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EC Tree
The taxonomic range for the selected organisms is: Pseudomonas putida The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
creatininase, creatinine amidohydrolase, creatinine hydrolase,
more
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creatinine amidohydrolase
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creatinine hydrolase
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creatinine + H2O = creatine
creatinine + H2O = creatine
mechanism
creatinine + H2O = creatine
mechanism
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creatinine + H2O = creatine
catalytic mechanism: a first water molecule is a hydroxide ion that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. A second water molecule is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis
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hydrolysis of peptide bond
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creatinine amidohydrolase
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creatinine + H2O
creatine
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?
creatinine + H2O
?
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-
-
-
?
creatinine + H2O
creatine
creatinine + H2O
creatine
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?
creatinine + H2O
creatine
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-
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?
creatinine + H2O
creatine
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r
creatinine + H2O
creatine
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r
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creatinine + H2O
?
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?
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Mn2+
may substitute for Zn2+
Zinc
removal of the zinc ions by dialyzing against buffers containing o-phenanthroline results in a partial decrease of 10-30% in activity and zinc content. Wild-type enzyme, purified in the presence of EDTA, shows only half the activity of the wild-type enzyme, the activity is recovered by the addition of ZnCl2 or MnCl2
Zn2+
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Zn2+
one zinc centre per monomer
Zn2+
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one gatom Zn2+ per mol of subunit monomer
Zn2+
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contains one zinc ion per subunit
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EDTA
wild-type enzyme, purified in the presence of EDTA, shows only half the activity of the wild-type enzyme, the activity is recovered by the addition of ZnCl2 or MnCl2
ethoxyformic anhydride
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additional information
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44
creatinine
wild-type, presence of Zn2+, pH not specified in the publication, temperature not specified in the publication
56
creatinine
mutant E122Q, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
77
creatinine
wild-type, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
90
creatinine
mutant W174F, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
150
creatinine
wild-type, presence of EDTA, semi-apo-enzyme, pH not specified in the publication, temperature not specified in the publication
160
creatinine
mutant Y121A, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
220
creatinine
mutant E122Q, presence of Zn2+, pH not specified in the publication, temperature not specified in the publication
350
creatinine
mutant W154F, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
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1.5
creatinine
mutant E122Q, presence of Zn2+, pH not specified in the publication, temperature not specified in the publication
11
creatinine
mutant E122Q, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
18
creatinine
mutant W154F, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
86
creatinine
mutant Y121A, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
222
creatinine
wild-type, presence of EDTA, semi-apo-enzyme, pH not specified in the publication, temperature not specified in the publication
252
creatinine
wild-type, presence of Zn2+, pH not specified in the publication, temperature not specified in the publication
688
creatinine
mutant W174F, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
1340
creatinine
wild-type, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
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0.008
creatinine
mutant E122Q, presence of Zn2+, pH not specified in the publication, temperature not specified in the publication
0.051
creatinine
mutant W154F, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
0.2
creatinine
mutant E122Q, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
0.54
creatinine
mutant Y121A, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
1.48
creatinine
wild-type, presence of EDTA, semi-apo-enzyme, pH not specified in the publication, temperature not specified in the publication
5.73
creatinine
wild-type, presence of Zn2+, pH not specified in the publication, temperature not specified in the publication
7.64
creatinine
mutant W174F, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
17.4
creatinine
wild-type, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
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0.13
mutant E122Q, presence of Zn2+, pH not specified in the publication, temperature not specified in the publication
13.9
mutant E122Q, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
140
mutant Y121A, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
1560
wild-type, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
178
wild-type, presence of EDTA, semi-apo-enzyme, pH not specified in the publication, temperature not specified in the publication
370
wild-type, presence of Zn2+, pH not specified in the publication, temperature not specified in the publication
4.9
mutant W154F, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
740
mutant W174F, presence of Mn2+, pH not specified in the publication, temperature not specified in the publication
additional information
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by Escherichia coli transformants pCRN741 without and with IPTG 0.678 U per ml
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SwissProt
brenda
