Information on EC 5.1.1.8 - 4-hydroxyproline epimerase

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

EC NUMBER
COMMENTARY hide
5.1.1.8
-
RECOMMENDED NAME
GeneOntology No.
4-hydroxyproline epimerase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
trans-4-Hydroxy-L-proline = cis-4-hydroxy-D-proline
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
epimerization
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Arginine and proline metabolism
-
-
trans-4-hydroxy-L-proline degradation II
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-
SYSTEMATIC NAME
IUBMB Comments
4-hydroxyproline 2-epimerase
Also interconverts trans-4-hydroxy-D-proline and cis-4-hydroxy-L-proline.
CAS REGISTRY NUMBER
COMMENTARY hide
9024-23-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
strain 16M
SwissProt
Manually annotated by BRENDA team
strain 16M
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
gene hypRE or smb20268
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
a hypRE mutant grows with cis-4-hydroxy-D-proline but not with trans-4-hydroxy-L-proline, and it exhibits wild-type growth on succinate and proline, mutant complementation with the native smb20268 gene
metabolism
-
the enzyme performs the first step of hydroxyproline catabolism, regulation, hyp cluster promoters are regulated by HypR, overview. cis-4-Hydroxy-D-proline is the effector that interacts with HypR and the results suggest a role of positive feedback in the regulation of hyp catabolism, as cis-4-hydroxy-D-proline (or a downstream catabolite) is a stronger inducer than the initial substrate trans-4-hydroxy-L-proline
physiological function
-
the enzyme is absolutely required for growth on trans-4-hydroxy-L-proline
additional information
-
the reaction mechanism requires two active site cysteines
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-hydroxy-L-Pro
3-hydroxy-D-Pro
show the reaction diagram
-
-
-
-
Allohydroxy-D-Pro
Allohydroxy-L-Pro
show the reaction diagram
cis-3-hydroxy-L-proline
trans-3-hydroxy-D-proline
show the reaction diagram
-
-
-
?
cis-4-hydroxy-D-proline
trans-4-hydroxy-L-proline
show the reaction diagram
the reaction is completely bi-directional. The specific activity with cis-4-hydroxy-D-proline is 160% compared to the L-enantiomer. The bifunctional enzyme reversibly catalyzes the racemization of proline and the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. The catalytic efficiency (kcat/Km) values for L-proline and trans-4-hydroxy-D-proline are similar, whereas a preference for D-proline over cis-4-hydroxy-D-proline (45fold) is identified and is caused by a 75fold lower Km for D-proline. Catalysis is based on the same 1,1-proton transfer mechanism using two general acidic/basic cysteine residues located on opposite faces of the active site
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-
r
cis-4-hydroxy-L-proline
trans-4-hydroxy-D-proline
show the reaction diagram
the reaction is completely bi-directional. The specific activity with trans-4-hydroxy-D-proline is 97% compared to the L-enantiomer. The bifunctional enzyme reversibly catalyzes the racemization of proline and the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Catalysis is based on the same 1,1-proton transfer mechanism using two general acidic/basic cysteine residues located on opposite faces of the active site
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-
r
D-Ala
L-Ala
show the reaction diagram
-
at 1-2% of the activity relative to allo-D-hydroxy-proline epimerization
-
-
Hydroxy-L-Pro
?
show the reaction diagram
L-azetidine-2-carboxylate
D-azetidine-2-carboxylate
show the reaction diagram
low activity
-
-
?
L-pipecolate
D-pipecolate
show the reaction diagram
low activity
-
-
?
trans-3-hydroxy-L-proline
trans-3-hydroxy-D-proline
show the reaction diagram
-
-
-
?
trans-4-hydroxy-D-Pro
cis-4-hydroxy-L-Pro
show the reaction diagram
-
-
-
?
trans-4-hydroxy-D-proline
cis-4-hydroxy-L-proline
show the reaction diagram
trans-4-hydroxy-L-Pro
cis-4-hydroxy-D-Pro
show the reaction diagram
trans-4-hydroxy-L-proline
cis-4-hydroxy-D-proline
show the reaction diagram
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
Hydroxy-L-Pro
?
