Information on EC 5.1.99.5 - hydantoin racemase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
5.1.99.5
-
RECOMMENDED NAME
GeneOntology No.
hydantoin racemase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-5-monosubstituted hydantoin = L-5-monosubstituted hydantoin
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
D-5-monosubstituted-hydantoin racemase
This enzyme, along with N-carbamoylase (EC 3.5.1.77 and EC 3.5.1.87) and hydantoinase, forms part of the reaction cascade known as the "hydantoinase process", which allows the total conversion of D,L-5-monosubstituted hydantoins into optically pure D- or L-amino acids [7]. The enzyme from Pseudomonas sp. (HyuE) has a preference for hydantoins with aliphatic substituents, e.g. D- and L-5-(2-methylthioethyl)hydantoin, whereas that from Arthrobacter aurescens shows highest activity with arylalkyl substituents, especially 5-benzylhydantoin, at the 5-position [2]. In the enzyme from Sinorhizobium meliloti, Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization [6].
CAS REGISTRY NUMBER
COMMENTARY hide
111310-51-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain DSM 3747
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Manually annotated by BRENDA team
strain AJ 3912
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Manually annotated by BRENDA team
strain AJ3912
-
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Manually annotated by BRENDA team
strain CECT 4114
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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
(5S)-5-(1H-indol-2-ylmethyl)-3-methylimidazolidine-2,4-dione
(5R)-5-(1H-indol-2-ylmethyl)-3-methylimidazolidine-2,4-dione
show the reaction diagram
-
20.2% of the activity with (5S)-5-(1H-indol-2-ylmethyl)imidazolidine-2,4-dione
-
-
r
(5S)-5-(1H-indol-2-ylmethyl)imidazolidine-2,4-dione
(5R)-5-(1H-indol-2-ylmethyl)imidazolidine-2,4-dione
show the reaction diagram
-
-
-
-
r
(5S)-5-(2-methylpropyl)imidazolidine-2,4-dione
(5S)-5-(2-methylpropyl)imidazolidine-2,4-dione
show the reaction diagram
-
9.8% of the activity with (5S)-5-(1H-indol-2-ylmethyl)imidazolidine-2,4-dione
-
-
r
(5S)-5-(4-hydroxybenzyl)imidazolidine-2,4-dione
(5R)-5-(4-hydroxybenzyl)imidazolidine-2,4-dione
show the reaction diagram
-
76.7% of the activity with (5S)-5-(1H-indol-2-ylmethyl)imidazolidine-2,4-dione
-
-
r
(5S)-5-benzylimidazolidine-2,4-dione
(5R)-5-benzylimidazolidine-2,4-dione
show the reaction diagram
-
62.7% of the activity with (5S)-5-(1H-indol-2-ylmethyl)imidazolidine-2,4-dione
-
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r
(5S)-5-[2-(methylsulfanyl)ethyl]imidazolidine-2,4-dione
(5R)-5-[2-(methylsulfanyl)ethyl]imidazolidine-2,4-dione
show the reaction diagram
-
20.4% of the activity with (5S)-5-(1H-indol-2-ylmethyl)imidazolidine-2,4-dione
-
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r
D-5-isobutylhydantoin
L-5-isobutylhydantoin
show the reaction diagram
D-5-methylhydantoin
L-5-methylhydantoin
show the reaction diagram
D-benzylhydantoin
L-benzylhydantoin
show the reaction diagram
D-benzylhydantoin
R-benzylhydantoin
show the reaction diagram
D-isobutylhydantoin
L-isobutylhydantoin
show the reaction diagram
L-5-(2-methylthioethyl)hydantoin
D-5-(2-methylthioethyl)hydantoin
show the reaction diagram
L-5-methylhydantoin
D-5-methylhydantoin
show the reaction diagram
L-5-methylthioethylhydantoin
D-5-methylthioethylhydantoin
show the reaction diagram
L-benzylhydantoin
D-benzylhydantoin
show the reaction diagram
L-ethylhydantoin
D-ethylhydantoin
show the reaction diagram
L-isobutylhydantoin
D-isobutylhydantoin
show the reaction diagram
L-isopropylhydantoin
D-isopropylhydantoin
show the reaction diagram
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-
-
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r
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
slight stimulation
Mn2+
slight stimulation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Co2+
-
slight inhibition
D-5-methylthioethyl-hydantoin
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determination of thermodynamic binding parameters. Binding of the inhibitor is entropically and thermodynamically favoured
DL-5-isopropylhydantoin
complete inhbition, pre-incubation with DL-5-(2-methylthioethyl)hydantoin protects
iodoacetamide
L-5-methylthioethyl-hydantoin
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determination of thermodynamic binding parameters. Binding of the inhibitor is entropically and thermodynamically favoured
N-ethylmaleimide
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Ni2+
