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Information on EC 3.5.4.23 - blasticidin-S deaminase and Organism(s) Aspergillus terreus and UniProt Accession P0C2P0
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3.5.4.23 blasticidin-S deaminaseIUBMB Comments
Catalyses the deamination of the cytosine moiety of the antibiotics blasticidin S, cytomycin and acetylblasticidin S.
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The taxonomic range for the selected organisms is: Aspergillus terreus
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
bsd, blasticidin s deaminase, bs deaminase, blasticidin-s deaminase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
blasticidin S + H2O = deaminohydroxyblasticidin S + NH3
the C-terminal flexible region appears to be directly involved in enzyme-substrate interaction facilitating the access of substrate to the active site, Tyr126, Trp128, and Gly130 are key residues
blasticidin S + H2O = deaminohydroxyblasticidin S + NH3
catalyses the deamination of the cytosine moiety of the antibiotics blasticidin S, cytomycin and acetylblasticidin S, Glu56 is required for catalysis, and Cys91 is required for structural maintenance
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
amidine hydrolysis
amidine hydrolysis
-
-
-
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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
54576-55-5
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
blasticidin S + H2O
deaminohydroxyblasticidin S + NH3
-
-
-
?
acetylblasticidin S + H2O
acetyldeaminohydroxyblasticidin S + NH3
-
-
-
-
?
blasticidin S + H2O
deaminohydroxyblasticidin S + NH3
-
-
-
-
?
blasticidin S methylester + H2O
deaminohydroxyblasticidin S methylester + NH3
-
only slightly deaminated
-
-
?
pyrimidinoblasticidin S + H2O
pyrimidinodeaminohydroxyblasticidin S + NH3
-
-
-
-
?
additional information
?
-
-
cytosine, cytidine, deoxycytidine, CMP, d-CMP or purine nucleosides are no substrates
-
-
?
additional information
?
-
-
cytosine, cytosinine, CMP, d-CMP, adenine, adenosine, AMP, guanine, guanosine, GMP or other purine bases or their nucleosides are no substrates
-
-
?
additional information
?
-
-
cytosine, CMP, d-CMP, adenine, guanine and their nucleosides and nucleotides are no substrates
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Zn2+
Zn2+
-
tightly bound catalytic zinc, 1 zinc ion per subunit, enzyme contains the catalytic zinc-coordinating sequence motif, role in enzyme tetrameric structure stabilization and protein folding, overview
Zn2+
tightly bound catalytic zinc, 1 zinc ion per subunit, coordinative role in enzyme tetrameric structure stabilization and protein folding, overview
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
p-Hydroxymercuriphenylsulfonic acid
-
leads to destabilization of the enzyme structure due to removal of required zinc
Rose bengal
-
0.01%, enzyme loses more than 80% of its activity, photooxidation
additional information
the wild-type and deletion mutant enzymes are highly resistant to protease digestions
-
additional information
-
the wild-type and deletion mutant enzymes are highly resistant to protease digestions
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
20
blasticidin S
deletion mutants DELTA128-130, DELTA127-130, DELTA126-130, 30°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). BSD_ASPTE
130
0
13468
Swiss-Prot
other Location (Reliability: 1)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
13340
-
recombinant enzyme, calculated by amino acid residues per subunit
49000
54000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
tetramer
the 4 polypeptides are tightly coordinated to the structurally and catalytically important zinc atom of each subunit
tetramer
additional information
additional information
-
zinc has a role in enzyme tetrameric structure stabilization and protein folding
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
using 20% (w/v) PEG8000, 50 mM magnesium chloride, and 0.1 M sodium cacodylate at pH 7.0 as precipitant
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
R90K
has a similar structure to the native enzyme except for the tetramer interface region
C91A
-
site-directed mutagenesis, inactive mutant, gross perturbation of the enzyme structure
C91H
-
site-directed mutagenesis, inactive mutant, gross perturbation of the enzyme structure
C91S
-
site-directed mutagenesis, inactive mutant, gross perturbation of the enzyme structure
additional information
additional information
construction of 5 C-terminal deletion mutants, i.e. DELTA130, DELTA126-130, DELTA127-130, DELTA128-130, and DELTA129-130
additional information
-
construction of 5 C-terminal deletion mutants, i.e. DELTA130, DELTA126-130, DELTA127-130, DELTA128-130, and DELTA129-130
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
60 - 80
-
reaction proceeds most rapidly at 60-70°C, occurs only during a short initial period at 80°C
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
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 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
-
-20°C, 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
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of deletion mutants as soluble enzymes in Escherichia coli
bsd overexpressed in Escherichia coli BL 21
-
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
-
overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3), Cys91 mutants are expressed in inclusion bodies
-
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
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pTPBS1, Arabidopsis thaliana and Nicotiana tabacum transformed to blasicidin S resistance
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
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
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
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
molecular biology
molecular biology
-
new selection marker for transformation of Arabidopsis thaliana and Nicotiana tabacum
molecular biology
-
BSD gene will be useful as a new dominant selectable marker for eukaryotes, first sucessful transformation with a drug resistance gene originating from a eukaryote by selecting for detoxification of the drug
molecular biology
-
generation of bicistronic expression vector mediating BSD resistance, blastidicin S selection of cells
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Yamaguchi, I.; Shibata, H.; Seto, H.; Misato, T.
