Cloned (Comment) | Organism |
---|---|
recombinant expression of the holo form of MBP-tagged TtProDH in Escherichia coli TOP10 cells, the recombinant enzyme contains about three times more FMN than FAD. The apoenzyme is produced by riboflavin auxotrophic Escherichia coli strain BSV11 and can be successfully reconstituted with FAD or FMN. The recombinant apo-enzyme reconstituted with FAD or FMN shows equal specific activities as holo-enzyme | Thermus thermophilus |
Crystallization (Comment) | Organism |
---|---|
purified TtProDH mutant variant DELTAABC lacking helices alphaA, alphaB and alphaC, X-ray diffraction structure determination and analysis at 2.2 A resolution | Thermus thermophilus |
Protein Variants | Comment | Organism |
---|---|---|
F10E/L12E | construction of a variant with a more polar N-terminus, mutant F10E/L12E, because Thermus thermophilus ProDH (TtProDH), produced through fusion with maltose-binding protein (MBP) appears to be prone to aggregation. Preparation of TtProDH mutant variant DELTAABC, which lacks helices alphaA, alphaB and alphaC, the mutant shows no electron density for an AMP moiety of the cofactor | Thermus thermophilus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
36.6 | - |
L-proline | recombinant apo-enzyme reconstituted with FAD, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus | |
50.1 | - |
L-proline | recombinant apo-enzyme reconstituted with FMN, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus | |
76.6 | - |
L-proline | recombinant holo-enzyme, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-proline + a quinone | Thermus thermophilus | the reverse reaction is catalyzed by pyrroline-5-carboxylate reductase, EC 1.5.1.2 | (S)-1-pyrroline-5-carboxylate + a quinol | - |
ir | |
L-proline + a quinone | Thermus thermophilus HB27 / ATCC BAA-163 / DSM 7039 | the reverse reaction is catalyzed by pyrroline-5-carboxylate reductase, EC 1.5.1.2 | (S)-1-pyrroline-5-carboxylate + a quinol | - |
ir |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermus thermophilus | Q72IB8 | - |
- |
Thermus thermophilus HB27 / ATCC BAA-163 / DSM 7039 | Q72IB8 | - |
- |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.84 | - |
purified recombinant apo-enzyme, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus |
4.74 | - |
purified recombinant holo-enzyme, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus |
4.83 | - |
purified recombinant apo-enzyme reconstituted with FAD, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus |
4.95 | - |
purified recombinant apo-enzyme reconstituted with FMN, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-proline + 2,6-dichlorphenol-indophenol | - |
Thermus thermophilus | (S)-1-pyrroline-5-carboxylate + reduced 2,6-dichlorphenol-indophenol | - |
? | |
L-proline + 2,6-dichlorphenol-indophenol | - |
Thermus thermophilus HB27 / ATCC BAA-163 / DSM 7039 | (S)-1-pyrroline-5-carboxylate + reduced 2,6-dichlorphenol-indophenol | - |
? | |
L-proline + a quinone | the reverse reaction is catalyzed by pyrroline-5-carboxylate reductase, EC 1.5.1.2 | Thermus thermophilus | (S)-1-pyrroline-5-carboxylate + a quinol | - |
ir | |
L-proline + a quinone | the reverse reaction is catalyzed by pyrroline-5-carboxylate reductase, EC 1.5.1.2 | Thermus thermophilus HB27 / ATCC BAA-163 / DSM 7039 | (S)-1-pyrroline-5-carboxylate + a quinol | - |
ir |
Subunits | Comment | Organism |
---|---|---|
dimer | 2 * 74401, recombinant MBP-tagged enzyme, sequence calculation, 2 * 71899, recombinant enzyme mutant DELTAABC, sequence calculation | Thermus thermophilus |
Synonyms | Comment | Organism |
---|---|---|
TtProDH | - |
Thermus thermophilus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Thermus thermophilus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
7.2 | - |
L-proline | recombinant apo-enzyme reconstituted with FAD, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus | |
7.9 | - |
L-proline | recombinant apo-enzyme reconstituted with FMN, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus | |
9.8 | - |
L-proline | recombinant holo-enzyme, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Thermus thermophilus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor, redox active prosthetic group | Thermus thermophilus | |
FMN | proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor, redox active prosthetic group | Thermus thermophilus | |
additional information | the recombinant holo form of MBP-tagged TtProDH, as produced in Escherichia coli TOP10 cells, contains about three times more FMN than FAD. The crystal structure of TtProDH variant DELTAABC, which lacks helices alphaA, alphaB and alphaC, shows no electron density for an AMP moiety of the cofactor. ProDH adopts a distorted (betalpha)8 TIM-barrel fold and is the only known TIM-barrel enzyme that contains an FAD cofactor. One cofactor molecule per enzyme subunit | Thermus thermophilus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
146 | - |
L-proline | recombinant holo-enzyme, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus | |
158 | - |
L-proline | recombinant apo-enzyme reconstituted with FMN, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus | |
198 | - |
L-proline | recombinant apo-enzyme reconstituted with FAD, pH 7.4, 25°C, with 2,6-dichlorphenol-indophenol | Thermus thermophilus |