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CRNA_PSEPU
260
0
28569
Swiss-Prot
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173000
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dynamic light scattering
175000
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ultracentrifugal analysis
23000
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8 * 23000, SDS-PAGE
28399
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6 * 28399, calculated from amino acid sequence
28400
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6 * 28400, MALDI-TOF mass spectrometry
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hexamer
dimer of trimers, crystallization data
hexamer
trimer of dimers, crystallization data
hexamer
trimer of dimers, Mn2+-activated creatininase-creatine complex, disc-shaped
homohexamer
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6 * 28399, calculated from amino acid sequence
homohexamer
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6 * 28400, MALDI-TOF mass spectrometry
octamer
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8 * 23000, SDS-PAGE
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glycoprotein
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3.5% glucose
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crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex exist as the closed form. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex reveal that the drastic decrease of the activity of the E122Q is caused by the loss of one Zn ion at the Metal1 site and a critical function of Glu122
crystal structures of the native and the Mn2+-activated enzyme determined by a difference Fourier method, crystal structure of Mn2+-activated enzyme determined by the single isomorphous replacement method
hanging drop vapour diffusion method with 1.5 M lithium sulfate in 0.1 M Na-HEPES pH 7.5
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sitting drop vapour diffusion method with 22% ethanol, 20% PEG 8000, and 0.1 M MOPS pH 7.5
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E122Q
almost complete loss of activity
E183Q
complete loss of activity
H178A
complete loss of activity
W154A
complete loss of activity
W154F
almost complete loss of activity
W174A
almost complete loss of activity
W174F
about 50% of wild-type activity
Y121A
about 10% of wild-type catalytic efficiency
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6 - 12
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for 3 h at 30°C
172079
6 - 9
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the enzyme tends to precipitate at pH values of under 6.0 and over 9.0 corresponding to the stability range of the enzyme
677306
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55
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30 min at pH 7.1, apoenzyme
70
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30 min at pH 7.1, native enzyme
75
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30 min, activity still 40%
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unstable to photooxidation
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172079
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native and Mn2+-activated enzymes purified, Toyopearl HW65C column and DEAE-Toyopearl column
ammonium sulfate fractionation, DEAE Sepharose CL-6B and Sepharose CL-6B column chromatographies, 50% total activity
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purified by column chromarographies on sarcosine-HM-Sepharose and DEAE-cellulose, and gel filtration on Sephadex G-200
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expression in Escherichia coli
cloned, sequenced and expressed in Escherichia coli DH5?, vector pCRN741, creatininase gene consists of 771 bp, encodes 257 amino acids, similarity of RS65 to PS-7 72.8% and Arthrobacter sp. TE1826 43.2%
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expressed in Escherichia coli
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wild-type enzyme, purified in the presence of EDTA, shows only half the activity of the wild-type enzyme, the activity is recovered by the addition of ZnCl2 or MnCl2
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analysis
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analysis of serum creatinine concentration
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Tang, T.Y.; Wen, C.J.
Expression of the creatininase gene from Pseudomonas putida RS65 in Escherichia coli
J. Ind. Microbiol.
24
2-6
2000
Alcaligenes sp., Pseudomonas putida
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brenda
Rikitake, K.; Oka, I.; Ando, M.; Yoshimoto, T.; Tsuru, D.
Creatinine amidohydrolase (creatininase) from Pseudomonas putida. Purification and some properties
J. Biochem.
86
1109-1117
1979
Pseudomonas putida
brenda
Tsuru, D.; Oka, I.; Yoshimoto, T.
Creatinine decomposing enzymes in Pseudomonas putida
Agric. Biol. Chem.
40
1011-1018
1976
Paenarthrobacter ureafaciens, Pseudomonas aeruginosa, Pseudomonas putida
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brenda
Beuth, B.; Niefind, K.; Schomburg, D.
Crystal structure of creatininase from Pseudomonas putida: a novel fold and a case of convergent evolution
J. Mol. Biol.
332
287-301
2003
Pseudomonas putida (P83772), Pseudomonas putida
brenda
Yoshimoto, T.; Tanaka, N.; Kanada, N.; Inoue, T.; Nakajima, Y.; Haratake, M.; Nakamura, K.T.; Xu, Y.; Ito, K.
Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism
J. Mol. Biol.
337
399-416
2004
Pseudomonas putida (P83772), Pseudomonas putida
brenda
Beuth, B.; Niefind, K.; Schomburg, D.
Crystallization and preliminary crystallographic analysis of creatininase from Pseudomonas putida
Acta Crystallogr. Sect. D
58
1356-1358
2002
Pseudomonas putida
brenda
Ito, K.; Kanada, N.; Inoue, T.; Furukawa, K.; Yamashita, K.; Tanaka, N.; Nakamura, K.T.; Nishiya, Y.; Sogabe, A.; Yoshimoto, T.
Preliminary crystallographic studies of the creatinine amidohydrolase from Pseudomonas putida
Acta Crystallogr. Sect. D
58
2180-2181
2002
Pseudomonas putida
brenda
Yamashita, K.; Nakajima, Y.; Matsushita, H.; Nishiya, Y.; Yamazawa, R.; Wu, Y.F.; Matsubara, F.; Oyama, H.; Ito, K.; Yoshimoto, T.
Substitution of Glu122 by glutamine revealed the function of the second water molecule as a proton donor in the binuclear metal enzyme creatininase
J. Mol. Biol.
396
1081-1096
2010
Pseudomonas putida (P83772)
brenda