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Literature summary for 1.5.99.B2 extracted from

  • Huijbers, M.; van Berkel, W.
    A more polar N-terminal helix releases MBP-tagged Thermus thermophilus proline dehydrogenase from tetramer-polymer self-association (2016), J. Mol. Catal. B, 134, 340-346 .
No PubMed abstract available
Search for more literature for 1.5.99.B2

Cloned(Commentary)

Cloned (Comment) Organism
recombinant overexpression of wild-type and mutant MBP-fusion enzyme in Escherichia coli TOP 10 cells, MBP-TtProDH and MBP-clipped TtProDH are proneto aggregation through non-native self-association Thermus thermophilus

Crystallization (Commentary)

Crystallization (Comment) Organism
analysis of the three-dimensional model of TtProDH crystal structure, PDB ID 2G37 Thermus thermophilus

Protein Variants

Protein Variants Comment Organism
F10E/L12E two point mutations in helix alphaA. This helix contains several hydrophobic residues. The recombinant MBP-tagged mutant variant exclusively forms tetramers and exhibits excellent catalytic features over a wide range of temperatures Thermus thermophilus

Inhibitors

Inhibitors Comment Organism Structure
L-proline substrate inhibition Thermus thermophilus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten and Haldane kinetics Thermus thermophilus
6.5
-
 L-proline pH 7.4, 45°C, recombinant wild-type enzyme, Haldane kinetics Thermus thermophilus
11.5
-
 L-proline pH 7.4, 25°C, recombinant wild-type enzyme, Michaelis-Menten kinetics Thermus thermophilus
16.5
-
 L-proline pH 7.4, 25°C, recombinant wild-type enzyme, Haldane kinetics Thermus thermophilus
33.8
-
 L-proline pH 7.4, 45°C, recombinant mutant F10E/L12E, Haldane kinetics Thermus thermophilus
67.6
-
 L-proline pH 7.4, 25°C, recombinant mutant F10E/L12E, Michaelis-Menten kinetics Thermus thermophilus
175.1
-
 L-proline pH 7.4, 25°C, recombinant mutant F10E/L12E, Haldane kinetics Thermus thermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-proline + acceptor Thermus thermophilus
-
 (S)-1-pyrroline-5-carboxylate + reduced acceptor
-
 ?

Organism

Organism UniProt Comment Textmining
Thermus thermophilus Q72IB8
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-proline + 2,6-dichlorophenolindophenol artificial electron acceptor Thermus thermophilus (S)-1-pyrroline-5-carboxylate + reduced 2,6-dichlorophenolindophenol
-
 ?
L-proline + acceptor
-
 Thermus thermophilus (S)-1-pyrroline-5-carboxylate + reduced acceptor
-
 ?

Subunits

Subunits Comment Organism
More MBP-TtProDH and MBP-clipped TtProDH are prone to aggregation through non-native self-association. The hydrophobic N-terminal helix of TtProDH is responsible for the self-association process Thermus thermophilus
tetramer mutant F10E/L12E, mass spectrometry and gel filtration Thermus thermophilus

Synonyms

Synonyms Comment Organism
PRODH
-
 Thermus thermophilus
TtProDH
-
 Thermus thermophilus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
45
-
 assay at, both for recombinant MBP-tagged wild-type and mutant enzyme, kcat increases when the temperature is raised from 25°C to 45°C, the temperature is not increased further due to the instability of the MBP-tag Thermus thermophilus

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
25 45 remarkable 10fold increase in catalytic efficiency from 25°C to 45°C Thermus thermophilus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.7
-
 L-proline pH 7.4, 25°C, recombinant wild-type enzyme, Michaelis-Menten kinetics Thermus thermophilus
4.4
-
 L-proline pH 7.4, 25°C, recombinant wild-type enzyme, Haldane kinetics Thermus thermophilus
9.8
-
 L-proline pH 7.4, 25°C, recombinant mutant F10E/L12E, Michaelis-Menten kinetics Thermus thermophilus
19.4
-
 L-proline pH 7.4, 45°C, recombinant wild-type enzyme, Haldane kinetics Thermus thermophilus
20
-
 L-proline pH 7.4, 25°C, recombinant mutant F10E/L12E, Haldane kinetics Thermus thermophilus
42.1
-
 L-proline pH 7.4, 45°C, recombinant mutant F10E/L12E, Haldane kinetics Thermus thermophilus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
 assay at Thermus thermophilus

Cofactor

Cofactor Comment Organism Structure
FAD
-
 Thermus thermophilus

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
171
-
 L-proline pH 7.4, 45°C, recombinant mutant F10E/L12E, Haldane kinetics Thermus thermophilus
206
-
 L-proline pH 7.4, 25°C, recombinant mutant F10E/L12E, Haldane kinetics Thermus thermophilus
306
-
 L-proline pH 7.4, 45°C, recombinant wild-type enzyme, Haldane kinetics Thermus thermophilus
825
-
 L-proline pH 7.4, 25°C, recombinant wild-type enzyme, Haldane kinetics Thermus thermophilus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.114
-
 L-proline pH 7.4, 25°C, recombinant mutant F10E/L12E, Haldane kinetics Thermus thermophilus
0.146
-
 L-proline pH 7.4, 25°C, recombinant mutant F10E/L12E, Michaelis-Menten kinetics Thermus thermophilus
0.269
-
 L-proline pH 7.4, 25°C, recombinant wild-type enzyme, Haldane kinetics Thermus thermophilus
0.325
-
 L-proline pH 7.4, 25°C, recombinant wild-type enzyme, Michaelis-Menten kinetics Thermus thermophilus
1.247
-
 L-proline pH 7.4, 45°C, recombinant mutant F10E/L12E, Haldane kinetics Thermus thermophilus
2.979
-
 L-proline pH 7.4, 45°C, recombinant wild-type enzyme, Haldane kinetics Thermus thermophilus