Information on EC 3.5.2.16 - maleimide hydrolase

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

EC NUMBER
COMMENTARY hide
3.5.2.16
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RECOMMENDED NAME
GeneOntology No.
maleimide hydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
maleimide + H2O = maleamic acid
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of imide bond
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SYSTEMATIC NAME
IUBMB Comments
cyclic-imide amidohydrolase (decyclizing)
Succinimide and glutarimide, and sulfur-containing cyclic imides, such as rhodanine, can also act as substrates for the enzyme from Blastobacter sp. A17p-4. The reverse, cyclization, reaction is also catalysed, but much more slowly. It has lower activity towards cyclic ureides, which are the substrates of EC 3.5.2.2, dihydropyrimidinase.
CAS REGISTRY NUMBER
COMMENTARY hide
9030-74-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
NRRL B1, recombinant enzyme expressed in Escherichia coli
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Manually annotated by BRENDA team
thermophilic enzyme
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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
2,3-pyridinedicarboximide + H2O
3-carbamoyl-alpha-picolinic acid
show the reaction diagram
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94.5% regioisomeric purity
?
2,4-thiazolidinedione + H2O
?
show the reaction diagram
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-
-
-
?
3,4-pyridinedicarboximide + H2O
4-carbamoyl-alpha-picolinic acid
show the reaction diagram
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-
-
?
6-methyldihydrouracil + H2O
3-ureidobutanoic acid
show the reaction diagram
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-
-
?
dihydrothymine + H2O
?
show the reaction diagram
dihydrouracil + H2O
N-carbamoyl-beta-alanine
show the reaction diagram
DL-5-hydantoin + H2O
hydantoic acid monoamide
show the reaction diagram
glutarimide + H2O
glutaric acid monoamide
show the reaction diagram
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-
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?
hydantoin + H2O
hydantoic acid monoamide
show the reaction diagram
maleimide + H2O
maleic acid
show the reaction diagram
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?
maleimide + H2O
maleic acid monoamide
show the reaction diagram
parabanic acid + H2O
?
show the reaction diagram
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-
-
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?
phtalimide + H2O
2-(aminocarbonyl)benzoic acid
show the reaction diagram
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?
phthalimide + H2O
2-(aminocarbonyl)benzoic acid
show the reaction diagram
rhodanine + H2O
?
show the reaction diagram
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?
succinimide + H2O
succinamic acid monoamide
show the reaction diagram
thiohydantoin + H2O
?
show the reaction diagram
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?
additional information
?
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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
6-methyldihydrouracil + H2O
3-ureidobutanoic acid
show the reaction diagram
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-
?
dihydrothymine + H2O
?
show the reaction diagram
dihydrouracil + H2O
N-carbamoyl-beta-alanine
show the reaction diagram
additional information
?
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the enzyme plays a role in pyrimidine catabolism
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cd2+
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apoenzyme reconstituted with Co2+ has 15.8fold lower activity than the native Zn-containing enzyme
Co2+
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apoenzyme reconstituted with Co2+ has about 2fold higher activity than the native Zn-containing enzyme
Cobalt
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purified imidase, less the addition of any metals, reveals 0.514 mol zinc, 0.102 mol cobalt, and 0.179 mol nickel per subunit
Mg2+
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only the two physiological substrates dihydrouracil and dihydrothymine are hydrolyzed at a greater rate in the presence of 5 mM MgSO4
Mn2+
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apoenzyme reconstituted with Co2+ has 4.4fold lower activity than the native Zn-containing enzyme
Nickel
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purified imidase, less the addition of any metals, reveals 0.514 mol zinc, 0.102 mol cobalt, and 0.179 mol nickel per subunit
Zn2+
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residue C7 is required or binding of Zn2+ and maintaining the stability of the enzyme
additional information
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enzyme reconstituted with Cu2+ and Ni2+ has no enzymatic activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8-hydroxyquinoline
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5 mM, 9.4% residual activity
EDTA
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5 mM, 43.6% residual activity
Heavy metal ions
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N-Carbamoyl-beta-alanine
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Pyridine-2,4-dicarboxylate
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5 mM, 20.8% residual activity
sulfhydryl reagents
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inhibition may be caused by modification of sulfhydryl residues involved in activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
33
2,4-Thiazolidinedione
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0.5
6-methyldihydrouracil
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pH 9.5, 30°C
9.4
beta-ureidopropionate
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0.2 - 0.35
Dihydrothymine
2.2 - 74.3
dihydrouracil
4.5
glutarimide
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5.7 - 53.5
hydantoin
0.34 - 200
Maleimide
2
N-Carbamoyl-beta-alanine
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pH 5.4, 30°C
62
parabanic acid
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1 - 7.7
