Cloned (Comment) | Organism |
---|---|
gene 6pgdh, recombinant expression in Escherichia coli TOP10, recombinant overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Moorella thermoacetica |
Protein Variants | Comment | Organism |
---|---|---|
A30C/R31I/T32K | saturation mutagenesis, the mutant shows better kinetics and increased activity for NAD+ compared to the wild-type enzyme | Moorella thermoacetica |
A30D/R31I/T32I | saturation mutagenesis, the mutant shows much better kinetics and strongly increased activity for NAD+ compared to the wild-type enzyme | Moorella thermoacetica |
A30E/R31I/T32D | saturation mutagenesis, the mutant shows better kinetics and increased activity for NAD+ compared to the wild-type enzyme | Moorella thermoacetica |
additional information | high-throughput screening of the two-site saturation mutagenesis library A30/T32 in Escherichia coli TOP10 by using a pair of degenerate primers 30_32_NNK_F/30_32_NNK_R, enzyme mutant identification and selection by heat treatment, usage of the dual T7-tac promoter and a colorimetric assay, method optimization, overview | Moorella thermoacetica |
R31I | saturation mutagenesis, the mutant shows slightly better kinetics and slightly increased activity for NAD+ compared to the wild-type enzyme | Moorella thermoacetica |
R31I/T32G | saturation mutagenesis, the mutant shows slightly better kinetics and slightly increased activity for NAD+ compared to the wild-type enzyme | Moorella thermoacetica |
R31T | saturation mutagenesis, the mutant shows slightly better kinetics and slightly increased activity for NAD+ compared to the wild-type enzyme | Moorella thermoacetica |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0119 | - |
NAD+ | recombinant His-tagged mutant A30D/R31I/T32I, pH 7.5, 50°C | Moorella thermoacetica | |
0.0139 | - |
NADP+ | recombinant His-tagged wild-type enzyme, pH 7.5, 50°C | Moorella thermoacetica | |
0.0265 | - |
NADP+ | recombinant His-tagged mutant R31I, pH 7.5, 50°C | Moorella thermoacetica | |
0.0355 | - |
NADP+ | recombinant His-tagged mutant R31T, pH 7.5, 50°C | Moorella thermoacetica | |
0.1044 | - |
NADP+ | recombinant His-tagged mutant R31I/T32G, pH 7.5, 50°C | Moorella thermoacetica | |
0.127 | - |
NAD+ | recombinant His-tagged mutant A30E/R31I/T32D, pH 7.5, 50°C | Moorella thermoacetica | |
0.228 | - |
NADP+ | recombinant His-tagged mutant A30D/R31I/T32I, pH 7.5, 50°C | Moorella thermoacetica | |
0.354 | - |
NAD+ | recombinant His-tagged mutant R31I, pH 7.5, 50°C | Moorella thermoacetica | |
0.362 | - |
NAD+ | recombinant His-tagged mutant R31I/T32G, pH 7.5, 50°C | Moorella thermoacetica | |
0.404 | - |
NAD+ | recombinant His-tagged mutant A30C/R31I/T32K, pH 7.5, 50°C | Moorella thermoacetica | |
0.605 | - |
NAD+ | recombinant His-tagged mutant R31T, pH 7.5, 50°C | Moorella thermoacetica | |
0.66 | - |
NADP+ | recombinant His-tagged mutant A30E/R31I/T32D, pH 7.5, 50°C | Moorella thermoacetica | |
0.698 | - |
NADP+ | recombinant His-tagged mutant A30C/R31I/T32K, pH 7.5, 50°C | Moorella thermoacetica | |
1.397 | - |
NAD+ | recombinant His-tagged wild-type enzyme, pH 7.5, 50°C | Moorella thermoacetica |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Moorella thermoacetica | |
Mn2+ | required | Moorella thermoacetica |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
6-phospho-D-gluconate + NADP+ | Moorella thermoacetica | - |
D-ribulose 5-phosphate + CO2 + NADPH + H+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Moorella thermoacetica | Q2RIZ2 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Moorella thermoacetica |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
6-phospho-D-gluconate + NAD+ | very low activity with NAD+ with the wild-type enzyme, low activity with mutants R31T, R31I, and R31I/T32G, higher activity with mutants A30C/R31I/T32K and A30E/R31I/T32D, and high activity with mutant A30D/R31I/T32I | Moorella thermoacetica | D-ribulose 5-phosphate + CO2 + NADH + H+ | - |
? | |
6-phospho-D-gluconate + NADP+ | - |
