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Literature summary for 5.1.1.8 extracted from

  • Watanabe, S.; Tanimoto, Y.; Nishiwaki, H.; Watanabe, Y.
    Identification and characterization of bifunctional proline racemase/hydroxyproline epimerase from archaea: discrimination of substrates and molecular evolution (2015), PLoS One, 10, e0120349.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 5.1.1.8

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Thermococcus litoralis
expression in Escherichia coli Ferroplasma acidarmanus

Protein Variants

Protein Variants Comment Organism
F240W mutant enzyme shows similar kinetic constants for proline and hydroxyproline as compared to the wild-type enzyme Ferroplasma acidarmanus
W241F the mutant enzyme shows 5.3fold, 430fold, and 5.8fold lower kcat/Km values for trans-4-hydroxy-L-proline, trans-3-hydroxy-L-proline, and cis-4-hydroxy-D-proline respectively, mainly due to a marked decrease in kcat values, whereas no significant effects were found in the kinetic constants of proline, suggesting that this (hydrophobic and bulky) tryptophan residue plays an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline) Thermococcus litoralis

Inhibitors

Inhibitors Comment Organism Structure
pyrrole-2-carboxylate
-
 Ferroplasma acidarmanus
pyrrole-2-carboxylate
-
 Thermococcus litoralis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.502
-
 trans-3-hydroxy-L-proline pH 8.0, 50°C, wild-type enzyme Thermococcus litoralis
0.905
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme F62V Thermococcus litoralis
2.17
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme F420W Ferroplasma acidarmanus
3.14
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, mutant enzyme F62S Thermococcus litoralis
4.18
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, mutant enzyme L221H Thermococcus litoralis
4.28
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme W241F Thermococcus litoralis
6.08
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, wild-type enzyme Thermococcus litoralis
6.12
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, wild-type enzyme Ferroplasma acidarmanus
6.22
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme F62S Thermococcus litoralis
6.5
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme L221H Thermococcus litoralis
6.65
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, wild-type enzyme Thermococcus litoralis
6.7
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, mutant enzyme W241F Thermococcus litoralis
16.4
-
 trans-3-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme W241F Thermococcus litoralis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
40000
-
 x * 40000, SDS-PAGE Thermococcus litoralis
40000
-
 x * 40000, SDS-PAGE Ferroplasma acidarmanus
80000
-
 gel filtration Thermococcus litoralis
80000
-
 gel filtration Ferroplasma acidarmanus

Organism

Organism UniProt Comment Textmining
Ferroplasma acidarmanus S0APF4
-
-
Thermococcus litoralis H3ZMH5
-
-
Thermococcus litoralis DSM 5473 H3ZMH5
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Thermococcus litoralis
-
 Ferroplasma acidarmanus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
cis-3-hydroxy-L-proline
-
 Thermococcus litoralis trans-3-hydroxy-D-proline
-
 ?
cis-4-hydroxy-D-proline 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 Thermococcus litoralis trans-4-hydroxy-L-proline
-
 r
cis-4-hydroxy-L-proline 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 Thermococcus litoralis trans-4-hydroxy-D-proline
-
 r
L-azetidine-2-carboxylate low activity Thermococcus litoralis D-azetidine-2-carboxylate
-
 ?
L-pipecolate low activity Thermococcus litoralis D-pipecolate
-
 ?
trans-3-hydroxy-L-proline
-
 Thermococcus litoralis trans-3-hydroxy-D-proline
-
 ?
trans-4-hydroxy-D-proline 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 Thermococcus litoralis cis-4-hydroxy-L-proline
-
 r
trans-4-hydroxy-D-proline 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 Thermococcus litoralis DSM 5473 cis-4-hydroxy-L-proline
-
 r
trans-4-hydroxy-L-proline kcat/Km value for trans-4-hydroxy-L-proline is about 3fold lower than that for L-proline, which is attributed to a 17fold lower kcat value. The kinetic parameters of the epimerization of cis-4-hydroxy-D-proline can not be determined Ferroplasma acidarmanus cis-4-hydroxy-D-proline
-
 r
trans-4-hydroxy-L-proline 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 Thermococcus litoralis cis-4-hydroxy-D-proline
-
 r

Subunits

Subunits Comment Organism
? x * 40000, SDS-PAGE Thermococcus litoralis
? x * 40000, SDS-PAGE Ferroplasma acidarmanus

Synonyms

Synonyms Comment Organism
FaProR
-
 Ferroplasma acidarmanus
proline racemase/hydroxyproline epimerase bifunctional enzyme Thermococcus litoralis
ProR/HypE bifunctional enzyme Thermococcus litoralis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
 assay at Thermococcus litoralis
50
-
 assay at Ferroplasma acidarmanus
90 100
-
 Thermococcus litoralis
90 100
-
 Ferroplasma acidarmanus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.012
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme F420W Ferroplasma acidarmanus
0.014
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, mutant enzyme F62S Thermococcus litoralis
0.039
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme F62S Thermococcus litoralis
0.0395
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, wild-type enzyme Ferroplasma acidarmanus
0.11
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, mutant enzyme L221H Thermococcus litoralis
0.13
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme F62V Thermococcus litoralis
0.29
-
 trans-3-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme W241F Thermococcus litoralis
0.65
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme L221H Thermococcus litoralis
0.69
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, mutant enzyme W241F Thermococcus litoralis
1.53
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme W241F Thermococcus litoralis
3.6
-
 trans-3-hydroxy-L-proline pH 8.0, 50°C, wild-type enzyme Thermococcus litoralis
3.83
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, wild-type enzyme Thermococcus litoralis
12.13
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, wild-type enzyme Thermococcus litoralis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Thermococcus litoralis
8
-
 assay at Ferroplasma acidarmanus

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.217
-
 pH 8.0, 50°C Thermococcus litoralis pyrrole-2-carboxylate
0.58
-
 pH 8.0, 50°C Ferroplasma acidarmanus pyrrole-2-carboxylate

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0046
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, mutant enzyme F62S Thermococcus litoralis
0.0055
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme F420W Ferroplasma acidarmanus
0.0063
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme F62S Thermococcus litoralis
0.0065
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, wild-type enzyme Ferroplasma acidarmanus
0.027
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, mutant enzyme L221H Thermococcus litoralis
0.1
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme L221H Thermococcus litoralis
0.1
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, mutant enzyme W241F Thermococcus litoralis
0.14
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme F62V Thermococcus litoralis
0.18
-
 trans-3-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme W241F Thermococcus litoralis
0.36
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, mutant enzyme W241F Thermococcus litoralis
0.58
-
 cis-4-hydroxy-D-proline pH 8.0, 50°C, wild-type enzyme Thermococcus litoralis
2.03
-
 trans-4-hydroxy-L-proline pH 8.0, 50°C, wild-type enzyme Thermococcus litoralis
7.2
-
 trans-3-hydroxy-L-proline pH 8.0, 50°C, wild-type enzyme Thermococcus litoralis