Phthalimide
31
rhodanine
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0.94 - 29.2
succinimide
23
thiohydantoin
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.14
dihydrouracil
Pseudomonas putida
Q4JG22
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2.107 - 2.94
hydantoin
106.7 - 3950
Maleimide
7.67 - 428.3
Phthalimide
1494
succinimide
Pseudomonas putida
Q4JG22
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
13.3
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measured at 50°C, increasing the reaction temperature from 25 to 60°C leads to an 50 and 60fold increase in specific activity for dihydrouracil and dihydrothymine, respectively, similar activities are found in other mammals
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 8.5
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different pH profile for cyclic ureide and cyclic imide hydrolysis
7.5 - 9.5
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optimal pH correlates with pKa of the substrates amide proton
7.5 - 8.5
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shifts to more acidic pH at higher temperatures
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
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activity increases when temperature increases from 5 to 70°C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 55
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15°C: about 40% of maximal activity, 55°C: maximal activity, 65°C: no activity
25 - 60
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imidase acitivity increases with the elevation of temperature except for a reaction at pH 6
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36000
x * 36000, (His)6-tagged enzyme, SDS-PAGE
51000
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6 * 51000, SDS-PAGE
53000
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4 * 53000, SDS-PAGE
105000
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gel filtration
140000
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gel filtration, recombinant protein with His-tag and a thrombin site
141000
gel filtration
230000
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gel filtration
300000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 33712.6, calculated from sequence; x * 36000, (His)6-tagged enzyme, SDS-PAGE
hexamer
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6 * 51000, SDS-PAGE
tetramer
trimer
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3 * 35000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
to 1.9 A resolution, space group C2221
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
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more than 80% initial activity after incubation at 30°C for 30 min
209267
6 - 9
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below 40°C, less stable at 50 and 60°C, stability decreases in acidic or alkaline pH at 60°C
209266
6
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the stability decreases in the order pH 8, pH 7, pH 6, pH 9
654446
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 60
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more than 90% initial activity after incubation at 20-60°C for 30 min at pH 7.5, activity completely lost at 70°C
25 - 60
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at a pH around 7.5 and 8.5, imidase is stable at 50°C or at lower temperature for over 2 h, no activity above 70°C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-cellulose, polyethylene glycol precipitation, DEAE-Sephacel, isoelectric focusing, hydroxyapatite, DEAE-Sephacel, gel filtration
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recombinant enzyme
salt precipitation, octyl Sepharose, DEAE-Sephacel, chelating Sephacel, gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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overexpressed in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C108G
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72% of wild-type activity. Like wild-type, mutant forms tetramers. It has a high binding ability for Zn2+
C7G
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complete loss of activity. Mutant is a mixture of monomers and oligomers and displays decreased binding of Zn2+
C7G/C108G
H151G
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120% of wild-type activity
H247A
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decrease in absorbance at 500 nm by 32.4%. Less than 10% of wild-type activity
H86A
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decrease in absorbance at 500 nm by 14.7%. Less than 10% of wild-type activity
C108G
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72% of wild-type activity. Like wild-type, mutant forms tetramers. It has a high binding ability for Zn2+
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C7G
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complete loss of activity. Mutant is a mixture of monomers and oligomers and displays decreased binding of Zn2+
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C7G/C108G
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complete loss of activity. Mutants is a multimer, with decreased binding ability for Zn2+; decrease in absorbance at 500 nm by 12.8%. Less than 10% of wild-type activity
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H151G
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120% of wild-type activity
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H247A
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decrease in absorbance at 500 nm by 32.4%. Less than 10% of wild-type activity
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H86A
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decrease in absorbance at 500 nm by 14.7%. Less than 10% of wild-type activity
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the activity of the apo-imidase depleted of zinc is recovered with the addition of zinc in the lower concentration of 0-20 micorM, whereas the enzymatic activity is decreased in the presence of high concentration of zinc above 100 microM
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