Moorella thermoacetica | D-ribulose 5-phosphate + CO2 + NADPH + H+ | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | three-dimensional structure homology modeling of wild-type 6PGDH from Moorella thermoacetica and mutants based on the structure of human enzyme 6PGDH, PDB ID 2JKV | Moorella thermoacetica |
Synonyms | Comment | Organism |
---|---|---|
6-phosphogluconate dehydrogenase | - |
Moorella thermoacetica |
6PGDH | - |
Moorella thermoacetica |
Moth_1283 | - |
Moorella thermoacetica |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
assay at | Moorella thermoacetica |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.81 | - |
NADP+ | recombinant His-tagged mutant A30D/R31I/T32I, pH 7.5, 50°C | Moorella thermoacetica | |
3.1 | - |
NADP+ | recombinant His-tagged mutant A30E/R31I/T32D, pH 7.5, 50°C | Moorella thermoacetica | |
5.75 | - |
NAD+ | recombinant His-tagged mutant A30D/R31I/T32I, pH 7.5, 50°C | Moorella thermoacetica | |
6 | - |
NAD+ | recombinant His-tagged mutant A30C/R31I/T32K, pH 7.5, 50°C | Moorella thermoacetica | |
6.23 | - |
NADP+ | recombinant His-tagged mutant A30C/R31I/T32K, pH 7.5, 50°C | Moorella thermoacetica | |
8.73 | - |
NADP+ | recombinant His-tagged wild-type enzyme, pH 7.5, 50°C | Moorella thermoacetica | |
9.12 | - |
NAD+ | recombinant His-tagged mutant R31T, pH 7.5, 50°C | Moorella thermoacetica | |
10.8 | - |
NAD+ | recombinant His-tagged mutant A30E/R31I/T32D, pH 7.5, 50°C | Moorella thermoacetica | |
10.83 | - |
NADP+ | recombinant His-tagged mutant R31T, pH 7.5, 50°C | Moorella thermoacetica | |
11.53 | - |
NADP+ | recombinant His-tagged mutant R31I, pH 7.5, 50°C | Moorella thermoacetica | |
11.9 | - |
NADP+ | recombinant His-tagged mutant R31I/T32G, pH 7.5, 50°C | Moorella thermoacetica | |
12.6 | - |
NAD+ | recombinant His-tagged wild-type enzyme, pH 7.5, 50°C | Moorella thermoacetica | |
12.94 | - |
NAD+ | recombinant His-tagged mutant R31I/T32G, pH 7.5, 50°C | Moorella thermoacetica | |
15.01 | - |
NAD+ | recombinant His-tagged mutant R31I, pH 7.5, 50°C | Moorella thermoacetica |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Moorella thermoacetica |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | active with enzyme mutants, not with the wild-type enzyme | Moorella thermoacetica | |
NADP+ | specific for, natural coenzyme is NADP+ | Moorella thermoacetica |
General Information | Comment | Organism |
---|---|---|
additional information | three-dimensional structure homology modeling of wild-type 6PGDH from Moorella thermoacetica and mutants based on the structure of human enzyme 6PGDH, PDB ID 2JKV | Moorella thermoacetica |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
4.7 | - |
NADP+ | recombinant His-tagged mutant A30E/R31I/T32D, pH 7.5, 50°C | Moorella thermoacetica | |
7.9 | - |
NADP+ | recombinant His-tagged mutant A30D/R31I/T32I, pH 7.5, 50°C | Moorella thermoacetica | |
8.9 | - |
NADP+ | recombinant His-tagged mutant A30C/R31I/T32K, pH 7.5, 50°C | Moorella thermoacetica | |
9 | - |
NAD+ | recombinant His-tagged wild-type enzyme, pH 7.5, 50°C | Moorella thermoacetica | |
14.8 | - |
NAD+ | recombinant His-tagged mutant A30C/R31I/T32K, pH 7.5, 50°C | Moorella thermoacetica | |
15 | - |
NAD+ | recombinant His-tagged mutant R31T, pH 7.5, 50°C | Moorella thermoacetica | |
35.8 | - |
NAD+ | recombinant His-tagged mutant R31I/T32G, pH 7.5, 50°C | Moorella thermoacetica | |
42.4 | - |
NAD+ | recombinant His-tagged mutant R31I, pH 7.5, 50°C | Moorella thermoacetica | |
85.1 | - |
NAD+ | recombinant His-tagged mutant A30E/R31I/T32D, pH 7.5, 50°C | Moorella thermoacetica | |
114 | - |
NADP+ | recombinant His-tagged mutant R31I/T32G, pH 7.5, 50°C | Moorella thermoacetica | |
305.6 | - |
NADP+ | recombinant His-tagged mutant R31T, pH 7.5, 50°C | Moorella thermoacetica | |
435 | - |
NADP+ | recombinant His-tagged mutant R31I, pH 7.5, 50°C | Moorella thermoacetica | |
484.2 | - |
NAD+ | recombinant His-tagged mutant A30D/R31I/T32I, pH 7.5, 50°C | Moorella thermoacetica | |
628.8 | - |
NADP+ | recombinant His-tagged wild-type enzyme, pH 7.5, 50°C | Moorella thermoacetica |