show the reaction diagram
trans-4-hydroxy-L-proline
cis-4-hydroxy-D-proline
show the reaction diagram
-
-
-
-
r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
the enzyme is pyridoxal 5'-phosphate-independent
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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-
2,2'-dipyridyl
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-
3-Pyrrolidinol
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5,5'-dithiobis(2-nitrobenzoate)
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Acriflavin
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Atebrin
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hydrazine
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hydroxylamine
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iodoacetate
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p-chloromercuribenzoate
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pyrrole 2-carboxylic acid
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selective inhibitor, competitive inhibition
pyrrole-2-carboxylate
sarcosine
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Semicarbazide
-
-
additional information
-
not inhibited by 5 mM trans-3-hydroxy-L-proline, P5C, and L-proline
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
pyridoxal 5'-phosphate is not involved in the reaction
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
24 - 42
2-2H-4-allohydroxy-D-Pro
11
2-2H-4-hydroxy-L-Pro
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solvent H2O
19
3-allohydroxy-D-Pro
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solvent 2H2O
29
3-allohydroxy-L-Pro
-
solvent H2O
37
3-Hydroxy-L-Pro
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solvent H2O
49
4-allohydroxy-D-Pro
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solvent H2O
32
4-allohydroxy-L-Pro
-
solvent H2O
13 - 14
4-hydroxy-L-Pro
53
allohydroxy-L-Pro
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-
3.14 - 8
cis-4-hydroxy-D-proline
22
hydroxy-L-Pro
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-
0.502 - 16.4
trans-3-hydroxy-L-proline
0.0016
trans-4-hydroxy-D-proline
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in 50 mM HEPES, pH 8.0, with 2 mM beta-mercaptoethanol,1 mM EDTA, at 37°C
7.6 - 14.4
trans-4-hydroxy-L-Pro
0.0024 - 10
trans-4-hydroxy-L-proline
additional information
additional information
-
Michaelis-Menten kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.014 - 3.83
cis-4-hydroxy-D-proline
0.29 - 3.6
trans-3-hydroxy-L-proline
0.012 - 12.13
trans-4-hydroxy-L-proline
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0046 - 0.58
cis-4-hydroxy-D-proline
45405
0.18 - 7.2
trans-3-hydroxy-L-proline
97611
0.0055 - 2.03
trans-4-hydroxy-L-proline
6646
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.3
pyrrole 2-carboxylic acid
Pseudomonas aeruginosa
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in 50 mM HEPES, pH 8.0, with 2 mM beta-mercaptoethanol,1 mM EDTA, at 37°C
0.217 - 0.58
pyrrole-2-carboxylate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
154
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hyperinducible strain M-4
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4 - 8
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allohydroxy-L-proline
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 30
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assay at
90 - 100
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Burkholderia multivorans (strain ATCC 17616 / 249)
Burkholderia multivorans (strain ATCC 17616 / 249)
Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5)
Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5)
Pseudomonas putida (strain ATCC 700007 / DSM 6899 / BCRC 17059 / F1)
Pseudomonas putida (strain ATCC 700007 / DSM 6899 / BCRC 17059 / F1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
17930
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low speed sedimentation without reaching equilibrium
18000
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approach to-equilibrium method
32000
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2 * 32000, SDS-PAGE
44000
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gel filtration
60000
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analytical ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 32000, SDS-PAGE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15°C, concentrated purified enzyme is stable for at least several weeks
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cobalt resin column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
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expression in Escherichia coli
gene hypRE, encoded in the Hyp catabolism locus, DNA and amino acid sequence determination and analysis, expression of the C-terminally His-tagged enzyme in Escherichia coli strain BL21/DE3/pET28 from the T7 promoter
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the enzyme is strongly induced by cis-4-hydroxy-D-proline, moderately by trans-4-hydroxy-L-proline
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F240W
mutant enzyme shows similar kinetic constants for proline and hydroxyproline as compared to the wild-type enzyme
C236S
radical loss of OH-L/D-Pro epimerization
C88S
radical loss of OH-L/D-Pro epimerization
V60F
radical loss of OH-L/D-Pro epimerization. V60 residue accounts for OH-Pro ligand specificity
V60G
almost complete loss of epimerization of trans-4-hydroxy-L-Pro, some residual activity on trans-4-hydroxy-D-Pro. V60 residue accounts for OH-Pro ligand specificity
W241F the mutant enzyme
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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spectrophotometric assay for hydroxyproline in collagenous tissue hydrolysates, with an enzymatic method using 4-hydroxyproline 2-epimerase,EC 5.1.1.8, D-amino acid oxidase, EC 1.4.3.3, and a colorimetric reagent of the mixture of Ti(IV) and 4-(2-pyridylazo)-resorcinol
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