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Literature summary for 1.7.1.17 extracted from

  • Nakanishi, M.; Yatome, C.; Ishida, N.; Kitade, Y.
    Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase (2001), J. Biol. Chem., 276, 46394-46399 .
No PubMed abstract available

Cloned(Commentary)

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

Inhibitors Comment Organism Structure
dicoumarol
-
Escherichia coli

KM Value [mM]

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 [Da] Molecular Weight Maximum [Da] Comment Organism
42000
-
gel filtration Escherichia coli
42000
-
gel fitlration Escherichia coli

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Escherichia coli P41407
-
-

Purification (Commentary)

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

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 [micromol/min/mg]

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 and Products (Substrate)

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

Subunits Comment Organism
? x * 23000, SDS-PAGE Escherichia coli
dimer 2 * 23000, SDS-PAGE Escherichia coli

Synonyms

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 [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
assay at Escherichia coli

Cofactor

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