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Literature summary for 1.1.1.44 extracted from

  • Huang, R.; Chen, H.; Zhong, C.; Kim, J.E.; Zhang, Y.H.
    High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP+ to NAD+ (2016), Sci. Rep., 6, 32644 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

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 [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

Metals/Ions Comment Organism Structure
Mg2+ required Moorella thermoacetica
Mn2+ required Moorella thermoacetica

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Moorella thermoacetica Q2RIZ2
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Moorella thermoacetica

Substrates and Products (Substrate)

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

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

Synonyms Comment Organism
6-phosphogluconate dehydrogenase
-
Moorella thermoacetica
6PGDH
-
Moorella thermoacetica
Moth_1283
-
Moorella thermoacetica

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
assay at Moorella thermoacetica

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Moorella thermoacetica

Cofactor

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

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 [mM/s]

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