Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes in Escherichia coli strain 0225 and BL21(DE3) | Anabaena sp. |
Crystallization (Comment) | Organism |
---|---|
purified recombinant mutant enzymes L263P, T155G/A160T, and T155G/A160T/L263P, hanging drop method, 0.002 ml of protein solution, containing 0.75 mM protein, 10 mM Tris-HCl, pH 8.0, plus 0.001 ml of unbuffered 5% w/v beta-octyl-glycoside solution, plus 0.002 ml reservoir solution, containing 18-20% w/v PEG 6000, 20 mM ammonium sulfate, 0.1 M MES-NaOH, pH 5.0, equilibration against 1 ml reservoir solution at 20°C, 1-7 days, phase separation caused by detergent, X-ray diffraction structure determination and analysis at 1.63-2.15 | Anabaena sp. |
Protein Variants | Comment | Organism |
---|---|---|
L263A | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
L263P | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
R224Q/R233L/Y235F | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
R233L/Y235F | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
S223D/R224Q/R233L/Y235F | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
S223D/R233L/Y235F | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
T155G | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
T155G/A160T | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
T155G/A160T/L263P | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
T155G/A160T/S223D/R224Q/R233L/Y235F | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
T155G/A160T/S223D/R224Q/R233L/Y235F/L263P | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
T155G/R224Q/R233L/Y235F | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
T155G/S223D/R224Q/R233L/Y235F | site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme | Anabaena sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics, wild-type and mutant enzymes | Anabaena sp. | |
0.006 | - |
NADPH | wild-type enzyme, pH 8.0 | Anabaena sp. | |
0.012 | - |
NADPH | mutant T155G/A160T/L263P, pH 8.0 | Anabaena sp. | |
0.015 | - |
NADPH | mutant L263A, pH 8.0 | Anabaena sp. | |
0.019 | - |
NADPH | mutant L263P, pH 8.0 | Anabaena sp. | |
0.022 | - |
NADPH | mutant T155G/A160T, pH 8.0 | Anabaena sp. | |
0.023 | - |
NADPH | mutant T155G, pH 8.0 | Anabaena sp. | |
0.18 | - |
NADH | mutant T155G, pH 8.0 | Anabaena sp. | |
0.39 | - |
NADH | mutant T155G/A160T/L263P, pH 8.0 | Anabaena sp. | |
0.51 | - |
NADH | mutant T155G/A160T, pH 8.0 | Anabaena sp. | |
0.63 | - |
NADH | mutant L263A, pH 8.0 | Anabaena sp. | |
0.65 | - |
NADH | mutant L263P, pH 8.0 | Anabaena sp. | |
0.8 | - |
NADH | wild-type enzyme, pH 8.0 | Anabaena sp. | |
1.5 | - |
NADH | mutant S223D/R233L/Y235F, pH 8.0 | Anabaena sp. | |
1.7 | - |
NADPH | mutant R233L/Y235F, pH 8.0 | Anabaena sp. | |
1.8 | - |
NADPH | above, mutant T155G/A160T/S223D/R224Q/R233L/Y235F/L263P, pH 8.0 | Anabaena sp. | |
2.3 | - |
NADH | mutant R233L/Y235F, pH 8.0 | Anabaena sp. | |
2.7 | - |
NADPH | mutant T155G/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
3 | - |
NADH | mutant T155G/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
3.6 | - |
NADPH | mutant R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
4.3 | - |
NADH | mutant R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
12 | - |
NADH | mutant T155G/A160T/S223D/R224Q/R233L/Y235F/L263P, pH 8.0 | Anabaena sp. |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Anabaena sp. | P21890 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli | Anabaena sp. |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
2 reduced ferredoxin + NADP+ + H+ = 2 oxidized ferredoxin + NADPH | coenzyme recognition and reaction mechanism | Anabaena sp. |
Source Tissue | Comment | Organism | Textmining |
---|
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
activity of wild-type and mutant enzymes with different cofactors | Anabaena sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | coenzyme binding causes structural rearrangements of the protein backbone | Anabaena sp. | ? | - |
? | |
NAD(P)H + H+ + oxidized 2,6-dichlorophenolindophenol | diaphorase activity, cofactor specificity, overview | Anabaena sp. | NAD(P)+ + reduced 2,6-dichlorophenolindophenol | - |
? | |
oxidized 2,6-dichlorophenolindophenol + NADH | - |
Anabaena sp. | NAD+ + reduced 2,6-dichlorophenolindophenol | - |
? |
Synonyms | Comment | Organism |
---|---|---|
ferredoxin-NADP+ reductase | - |
Anabaena sp. |
FNR | - |
Anabaena sp. |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.02 | - |
NADH | mutant T155G, pH 8.0 | Anabaena sp. | |
0.02 | - |
NADPH | mutant T155G/A160T/S223D/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
0.03 | - |
NADPH | mutant T155G/A160T/S223D/R224Q/R233L/Y235F/L263P, pH 8.0 | Anabaena sp. | |
0.04 | - |
NADH | mutant S223D/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
0.04 | - |
NADH | mutant T155G/A160T/S223D/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
0.05 | - |
NADH | mutant L263P, pH 8.0 | Anabaena sp. | |
0.05 | - |
NADPH | mutant S223D/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
0.06 | - |
NADPH | mutant T155G/S223D/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
0.06 | - |
NADH | mutant T155G/S223D/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
0.07 | - |
NADH | mutant T155G/A160T, pH 8.0 | Anabaena sp. | |
0.1 | - |
NADH | mutant S223D/R233L/Y235F, pH 8.0 | Anabaena sp. | |
0.13 | - |
NADH | mutant L263A, pH 8.0 | Anabaena sp. | |
0.16 | - |
NADH | wild-type enzyme, pH 8.0 | Anabaena sp. | |
0.2 | - |
NADPH | mutant T155G/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
0.28 | - |
NADH | mutant T155G/R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
0.33 | - |
NADH | mutant T155G/A160T/L263P, pH 8.0 | Anabaena sp. | |
0.86 | - |
NADH | mutant R233L/Y235F, pH 8.0 | Anabaena sp. | |
1.2 | - |
NADH | mutant R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
1.3 | - |
NADPH | mutant R224Q/R233L/Y235F, pH 8.0 | Anabaena sp. | |
2.1 | - |
NADH | mutant T155G/A160T/S223D/R224Q/R233L/Y235F/L263P, pH 8.0 | Anabaena sp. | |
17 | - |
NADPH | mutant L263P, pH 8.0 | Anabaena sp. | |
22 | - |
NADPH | mutant R233L/Y235F, pH 8.0 | Anabaena sp. | |
60 | - |
NADPH | mutant L263A, pH 8.0 | Anabaena sp. | |
72 | - |
NADPH | mutant T155G/A160T, pH 8.0 | Anabaena sp. | |
77 | - |
NADPH | mutant T155G/A160T/L263P, pH 8.0 | Anabaena sp. | |
81.5 | - |
NADPH | wild-type enzyme, pH 8.0 | Anabaena sp. | |
97 | - |
NADPH | mutant T155G, pH 8.0 | Anabaena sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Anabaena sp. |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | the coenzyme specificity determining structures are located in the 2'-phosphate NADP+ and pyrophosphate binding region of amino acid residues 155-160, 261-268, and S233, R224, R233, and Y235, coenzyme binding causes structural rearrangements of the protein backbone, binding and interaction mechanism with the enzyme, overview | Anabaena sp. | |
NADH | diaphorase activity | Anabaena sp. | |
NADPH | diaphorase activity | Anabaena sp. |