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Literature summary 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

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.B5 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

EC Number Protein Variants Comment Organism
1.5.1.B5 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
1.5.1.B5 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
1.5.1.B5 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.B5 0.944
-
 1-pyrroline-2-carboxylate recombinant wild-type enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis
1.5.1.B5 2.77
-
 1-pyrroline-2-carboxylate recombinant mutant V224D/A228K enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.B5 1-pyrroline-2-carboxylate + NADH + H+ Thermococcus litoralis
-
 L-proline + NAD+
-
 r
1.5.1.B5 1-pyrroline-2-carboxylate + NADH + H+ Thermococcus litoralis DSM 5473
-
 L-proline + NAD+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.B5 Thermococcus litoralis
-
-
-
1.5.1.B5 Thermococcus litoralis DSM 5473
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.B5 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]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.5.1.B5 23.9
-
 purified recombinant wild-type enzyme, pH 6.5, 50°C, substrate 1-pyrroline-2-carboxylate Thermococcus litoralis
1.5.1.B5 239
-
 purified recombinant mutant V224D/A228K enzyme, pH 6.5, 50°C, substrate 1-pyrroline-2-carboxylate Thermococcus litoralis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.B5 1-pyrroline-2-carboxylate + NADH + H+
-
 Thermococcus litoralis L-proline + NAD+
-
 r
1.5.1.B5 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
1.5.1.B5 1-pyrroline-2-carboxylate + NADH + H+
-
 Thermococcus litoralis DSM 5473 L-proline + NAD+
-
 r
1.5.1.B5 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
1.5.1.B5 cis-4-hydroxy-L-proline + NAD+
-
 Thermococcus litoralis DELTA1-pyrroline-4S-hydroxy-2-carboxylate + NADPH + H+
-
 r
1.5.1.B5 cis-4-hydroxy-L-proline + NAD+
-
 Thermococcus litoralis DSM 5473 DELTA1-pyrroline-4S-hydroxy-2-carboxylate + NADPH + H+
-
 r
1.5.1.B5 L-pipecolate + NAD+
-
 Thermococcus litoralis 1-piperideine-2-carboxylate + NADH + H+
-
 r
1.5.1.B5 L-pipecolate + NAD+
-
 Thermococcus litoralis DSM 5473 1-piperideine-2-carboxylate + NADH + H+
-
 r
1.5.1.B5 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 ?
-
 ?
1.5.1.B5 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 ?
-
 ?
1.5.1.B5 trans-4-hydroxy-L-proline + NAD+
-
 Thermococcus litoralis DELTA1-pyrroline-4R-hydroxy-2-carboxylate + NADPH + H+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.5.1.B5 NADH-dependent Pyr2C reductase
-
 Thermococcus litoralis
1.5.1.B5 Pyr2C reductase
-
 Thermococcus litoralis
1.5.1.B5 TlLhpI
-
 Thermococcus litoralis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.5.1.B5 50
-
 assay at Thermococcus litoralis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.5.1.B5 16.58
-
 1-pyrroline-2-carboxylate recombinant wild-type enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis
1.5.1.B5 558.3
-
 1-pyrroline-2-carboxylate recombinant mutant V224D/A228K enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.1.B5 6.5
-
 1-pyrroline-2-carboxylate reduction Thermococcus litoralis
1.5.1.B5 11.5
-
 L-proline oxidation Thermococcus litoralis

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.B5 NAD+ preferred cofactor Thermococcus litoralis
1.5.1.B5 NADH preferred cofactor Thermococcus litoralis

General Information

EC Number General Information Comment Organism
1.5.1.B5 evolution the enzyme belongs to the ornithine cyclodeaminase/l-crystallin (OCD/CRYM) superfamily Thermococcus litoralis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.5.1.B5 17.57
-
 1-pyrroline-2-carboxylate recombinant wild-type enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis
1.5.1.B5 201.6
-
 1-pyrroline-2-carboxylate recombinant mutant V224D/A228K enzyme, pH 6.5, 50°C, with NADH Thermococcus litoralis