Literature summary for 5.1.1.4 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.

Search for more literature for 5.1.1.4

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Clostridioides difficile
expression in Escherichia coli	Thermococcus litoralis
expression in Escherichia coli	Haloarcula japonica
expression in Escherichia coli	Ferroplasma acidarmanus

Protein Variants

Protein Variants	Comment	Organism
R240W	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 are 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	-	Clostridioides difficile
pyrrole-2-carboxylate	-	Ferroplasma acidarmanus
pyrrole-2-carboxylate	-	Haloarcula japonica
pyrrole-2-carboxylate	-	Thermococcus litoralis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.092	-	D-proline	pH 8.0, 50°C, wild-type enzyme	Thermococcus litoralis
0.156	-	D-proline	pH 8.0, 50°C, mutant enzyme F62V	Thermococcus litoralis
0.194	-	D-proline	pH 8.0, 50°C, mutant enzyme W241F	Thermococcus litoralis
0.612	-	D-proline	pH 8.0, 50°C, wild-type enzyme	Clostridioides difficile
1.17	-	D-proline	pH 8.0, 50°C, wild-type enzyme	Ferroplasma acidarmanus
1.21	-	L-proline	pH 8.0, 50°C, wild-type enzyme	Thermococcus litoralis
1.29	-	L-proline	pH 8.0, 50°C, mutant enzyme W241F	Thermococcus litoralis
1.93	-	L-proline	pH 8.0, 50°C, mutant enzyme F62V	Thermococcus litoralis
2.54	-	D-proline	pH 8.0, 50°C, mutant enzyme L221H	Thermococcus litoralis
4	-	D-proline	pH 8.0, 50°C, mutant enzyme F62S	Thermococcus litoralis
8.53	-	L-proline	pH 8.0, 50°C, wild-type enzyme	Clostridioides difficile
20.5	-	L-proline	pH 8.0, 50°C, mutant enzyme L221H	Thermococcus litoralis
31.6	-	L-proline	pH 8.0, 50°C, wild-type enzyme	Ferroplasma acidarmanus
53	-	L-proline	pH 8.0, 50°C, mutant enzyme F240W	Ferroplasma acidarmanus
154	-	L-proline	pH 8.0, 50°C, mutant enzyme F62S	Thermococcus litoralis

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
40000	-	x * 40000, SDS-PAGE	Clostridioides difficile
40000	-	x * 40000, SDS-PAGE	Thermococcus litoralis
40000	-	x * 40000, SDS-PAGE	Ferroplasma acidarmanus
42000	-	x * 42000, SDS-PAGE	Haloarcula japonica
80000	-	gel filtration	Clostridioides difficile
80000	-	gel filtration	Thermococcus litoralis
80000	-	gel filtration	Haloarcula japonica
80000	-	gel filtration	Ferroplasma acidarmanus

Organism

Organism	UniProt	Comment	Textmining
Clostridioides difficile	Q17ZY4	-	-
Ferroplasma acidarmanus	S0APF4	-	-
Haloarcula japonica	M0LMI3	-	-
Haloarcula japonica DSM 6131	M0LMI3	-	-
Thermococcus litoralis	H3ZMH5	-	-
Thermococcus litoralis DSM 5473	H3ZMH5	-	-

Purification (Commentary)

Purification (Comment)	Organism
-	Clostridioides difficile
-	Thermococcus litoralis
-	Haloarcula japonica
-	Ferroplasma acidarmanus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
D-proline	the reaction is completely bi-directional, and the reverse reactions. The specific activity with D-proline is 130% 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	L-proline	-	?
D-proline	the reaction is completely bi-directional, and the reverse reactions. The specific activity with D-proline is 130% 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	L-proline	-	?
L-azetidine-2-carboxylate	low activity	Thermococcus litoralis	D-azetidine-2-carboxylate	-	?
L-azetidine-2-carboxylate	low activity	Thermococcus litoralis DSM 5473	D-azetidine-2-carboxylate	-	?
L-pipecolate	low activity	Thermococcus litoralis	D-pipecolate	-	?
L-pipecolate	low activity	Thermococcus litoralis DSM 5473	D-pipecolate	-	?
L-proline	-	Clostridioides difficile	D-proline	-	r
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	D-proline	-	r
L-proline	the enzyme can utilize both proline and hydroxyprolines as substrate	Haloarcula japonica	D-proline	-	r
L-proline	the reaction is completely bi-directional, and the reverse reactions. The specific activity with D-proline is 130% 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	D-proline	-	r
L-proline	the reaction is completely bi-directional, and the reverse reactions. The specific activity with D-proline is 130% 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	D-proline	-	r
L-proline	the enzyme can utilize both proline and hydroxyprolines as substrate	Haloarcula japonica DSM 6131	D-proline	-	r

