Cloned (Comment) | Organism |
---|---|
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain C41(DE3) | Archaeoglobus fulgidus |
Protein Variants | Comment | Organism |
---|---|---|
I135A | site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme | Archaeoglobus fulgidus |
I135G | site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme | Archaeoglobus fulgidus |
I135V | site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme | Archaeoglobus fulgidus |
additional information | pre-steady-state data with F420 cofactor and NADPH for the enzyme Fno mutant variants reveal biphasic kinetics with a fast and slow phase, similar with wild-type Fno, overview | Archaeoglobus fulgidus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | substrate binding studies, steady-state and pre steady-state kinetic analysis with wild-type enzyme Fno and Ile135 Fno mutant variants, I135A, I135V, and I135G, overview. Steady-state kinetic analysis of wild-type Fno and the variants show classical Michaelis-Menten kinetics with varying FO concentrations. The data reveal a decreased kcat as side chain length decreased, with varying FO concentrations. The steady-state plots reveal non-Michaelis-Menten kinetic behavior when NADPH is varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs420 versus time trace reveals biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displays an increased rate constant as side chain length decreases. The rate constant for the second phase, remained about 2/s for each variant. Pre-steady-state data with F420 cofactor and NADPH for the enzyme Fno mutant variants reveal biphasic kinetics with a fast and slow phase, similar with wild-type Fno, overview | Archaeoglobus fulgidus | |
0.00027 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant mutant I135A | Archaeoglobus fulgidus | |
0.0007 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant mutant I135V | Archaeoglobus fulgidus | |
0.0023 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant wild-type enzyme | Archaeoglobus fulgidus | |
0.0029 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant mutant I135A | Archaeoglobus fulgidus | |
0.0036 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135A | Archaeoglobus fulgidus | |
0.0036 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135G | Archaeoglobus fulgidus | |
0.0037 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135V | Archaeoglobus fulgidus | |
0.004 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant wild-type enzyme | Archaeoglobus fulgidus | |
0.016 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant mutant I135G | Archaeoglobus fulgidus | |
0.051 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant mutant I135V | Archaeoglobus fulgidus | |
0.062 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant wild-type enzyme | Archaeoglobus fulgidus | |
0.654 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant mutant I135G | Archaeoglobus fulgidus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
reduced coenzyme F420 + NADP+ | Archaeoglobus fulgidus | - |
oxidized coenzyme F420 + NADPH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Archaeoglobus fulgidus | O29370 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from cell-free extracts of Escherichia coli strain C41(DE3) by heat treatment at 90°C for 30 min, ammonium sulfate fractionation with addition of 0.05% polyethylenimine, followed by an ion exchange chromatography, ultrafiltration, and gel filtration | Archaeoglobus fulgidus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | effects of side chain length of residue Il135 on the donor-acceptor distance between NADP+ and the F420 precursor, FO, overview | Archaeoglobus fulgidus | ? | - |
? | |
reduced coenzyme F420 + NADP+ | - |
Archaeoglobus fulgidus | oxidized coenzyme F420 + NADPH + H+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
F420H2:NADP+ oxidoreductase | - |
Archaeoglobus fulgidus |
Fno | - |
Archaeoglobus fulgidus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at | Archaeoglobus fulgidus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.11 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant mutant I135G | Archaeoglobus fulgidus | |
0.33 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant mutant I135G | Archaeoglobus fulgidus | |
0.7 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135G | Archaeoglobus fulgidus | |
0.91 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant mutant I135A | Archaeoglobus fulgidus | |
1.24 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant mutant I135A | Archaeoglobus fulgidus | |
1.5 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant mutant I135V | Archaeoglobus fulgidus | |
1.6 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135A | Archaeoglobus fulgidus | |
1.8 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135V | Archaeoglobus fulgidus | |
2.16 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant mutant I135V | Archaeoglobus fulgidus | |
4.16 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant wild-type enzyme | Archaeoglobus fulgidus | |
5.3 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant wild-type enzyme | Archaeoglobus fulgidus | |
5.41 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant wild-type enzyme | Archaeoglobus fulgidus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.5 | - |
assay at | Archaeoglobus fulgidus |
General Information | Comment | Organism |
---|---|---|
physiological function | residue I135 plays a key role in sustaining the donor-acceptor distance between the two cofactor substrates, thereby regulating the rate at which the hydride is transferred from FOH2 to NADP+. Fno is a dynamic enzyme that regulates NADPH production | Archaeoglobus fulgidus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.5 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant mutant I135G | Archaeoglobus fulgidus | |
6.8 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant mutant I135G | Archaeoglobus fulgidus | |
42 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant mutant I135V | Archaeoglobus fulgidus | |
88 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant wild-type enzyme | Archaeoglobus fulgidus | |
194.4 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135G | Archaeoglobus fulgidus | |
420 | - |
NADPH | phase II, pH 6.5, 22°C, recombinant mutant I135A | Archaeoglobus fulgidus | |
444.4 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135A | Archaeoglobus fulgidus | |
486.5 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135V | Archaeoglobus fulgidus | |
1325 | - |
oxidized coenzyme F420 | with F420 precursor, FO, pH 6.5, 22°C, recombinant wild-type enzyme | Archaeoglobus fulgidus | |
1800 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant wild-type enzyme | Archaeoglobus fulgidus | |
2100 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant mutant I135V | Archaeoglobus fulgidus | |
3400 | - |
NADPH | phase I, pH 6.5, 22°C, recombinant mutant I135A | Archaeoglobus fulgidus |