Literature summary for 1.5.1.B5 extracted from

Watanabe, S.; Tozawa, Y.; Watanabe, Y.
Ornithine cyclodeaminase/mu-crystallin homolog from the hyperthermophilic archaeon Thermococcus litoralis functions as a novel DELTA(1)-pyrroline-2-carboxylate reductase involved in putative trans-3-hydroxy-L-proline metabolism (2014), FEBS Open Bio, 4, 617-626.

Cloned(Commentary)

Cloned (Comment)	Organism
His-tagged protein is expressed in Escherichia coli BL21. TlLhpI is a fusion of OCC_00362 and OCC_00367 genes by PCR. The gene from Thermococcus litoralis DSM 5473 consists of two separate open-reading frames (ORFs): OCC_00362 and OCC_00367. When a single A is inserted between A227646 and T227647 in the original genome sequence of T. litoralis DSM 5473, the separate ORFs fuse into a single ORF, and the putative amino acid sequence of is homologous to that of the Thermococcus sibiricus TSIB_0634 gene. Although there is a possibility that this OCD-like protein from Thermococcus litoralis DSM 5473 forms a heterooligomeric structure, the TlLhpI gene is synthesized as a single ORF by sequential steps of PCR using sense and antisense primers and genome DNA of Thermococcus litoralis DSM 5473 as a template	Thermococcus litoralis

Cloned (Comment)

Organism

His-tagged protein is expressed in Escherichia coli BL21. TlLhpI is a fusion of OCC_00362 and OCC_00367 genes by PCR. The gene from Thermococcus litoralis DSM 5473 consists of two separate open-reading frames (ORFs): OCC_00362 and OCC_00367. When a single A is inserted between A227646 and T227647 in the original genome sequence of T. litoralis DSM 5473, the separate ORFs fuse into a single ORF, and the putative amino acid sequence of is homologous to that of the Thermococcus sibiricus TSIB_0634 gene. Although there is a possibility that this OCD-like protein from Thermococcus litoralis DSM 5473 forms a heterooligomeric structure, the TlLhpI gene is synthesized as a single ORF by sequential steps of PCR using sense and antisense primers and genome DNA of Thermococcus litoralis DSM 5473 as a template

Thermococcus litoralis

Protein Variants

Protein Variants	Comment	Organism
L52E/V224D/A228K	DELTA1-pyrroline-2-carboxylate reductase activity is the same as in wild-type enzyme, no alanine dehydrogenase activity	Thermococcus litoralis
L52R/V224D/A228K	DELTA1-pyrroline-2-carboxylate reductase activity is the same as in wild-type enzyme, no alanine dehydrogenase activity	Thermococcus litoralis
additional information	TlLhpI is a fusion of OCC_00362 and OCC_00367 genes by PCR. The gene from Thermococcus litoralis DSM 5473 consistsof two separate open-reading frames (ORFs): OCC_00362 and OCC_00367. When a single A is inserted between A227646 and T227647 in the original genome sequence of T. litoralis DSM 5473, the separate ORFs fuse into a single ORF, and the putative amino acid sequence of is homologous to that of the Thermococcus sibiricus TSIB_0634 gene. Although there is a possibility that this OCD-like protein from Thermococcus litoralis DSM 5473 forms a heterooligomeric structure, the TlLhpI gene is synthesized as a single ORF by sequential steps of PCR using sense and antisense primers and genome DNA of Thermococcus litoralis DSM 5473 as a template	Thermococcus litoralis
V224D/A228K	12fold enhancement of DELTA1-pyrroline-2-carboxylate reductase activity, 6.7fold enhancement for the oxidization activity for L-proline, no alanine dehydrogenase activity	Thermococcus litoralis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.444	-	L-proline	pH 6.0, 50°C, mutant enzyme V224D/A228K	Thermococcus litoralis
0.608	-	L-pipecolate	pH 6.0, 50°C, wild-type enzyme	Thermococcus litoralis
0.944	-	DELTA1-pyrroline-2-carboxylate	pH 6.0, 50°C, wild-type enzyme	Thermococcus litoralis
1.12	-	L-proline	pH 6.0, 50°C, wild-type enzyme	Thermococcus litoralis
2.77	-	DELTA1-pyrroline-2-carboxylate	pH 6.0, 50°C, mutant enzyme V224D/A228K	Thermococcus litoralis

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
40000	-	2 * 40000, SDS-PAGE	Thermococcus litoralis
72000	-	gel filtration	Thermococcus litoralis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
DELTA1-pyrroline-2-carboxylate + NADH + H+	Thermococcus litoralis	the enzyme is involved in the trans-3-hydroxy-L-proline metabolism	L-proline + NAD+	-	?
DELTA1-pyrroline-2-carboxylate + NADH + H+	Thermococcus litoralis DSM 5473	the enzyme is involved in the trans-3-hydroxy-L-proline metabolism	L-proline + NAD+	-	?

Organism

Organism	UniProt	Comment	Textmining
Thermococcus litoralis	H3ZMH3 and H3ZMH4	TlLhpI is a fusion of OCC_00362 and OCC_00367 genes by PCR. The gene from Thermococcus litoralis DSM 5473 consists of two separate open-reading frames (ORFs): OCC_00362 and OCC_00367. When a single A is inserted between A227646 and T227647 in the original genome sequence of T. litoralis DSM 5473, the separate ORFs fuse into a single ORF, and the putative amino acid sequence of is homologous to that of the Thermococcus sibiricus TSIB_0634 gene. Although there is a possibility that this OCD-like protein from Thermococcus litoralis DSM 5473 forms a heterooligomeric structure, the TlLhpI gene is synthesized as a single ORF by sequential steps of PCR using sense and antisense primers and genome DNA of Thermococcus litoralis DSM 5473 as a template	-
Thermococcus litoralis DSM 5473	H3ZMH3 and H3ZMH4	TlLhpI is a fusion of OCC_00362 and OCC_00367 genes by PCR. The gene from Thermococcus litoralis DSM 5473 consists of two separate open-reading frames (ORFs): OCC_00362 and OCC_00367. When a single A is inserted between A227646 and T227647 in the original genome sequence of T. litoralis DSM 5473, the separate ORFs fuse into a single ORF, and the putative amino acid sequence of is homologous to that of the Thermococcus sibiricus TSIB_0634 gene. Although there is a possibility that this OCD-like protein from Thermococcus litoralis DSM 5473 forms a heterooligomeric structure, the TlLhpI gene is synthesized as a single ORF by sequential steps of PCR using sense and antisense primers and genome DNA of Thermococcus litoralis DSM 5473 as a template	-

Purification (Commentary)

Purification (Comment)	Organism
-	Thermococcus litoralis

Reaction

Reaction	Comment	Organism	Reaction ID
L-pipecolate + NAD+ = DELTA1-piperideine-2-carboxylate + NADH + H+	(1)	Thermococcus litoralis	a
L-proline + NAD+ = DELTA1-pyrroline-2-carboxylate + NADH + H+	(2)	Thermococcus litoralis	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
cis-4-hydroxy-L-proline + NAD+	activity is 85% compared to the activity with L-proline	Thermococcus litoralis	?	-	?
cis-4-hydroxy-L-proline + NAD+	activity is 85% compared to the activity with L-proline	Thermococcus litoralis DSM 5473	?	-	?
DELTA1-pyrroline-2-carboxylate + NADH + H+	the enzyme is involved in the trans-3-hydroxy-L-proline metabolism	Thermococcus litoralis	L-proline + NAD+	-	?
DELTA1-pyrroline-2-carboxylate + NADH + H+	preference of the reaction equilibrium in the direction toward NADH-dependent reduction	Thermococcus litoralis	L-proline + NAD+	-	r
DELTA1-pyrroline-2-carboxylate + NADH + H+	the enzyme is involved in the trans-3-hydroxy-L-proline metabolism	Thermococcus litoralis DSM 5473	L-proline + NAD+	-	?
DELTA1-pyrroline-2-carboxylate + NADH + H+	preference of the reaction equilibrium in the direction toward NADH-dependent reduction	Thermococcus litoralis DSM 5473	L-proline + NAD+	-	r
L-pipecolate + NAD+	activity is 49% compared to the activity with L-proline	Thermococcus litoralis	DELTA1-piperideine-2-carboxylate + NADH + H+	-	?
L-pipecolate + NAD+	activity is 49% compared to the activity with L-proline	Thermococcus litoralis DSM 5473	DELTA1-piperideine-2-carboxylate + NADH + H+	-	?
L-proline + NAD+	preference of the reaction equilibrium in the direction toward NADH-dependent reduction	Thermococcus litoralis	DELTA1-pyrroline-2-carboxylate + NADH + H+	-	r
additional information	the enzyme also shows alanine dehydrogenase activity (EC 1.4.1.1) and N-methyl-L-alanine dehydrogenase activity (EC 1.4.1.17)	Thermococcus litoralis	?	-	?
additional information	the enzyme also shows alanine dehydrogenase activity (EC 1.4.1.1) and N-methyl-L-alanine dehydrogenase activity (EC 1.4.1.17)	Thermococcus litoralis DSM 5473	?	-	?
trans-4-hydroxy-L-proline + NAD+	activity is 70% compared to the activity with L-proline	Thermococcus litoralis	?	-	?

Subunits

Subunits	Comment	Organism
dimer	2 * 40000, SDS-PAGE	Thermococcus litoralis

Synonyms

Synonyms	Comment	Organism
DELTA1-pyrroline-2-carboxylate reductase	misleading	Thermococcus litoralis
Pyr2C reductase	-	Thermococcus litoralis
TlLhpI	-	Thermococcus litoralis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
50	-	assay at	Thermococcus litoralis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.2	-	L-pipecolate	pH 6.0, 50°C, wild-type enzyme	Thermococcus litoralis
0.23	-	L-proline	pH 6.0, 50°C, wild-type enzyme	Thermococcus litoralis
0.61	-	L-proline	pH 6.0, 50°C, mutant enzyme V224D/A228K	Thermococcus litoralis
16.6	-	DELTA1-pyrroline-2-carboxylate	pH 6.0, 50°C, wild-type enzyme	Thermococcus litoralis
558	-	DELTA1-pyrroline-2-carboxylate	pH 6.0, 50°C, mutant enzyme V224D/A228K	Thermococcus litoralis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
4	6	substrates: DELTA1-pyrroline-2-carboxylate + NADH + H+	Thermococcus litoralis
11	-	substrates: L-proline + NAD+	Thermococcus litoralis

pH Range

pH Minimum	pH Maximum	Comment	Organism
5	11	pH 5.0: about 40% of maximal activity, pH 11.0: about 80% of maximal activity	Thermococcus litoralis
10	12	pH 10.0: about 75% of maximal activity, pH 12.0: about 85% of maximal activity	Thermococcus litoralis

Cofactor

Cofactor	Comment	Organism	Structure
NAD+	the kcat/Km value for proline in the presence of NADP+ is about 50fold lower than that in the presence of NAD+	Thermococcus litoralis
NADH	no activity with NADPH	Thermococcus litoralis

General Information

General Information	Comment	Organism
metabolism	the enzyme is involved in the trans-3-hydroxy-L-proline metabolism	Thermococcus litoralis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.2	-	L-proline	pH 6.0, 50°C, wild-type enzyme	Thermococcus litoralis
0.33	-	L-pipecolate	pH 6.0, 50°C, wild-type enzyme	Thermococcus litoralis
1.4	-	L-proline	pH 6.0, 50°C, mutant enzyme V224D/A228K	Thermococcus litoralis
17.6	-	DELTA1-pyrroline-2-carboxylate	pH 6.0, 50°C, wild-type enzyme	Thermococcus litoralis
201	-	DELTA1-pyrroline-2-carboxylate	pH 6.0, 50°C, mutant enzyme V224D/A228K	Thermococcus litoralis