Subunits

Subunits	Comment	Organism
?	x * 42000, SDS-PAGE	Haloarcula japonica
?	x * 40000, SDS-PAGE	Clostridioides difficile
?	x * 40000, SDS-PAGE	Thermococcus litoralis
?	x * 40000, SDS-PAGE	Ferroplasma acidarmanus

Synonyms

Synonyms	Comment	Organism
CdProR	-	Clostridioides difficile
FaProR	-	Ferroplasma acidarmanus
HjProR	-	Haloarcula japonica
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	Clostridioides difficile
50	-	assay at	Thermococcus litoralis
50	-	assay at	Haloarcula japonica
50	-	assay at	Ferroplasma acidarmanus
90	100	-	Clostridioides difficile
90	100	-	Thermococcus litoralis
90	100	-	Haloarcula japonica
90	100	-	Ferroplasma acidarmanus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.0235	-	D-proline	pH 8.0, 50°C, mutant enzyme F62V	Thermococcus litoralis
0.026	-	D-proline	pH 8.0, 50°C, mutant enzyme F62S	Thermococcus litoralis
0.092	-	L-proline	pH 8.0, 50°C, mutant enzyme F62V	Thermococcus litoralis
0.119	-	D-proline	pH 8.0, 50°C, wild-type enzyme	Ferroplasma acidarmanus
0.2	-	L-proline	pH 8.0, 50°C, mutant enzyme F62S	Thermococcus litoralis
0.29	-	D-proline	pH 8.0, 50°C, mutant enzyme L221H	Thermococcus litoralis
0.42	-	L-proline	pH 8.0, 50°C, mutant enzyme F240W	Ferroplasma acidarmanus
0.6	-	L-proline	pH 8.0, 50°C, wild-type enzyme	Ferroplasma acidarmanus
0.92	-	L-proline	pH 8.0, 50°C, mutant enzyme L221H	Thermococcus litoralis
2.22	-	D-proline	pH 8.0, 50°C, wild-type enzyme	Thermococcus litoralis
2.48	-	D-proline	pH 8.0, 50°C, wild-type enzyme	Clostridioides difficile
2.78	-	L-proline	pH 8.0, 50°C, wild-type enzyme	Thermococcus litoralis
3.17	-	L-proline	pH 8.0, 50°C, mutant enzyme W241F	Thermococcus litoralis
3.18	-	D-proline	pH 8.0, 50°C, mutant enzyme W241F	Thermococcus litoralis
14.15	-	L-proline	pH 8.0, 50°C, wild-type enzyme	Clostridioides difficile

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Clostridioides difficile
8	-	assay at	Thermococcus litoralis
8	-	assay at	Haloarcula japonica
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
0.269	-	pH 8.0, 50°C	Clostridioides difficile	pyrrole-2-carboxylate
0.295	-	pH 8.0, 50°C	Haloarcula japonica	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
0.0013	-	L-proline	pH 8.0, 50°C, mutant enzyme F62S	Thermococcus litoralis
0.0065	-	D-proline	pH 8.0, 50°C, mutant enzyme F62S	Thermococcus litoralis
0.0079	-	L-proline	pH 8.0, 50°C, mutant enzyme F240W	Ferroplasma acidarmanus
0.019	-	L-proline	pH 8.0, 50°C, wild-type enzyme	Ferroplasma acidarmanus
0.045	-	L-proline	pH 8.0, 50°C, mutant enzyme L221H	Thermococcus litoralis
0.048	-	L-proline	pH 8.0, 50°C, mutant enzyme F62V	Thermococcus litoralis
0.1	-	D-proline	pH 8.0, 50°C, wild-type enzyme	Ferroplasma acidarmanus
0.11	-	D-proline	pH 8.0, 50°C, mutant enzyme L221H	Thermococcus litoralis
0.15	-	D-proline	pH 8.0, 50°C, mutant enzyme F62V	Thermococcus litoralis
1.65	-	L-proline	pH 8.0, 50°C, wild-type enzyme	Clostridioides difficile
2.3	-	L-proline	pH 8.0, 50°C, wild-type enzyme	Thermococcus litoralis
2.45	-	L-proline	pH 8.0, 50°C, mutant enzyme W241F	Thermococcus litoralis
4.08	-	D-proline	pH 8.0, 50°C, wild-type enzyme	Clostridioides difficile
16.5	-	D-proline	pH 8.0, 50°C, mutant enzyme W241F	Thermococcus litoralis
26.2	-	D-proline	pH 8.0, 50°C, wild-type enzyme	Thermococcus litoralis

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Release 2023.1 (January 2023)