Isolation and purification of blasticidin S deaminase from Aspergillus terreus
J. Antibiot.
28
7-14
1975
Aspergillus flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus ochraceus, Aspergillus terreus, Aspergillus ustus, Aspergillus flavipes NHL 5013, Aspergillus ustus 1004, Aspergillus fumigatus NHL 5009, Aspergillus ochraceus 67-MI-87, Aspergillus terreus NHL 5037
Misato, T.; Yamaguchi, I.
Blasticidin S deaminase production by Aspergillus
Jpn. Kokai JP48099383 JP72-33359
19 pp
1973
Aspergillus terreus
-
Yamaguchi, I.; Misato, T.
Active center and mode of reaction of blasticidin S deaminase
Agric. Biol. Chem.
49
3355-3361
1985
Aspergillus terreus
-
Yamaguchi, I.; Sheto, H.; Misato, T.
Substrate binding by blasticidin S deaminase, an aminohydrolase for novel 4-aminopyrimidine nucleosides
Pestic. Biochem. Physiol.
25
54-62
1986
Aspergillus fumigatus, Aspergillus terreus, Bacillus sp. (in: Bacteria), Streptomyces sp.
-
Kimura, M.; Kamakura, T.; Tao, Q.Z.; Kaneko, I.; Yamaguchi, I.
Cloning of the blasticidin S deaminase from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae
Mol. Gen. Genet.
242
121-129
1994
Aspergillus terreus, Bacillus sp. (in: Bacteria)
Tamura, K.; Kimura, M.; Yamaguchi, I.
Blasticidin S deaminase gene(BSD): a new selection marker gene for tranformation of arabidopsis thaliana and Nicotiana tabacum
Biosci. Biotechnol. Biochem.
59
2336-2338
1995
Aspergillus terreus
Karremann, C.
New positive/negative selectable markers for mammalian cells on the basis of blasticidin deaminase-thymidine kinase fusions
Nucleic Acids Res.
26
2508-2510
1998
Aspergillus terreus
Nakasako, M.; Kimura, M.; Yamaguchi, I.
Crystallization and preliminary X-ray diffraction studies of blasticidin S deaminase from Aspergillus terreus
Acta Crystallogr. Sect. D
55
547-548
1999
Aspergillus terreus
Fukuda, H.; Kizaki, Y.
A new transformation system of Saccharomyces cerevisiae with blasticidin S deaminase gene
Biotechnol. Lett.
21
969-971
1999
Aspergillus terreus
-
Kimura, M.; Sekido, S.; Isogai, Y.; Yamaguchi, I.
Expression, purification, and characterization of blasticidin S deaminase (BSD) from Aspergillus terreus: The role of catalytic zinc in enzyme structure
J. Biochem.
127
955-963
2000
Aspergillus terreus, Bacillus cereus
Kimura, M.; Furuichi, M.; Yamamoto, M.; Kumasaka, T.; Mizuno, H.; Miyano, M.; Yamaguchi, I.
The flexible C-terminal region of Aspergillus terreus blasticidin S deaminase: identification of its functional roles with deletion enzymes
Biochem. Biophys. Res. Commun.
290
421-426
2002
Aspergillus terreus (P0C2P0), Aspergillus terreus
Kumasaka, T.; Yamamoto, M.; Furuichi, M.; Nakasako, M.; Teh, A.H.; Kimura, M.; Yamaguchi, I.; Ueki, T.
Crystal structures of blasticidin S deaminase (BSD): implications for dynamic properties of catalytic zinc
J. Biol. Chem.
282
37103-37111
2007
Aspergillus terreus (P0C2P0), Aspergillus terreus S-712 / ATCC 28865 (P0C2P0)
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