Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Ensifer adhaerens |
Crystallization (Comment) | Organism |
---|---|
hanging drop vapor diffusion method, enzyme crystallized in complex with the cofactor NADP(H) and its structure is determined to 2.2 A resolution using selenomethionine single-wavelength anomalous dispersion | Ensifer adhaerens |
Protein Variants | Comment | Organism |
---|---|---|
A13G | kcat/Km for reaction with 1,5-anhydro-D-fructose and NADPH is 1.8fold higher than wild type value. Mutant enzyme shows activity with NADH as cofactor | Ensifer adhaerens |
G206I | kcat/Km for reaction with 1,5-anhydro-D-fructose and NADPH is 1.4fold lower than wild type value | Ensifer adhaerens |
H180A | kcat/Km for reaction with 1,5-anhydro-D-fructose and NADPH is 61.9fold lower than wild type value | Ensifer adhaerens |
K94G | kcat/Km for reaction with 1,5-anhydro-D-fructose and NADPH is 137fold lower than wild type value | Ensifer adhaerens |
S10G | kcat/Km for reaction with 1,5-anhydro-D-fructose and NADPH is 1.3fold higher than wild type value | Ensifer adhaerens |
S10G/A13G | kcat/Km for reaction with 1,5-anhydro-D-fructose and NADPH is 2fold higher than wild type value. Mutant enzyme shows activity with NADH as cofactor | Ensifer adhaerens |
S176A | kcat/Km for reaction with 1,5-anhydro-D-fructose and NADPH is 1001fold lower than wild type value | Ensifer adhaerens |
S33D | no activity | Ensifer adhaerens |
S33D/A13G | kcat/Km for reaction with 1,5-anhydro-D-fructose and NADPH is 84fold lower than wild type value. Mutant enzyme shows activity with NADH as cofactor | Ensifer adhaerens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.02 | - |
NADPH | pH 6.5, mutant enzyme A13G | Ensifer adhaerens | |
0.06 | - |
NADPH | pH 6.5 recombinant wild-type enzyme | Ensifer adhaerens | |
0.06 | - |
NADPH | pH 6.5, mutant enzyme G206I | Ensifer adhaerens | |
0.1 | - |
NADPH | pH 6.5, native wild-type enzyme | Ensifer adhaerens | |
0.2 | - |
NADPH | pH 6.5, mutant enzyme K94G | Ensifer adhaerens | |
0.27 | - |
NADPH | pH 6.5, mutant enzyme S10G | Ensifer adhaerens | |
0.38 | - |
NADPH | pH 6.5, mutant enzyme S10G/A13G | Ensifer adhaerens | |
1 | - |
NADPH | pH 6.5, mutant enzyme S33D/A13G | Ensifer adhaerens | |
1.1 | - |
NADH | pH 6.5, mutant enzyme A13G | Ensifer adhaerens | |
1.1 | - |
NADH | pH 6.5, mutant enzyme S33D/A13G | Ensifer adhaerens | |
1.1 | - |
NADH | mutant enzyme A13G/S33D, at pH 6.5 and 30°C | Ensifer adhaerens | |
1.2 | - |
NADH | pH 6.5, mutant enzyme S10G/A13G | Ensifer adhaerens | |
1.2 | - |
NADH | mutant enzyme A13G/S10G, at pH 6.5 and 30°C | Ensifer adhaerens | |
3.2 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S33D/A13G, cofactor: NADH | Ensifer adhaerens | |
3.5 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S10G, cofactor: NADPH | Ensifer adhaerens | |
6.4 | - |
1,5-Anhydro-D-fructose | pH 6.5 recombinant wild-type enzyme, cofactor: NADPH | Ensifer adhaerens | |
7.1 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S10G/A13G, cofactor: NADPH | Ensifer adhaerens | |
8.3 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme G206I, cofactor: NADPH | Ensifer adhaerens | |
8.3 | - |
1,5-Anhydro-D-fructose | pH 6.5, native wild-type enzyme, cofactor: NADPH | Ensifer adhaerens | |
8.5 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme A13G, cofactor: NADPH | Ensifer adhaerens | |
8.9 | - |
1,5-Anhydro-D-fructose | pH 8.0, mutant enzyme H180A, cofactor: NADPH | Ensifer adhaerens | |
11.1 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme A13G, cofactor: NADH | Ensifer adhaerens | |
20.2 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S33D/A13G, cofactor: NADPH | Ensifer adhaerens | |
22.5 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme K94G, cofactor: NADPH | Ensifer adhaerens | |
39 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S10G/A13G, cofactor: NADH | Ensifer adhaerens | |
49 | - |
1,5-Anhydro-D-fructose | pH 7.5, mutant enzyme D176A, cofactor: NADPH | Ensifer adhaerens |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Ensifer adhaerens | Q2I8V6 | i.e. Ensifer adhaerens | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
1,5-anhydro-D-fructose + NADH + H+ | - |
Ensifer adhaerens | 1,5-anhydro-D-mannitol + NAD+ | - |
r | |
1,5-anhydro-D-fructose + NADPH + H+ | - |
Ensifer adhaerens | 1,5-anhydro-D-mannitol + NADP+ | - |
? |
Synonyms | Comment | Organism |
---|---|---|
1,5-anhydro-D-fructose reductase | - |
Ensifer adhaerens |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.3 | - |
1,5-Anhydro-D-fructose | pH 7.5, mutant enzyme D176A, cofactor: NADPH | Ensifer adhaerens | |
3.7 | - |
1,5-Anhydro-D-fructose | pH 8.0, mutant enzyme H180A, cofactor: NADPH | Ensifer adhaerens | |
4.2 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme K94G, cofactor: NADPH | Ensifer adhaerens | |
5.5 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S10G/A13G, cofactor: NADH | Ensifer adhaerens | |
6.3 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S33D/A13G, cofactor: NADPH | Ensifer adhaerens | |
12.4 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme A13G, cofactor: NADH | Ensifer adhaerens | |
13.5 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S33D/A13G, cofactor: NADH | Ensifer adhaerens | |
119 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S10G, cofactor: NADPH | Ensifer adhaerens | |
145 | - |
1,5-Anhydro-D-fructose | pH 6.5 recombinant wild-type enzyme, cofactor: NADPH | Ensifer adhaerens | |
156 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme G206I, cofactor: NADPH | Ensifer adhaerens | |
216 | - |
1,5-Anhydro-D-fructose | pH 6.5, native wild-type enzyme, cofactor: NADPH | Ensifer adhaerens | |
369 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme S10G/A13G, cofactor: NADPH | Ensifer adhaerens | |
405 | - |
1,5-Anhydro-D-fructose | pH 6.5, mutant enzyme A13G, cofactor: NADPH | Ensifer adhaerens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADH | wild-type enzyme and mutant enzymes S10G, S33D, K94G, D176A, H180A and G206I shows no activity with NADH. Mutant enzymes A13G, S10G/A13G and S33D/A13G are active with NADH | Ensifer adhaerens | |
NADPH | the N-terminal domain displays a Rossman fold and contains the cofactor binding site. The intact crystals contain the oxidized cofactor NADP+ | Ensifer adhaerens |