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slight inhibition
p-mercuribenzoate
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complete loss of activity
Zn2+
strong inhibition
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cysteine
-
slight stimulation
dithiothreitol
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up to 50% stimulation
EDTA
-
slight stimulation
Sodium dodecyl sulfate
-
slight stimulation
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
29.8
D-5-(2-methylthioethyl)hydantoin
pH 7.5, 30°C
4.47 - 6.3
D-5-methylthioethylhydantoin
4.71 - 20.8
D-benzylhydantoin
4.26 - 12.54
D-ethylhydantoin
3.76 - 6.79
D-isobutylhydantoin
79.7
L-5-(2-methylthioethyl)hydantoin
pH 7.5, 30°C
5.41 - 10.9
L-5-methylthioethylhydantoin
5.56 - 18.4
L-benzylhydantoin
4.45 - 19.42
L-ethylhydantoin
1.23 - 6.41
L-isobutylhydantoin
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.5
D-5-methylthioethylhydantoin
Agrobacterium tumefaciens
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pH 7.5, 40°C
0.46 - 2.29
D-benzylhydantoin
1.8
D-ethylhydantoin
Agrobacterium tumefaciens
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pH 7.5, 40°C
0.83 - 3.24
D-isobutylhydantoin
0.78
L-5-methylthioethylhydantoin
Agrobacterium tumefaciens
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pH 7.5, 40°C
0.18 - 1.94
L-benzylhydantoin
1.81 - 6.42
L-ethylhydantoin
0.48 - 2.12
L-isobutylhydantoin
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20.8
pH 7.5, 30°C
79
-
pH 8.0, 37°C, substrate D-benzylhydantoin
100
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pH 8.0, 37°C, substrate L-benzylhydantoin
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
11
95% of maximum activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5
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isoelectric focusing
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21000
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4 * 21000, SDS-PAGE
25412
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x * 25412, calculated
31000
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4 * 31000, SDS-PAGE
84000
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gel filtration
100000
117000
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gel filtration
190000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 25412, calculated
hexamer
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
active-site mutant C76A, in presence and absence of substrate D,L-5-isopropyl hydantoin, diffraction to 2.17 and 1.85 A, respectively. both crystals belong to space group R3
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
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685646
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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stable at temperatures below
45
in presence of substrate, stable
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is active after 10 freeze-thawing cycles
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the enzyme remains active after 10 freeze-thawing cycles
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 0.1 M Tris, 20% glycerol, pH 8.5, stable for at least 3 months
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-20°C, pH 8.0, 20% glycerol, stable for at least 6 months
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4°C, 0.1 M Tris, pH 8.5, stable for 10 weeks
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4°C, ammonium sulfate at 90% saturation, stable for 6 months
4°C, pH 8.0, stable for 8 weeks
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
concomitant expression of D-hydantoinase, D-carbamoylase, and hydantoin recemase in Escherichia coli
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expression in Escherichia coli
expression in Escherichia coli, together with D-specific hydantoinase, and N-carbamoyl-D-amino acid amidohydrolase
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expression with His-tag
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C181S
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less than 1% of wild-type activity
C76S
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less than 0.5% of wild-type activity
C76A
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active site mutant, crystallization data
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
thermodynamic parameters of the guanidinium hydrochloride-induced denaturation of wild-type and mutants C76A, C181A
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis