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Literature summary for 1.5.1.B5 extracted from

  • Watanabe, S.; Tozawa, Y.; Watanabe, Y.
    Ornithine cyclodeaminase/micro-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 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.5.1.B5

Cloned(Commentary)

Cloned (Comment) Organism
gene lphI, sequence comparisons and phylogenetic analysis, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)-RIL Thermococcus litoralis

Protein Variants

Protein Variants Comment Organism
L52E/V224D/A228K site-directed mutagenesis, the mutant shows 2fold increased activity and highly increased specifiicity for substrate DELTA1-pyrrolidine-2-carboxylate as compared to the wild-type enzyme Thermococcus litoralis
L52R/V224D/A228K site-directed mutagenesis, the mutant shows slightly increased activity and highly increased specifiicity for substrate DELTA1-pyrrolidine-2-carboxylate and no activity with other substrates in contrast to the wild-type enzyme Thermococcus litoralis
V224D/A228K site-directed mutagenesis, the mutant shows 10fold increased activity for substrate DELTA1-pyrrolidine-2-carboxylate as compared to the wild-type enzyme Thermococcus litoralis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.944
-
 1-pyrroline-2-carboxylate recombinant wild-type enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis
2.77
-
 1-pyrroline-2-carboxylate recombinant mutant V224D/A228K enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1-pyrroline-2-carboxylate + NADH + H+ Thermococcus litoralis
-
 L-proline + NAD+
-
 r
1-pyrroline-2-carboxylate + NADH + H+ Thermococcus litoralis DSM 5473
-
 L-proline + NAD+
-
 r

Organism

Organism UniProt Comment Textmining
Thermococcus litoralis
-
-
-
Thermococcus litoralis DSM 5473
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)-RIL by nickel affinity chromatography, ultrafiltration, and gel filtration Thermococcus litoralis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
23.9
-
 purified recombinant wild-type enzyme, pH 6.5, 50°C, substrate 1-pyrroline-2-carboxylate Thermococcus litoralis
239
-
 purified recombinant mutant V224D/A228K enzyme, pH 6.5, 50°C, substrate 1-pyrroline-2-carboxylate Thermococcus litoralis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1-pyrroline-2-carboxylate + NADH + H+
-
 Thermococcus litoralis L-proline + NAD+
-
 r
1-pyrroline-2-carboxylate + NADH + H+ highly preferred substrates, preference of the reaction equilibrium in the direction toward NADH-dependent reduction Thermococcus litoralis L-proline + NAD+
-
 r
1-pyrroline-2-carboxylate + NADH + H+
-
 Thermococcus litoralis DSM 5473 L-proline + NAD+
-
 r
1-pyrroline-2-carboxylate + NADH + H+ highly preferred substrates, preference of the reaction equilibrium in the direction toward NADH-dependent reduction Thermococcus litoralis DSM 5473 L-proline + NAD+
-
 r
cis-4-hydroxy-L-proline + NAD+
-
 Thermococcus litoralis DELTA1-pyrroline-4S-hydroxy-2-carboxylate + NADPH + H+
-
 r
cis-4-hydroxy-L-proline + NAD+
-
 Thermococcus litoralis DSM 5473 DELTA1-pyrroline-4S-hydroxy-2-carboxylate + NADPH + H+
-
 r
L-pipecolate + NAD+
-
 Thermococcus litoralis 1-piperideine-2-carboxylate + NADH + H+
-
 r
L-pipecolate + NAD+
-
 Thermococcus litoralis DSM 5473 1-piperideine-2-carboxylate + NADH + H+
-
 r
additional information trans-4-hydroxy-L-proline, L-2-aminovalerate, and N-methyl-L-alanine. TlLhpI shows NAD+-dependent oxidation of L-proline. The kcat/Km value in the presence of NADP+ is about 50fold lower than that in the presence of NAD+. In addition to L-proline, several L-proline analogues, including cis-4-hydroxy-L-proline (85%), trans-4-hydroxy-L-proline (70%), and L-pipecolate (49%), are active substrates (relative activity to L-proline in parentheses). The enzyme also shows low activity with pyruvate, oxaloacetate, 2-oxobutyrate, 2-oxovalerate, and 4-methyl-2-oxovalerate in the oxidation reaction direction, overview Thermococcus litoralis ?
-
 ?
additional information trans-4-hydroxy-L-proline, L-2-aminovalerate, and N-methyl-L-alanine. TlLhpI shows NAD+-dependent oxidation of L-proline. The kcat/Km value in the presence of NADP+ is about 50fold lower than that in the presence of NAD+. In addition to L-proline, several L-proline analogues, including cis-4-hydroxy-L-proline (85%), trans-4-hydroxy-L-proline (70%), and L-pipecolate (49%), are active substrates (relative activity to L-proline in parentheses). The enzyme also shows low activity with pyruvate, oxaloacetate, 2-oxobutyrate, 2-oxovalerate, and 4-methyl-2-oxovalerate in the oxidation reaction direction, overview Thermococcus litoralis DSM 5473 ?
-
 ?
trans-4-hydroxy-L-proline + NAD+
-
 Thermococcus litoralis DELTA1-pyrroline-4R-hydroxy-2-carboxylate + NADPH + H+
-
 r

Synonyms

Synonyms Comment Organism
NADH-dependent Pyr2C reductase
-
 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 Structure
16.58
-
 1-pyrroline-2-carboxylate recombinant wild-type enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis
558.3
-
 1-pyrroline-2-carboxylate recombinant mutant V224D/A228K enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5
-
 1-pyrroline-2-carboxylate reduction Thermococcus litoralis
11.5
-
 L-proline oxidation Thermococcus litoralis

Cofactor

Cofactor Comment Organism Structure
NAD+ preferred cofactor Thermococcus litoralis
NADH preferred cofactor Thermococcus litoralis

General Information

General Information Comment Organism
evolution the enzyme belongs to the ornithine cyclodeaminase/l-crystallin (OCD/CRYM) superfamily Thermococcus litoralis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
17.57
-
 1-pyrroline-2-carboxylate recombinant wild-type enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis
201.6
-
 1-pyrroline-2-carboxylate recombinant mutant V224D/A228K enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis