotin
-
enzyme induced by
IPTG
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inducer
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.021 - 20
blasticidin S
0.053
pyrimidinoblasticidin S
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-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
71 - 890
blasticidin S
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 11.5
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-
10
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Tris-HCl, glycinate or bicarbonate buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 11
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retains original activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
13000
-
SDS-PAGE, heat treatment
13340
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recombinant enzyme, calculated by amino acid residues per subunit
13470
-
calculated from amino acid sequence
29600
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sedimentation analysis
30000
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SDS-PAGE
36000
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SDS-PAGE without heat treatment
46000
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gel filtration
49000
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native PAGE; recombinant wild-type enzyme, native PAGE
52000
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ultracentrifugal sedimentation analysis
54000
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gel filtration; recombinant and native wild-type enzyme, gel filtration
55000
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gel filtration, buffer without sodium chloride
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
x-ray crystallography
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 types of crystals, thin plates and rhombic shapes
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using 20% (w/v) PEG8000, 50 mM magnesium chloride, and 0.1 M sodium cacodylate at pH 7.0 as precipitant
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 7
-
inactivation below pH 4 for 1h, but stable below pH 7
209568, 209569
4.5
-
rapid inactivation below
209567
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60 - 80
-
reaction proceeds most rapidly at 60-70C, occurs only during a short initial period at 80C
65
half-lives of wild-type and mutant enzymes
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the tetrameric enzyme form is very resistant against denaturation by SDS, 90% remaining activity at 2% SDS, diluted 10fold and incubated 2 h at room temperature
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SDS
-
quite resistant to denaturation
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 40% glycerol-5 mM Tris-HCl buffer, pH 7.5, 5 mM 2-mercaptoethanol, no detectable loss of activity over a period of 3 months
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-20C, 50% glycerol 5 mM mercaptoethanol, no appreciable loss of activity after 3 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; native enzyme from Aspergiluus terreus, recombinant His-tagged enzyme from Escherichia coli by nickel chelate affinity chromatography
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HisTrap column chromatography, and gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bsd overexpressed in Escherichia coli BL 21; overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3), Cys91 mutants are expressed in inclusion bodies
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bsd overexpressed in Escherichia coli BL 21; PCR amplification of the BSD gene, cloned from cDNA and expressed in Escherichia coli, also transformed into Schizosaccharomyces pombe and Pyricularia oryzae, P. oryzae could not be transformed with bsr, but with BSD
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bsd overexpressed in Escherichia coli BL 21; PCR amplification of the BSD gene, cloned from cDNA and expressed in Escherichia coli, also transformed into Schizosaccharomyces pombe and Pyricularia oryzae, P. oryzae could not be transformed with bsr, but with BSD; plasmid pAUR123-BSD, transformation of industrial yeast Saccharomyces cerevisiae X-2180AB; selectable marker for mammalian cells
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bsd overexpressed in Escherichia coli BL 21; pTPBS1, Arabidopsis thaliana and Nicotiana tabacum transformed to blasicidin S resistance
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bsd overexpressed in Escherichia coli BL 21; recombinant enzyme overexpressed in Escherichia coli
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bsr gene employed as marker gene for Escherichia coli, Bacillus subtilis, Nicotiana tabacum, Rhizopus niveus and cultured mammalian cells
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expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3)/pLysE cells
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expression of deletion mutants as soluble enzymes in Escherichia coli
generating of bicistronic vectors express both blastidicin S deaminase gene and a fusion gene consisting of a sarcomeric protein, selection with blasticidin S results in a relatively pure population of ES cell-derived cardiomycetes
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pBSR8 with structural gene bsr cloned and expressed in both Escherichia coli JM109 and Bacillus subtilis 168
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selectable marker for mammalian cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C91A
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site-directed mutagenesis, inactive mutant, gross perturbation of the enzyme structure
C91H
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site-directed mutagenesis, inactive mutant, gross perturbation of the enzyme structure
C91S
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site-directed mutagenesis, inactive mutant, gross perturbation of the enzyme structure
E56D
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site-directed mutagenesis, inactive mutant
H48Q
no catalytic activity
R90K
has a similar structure to the native enzyme except for the tetramer interface region
additional information
construction of 5 C-terminal deletion mutants, i.e. DELTA130, DELTA126-130, DELTA127-130, DELTA128-130, and DELTA129-130
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
reconstitution of the enzyme after denaturation with guanidine-HCl, acid, or p-hydroxymercuriphenylsulfonic acid, using different divalent metal ions, renaturation of the enzyme is completely blocked in presence of 1 mM EDTA, refolding of recombinant Cys91 mutant enzymes from inclusion bodies
-
wild-type and deletion mutant apoenzymes, prepared by zinc removal via EDTA, exhibit similar physical properties in thermodynamic refolding into the stable tetramer conformation, reconstitution with zinc
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology