Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Escherichia coli |
gene acpD, sequence comparisons, recombinant overexpression of the gene provides Escherichia coli strain JM109 with more than 800 times increased azoreductase activity | Escherichia coli |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
dicoumarol | - |
Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetic analysis | Escherichia coli | |
0.0179 | - |
2-(4-dimethylaminophenyl)diazenylbenzoate | recombinant enzyme, pH 7.4, 30°C | Escherichia coli | |
0.018 | - |
4'-(dimethylamino)-azobenzene-2-carboxylic acid | pH 7.4, 30°C | Escherichia coli | |
0.0316 | - |
NADH | recombinant enzyme, pH 7.4, 30°C | Escherichia coli | |
0.032 | - |
NADH | pH 7.4, 30°C | Escherichia coli |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
42000 | - |
gel filtration | Escherichia coli |
42000 | - |
gel fitlration | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+ | Escherichia coli | i.e. azo dye methyl red | anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P41407 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Escherichia coli |
native enzyme 4500fold by anion exchange chromatography, Blue Sepharose affinity chromtography, gel filtration, hydroxyapatite chromatography, again anion exchange chromatography and hydrophobic interaction chromatography. Recombinant enzyme AcpD from Escherichia coli, strain JM109 by anion exchange chromatography, Blue Sepharose affinity chromtography, gel filtration, and chromatography on GIGAPITE resin, the purified gene product exhibits activity corresponding to that of the native azoreductase | Escherichia coli |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ = 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+ | ping-pong reaction mechanism | Escherichia coli |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
63.9 | - |
pH 7.4, 30°C | Escherichia coli |
98.9 | - |
purified recombinant enzyme, pH 7.4, 30°C, methyl red with FAD | Escherichia coli |
188.6 | - |
purified recombinant enzyme, pH 7.4, 30°C, methyl red with FMN | Escherichia coli |
362.6 | - |
purified recombinant enzyme, pH 7.4, 30°C, menadione with FMN | Escherichia coli |
441 | - |
purified recombinant enzyme, pH 7.4, 30°C, ethyl red with FMN | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+ | i.e. azo dye methyl red | Escherichia coli | anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ | - |
? | |
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+ | i.e. methyl red | Escherichia coli | 2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+ | - |
? | |
ethyl red + 2 NADH + 2 H+ | - |
Escherichia coli | anthranilate + N,N-diethyl-1,4-phenylenediamine + 2 NAD+ | - |
? | |
menadione + 2 NADH + 2 H+ | - |
Escherichia coli | ? + 2 NAD+ | - |
? | |
additional information | no activity with Ponceau SX. Recombinant AcpD reaction follows a ping-pong mechanism requiring 2 mol of NADH to reduce 1 mol of methyl red into 2-aminobenzoic acid and N,N'-dimethyl-p-phenylenediamine. On the other hand, the gene product cannot convert holo-acyl carrier protein into the apo form under either in vitro or in vivo conditions. These data indicate that the acpD gene product is not acyl carrier protein phosphodiesterase but an azoreductase | Escherichia coli | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 23000, SDS-PAGE | Escherichia coli |
dimer | 2 * 23000, SDS-PAGE | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
acpD | - |
Escherichia coli |
azoreductase | - |
Escherichia coli |
FMN-dependent methyl red reductase | - |
Escherichia coli |
FMN-dependent NADH-azoreductase | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | can partially substitute for FMN | Escherichia coli | |
FAD | about 50% of the activity with FMN | Escherichia coli | |
FMN | - |
Escherichia coli | |
FMN | absolutely dependent on, no activity without FMN | Escherichia coli | |
additional information | no activity with NADPH | Escherichia coli | |
additional information | no cofactor: NADPH | Escherichia coli | |
NADH | - |
Escherichia coli |