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Literature summary 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)

EC Number Cloned (Comment) Organism
1.7.1.17 expression in Escherichia coli Escherichia coli
1.7.1.17 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

EC Number Inhibitors Comment Organism Structure
1.7.1.17 dicoumarol
-
 Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.7.1.17 additional information
-
 additional information steady-state kinetic analysis Escherichia coli
1.7.1.17 0.0179
-
 2-(4-dimethylaminophenyl)diazenylbenzoate recombinant enzyme, pH 7.4, 30°C Escherichia coli
1.7.1.17 0.018
-
 4'-(dimethylamino)-azobenzene-2-carboxylic acid pH 7.4, 30°C Escherichia coli
1.7.1.17 0.0316
-
 NADH recombinant enzyme, pH 7.4, 30°C Escherichia coli
1.7.1.17 0.032
-
 NADH pH 7.4, 30°C Escherichia coli

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.7.1.17 42000
-
 gel filtration Escherichia coli
1.7.1.17 42000
-
 gel fitlration Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.7.1.17 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

EC Number Organism UniProt Comment Textmining
1.7.1.17 Escherichia coli P41407
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.7.1.17
-
 Escherichia coli
1.7.1.17 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

EC Number Reaction Comment Organism Reaction ID
1.7.1.17 anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ = 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+ ping-pong reaction mechanism Escherichia coli
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.7.1.17 63.9
-
 pH 7.4, 30°C Escherichia coli
1.7.1.17 98.9
-
 purified recombinant enzyme, pH 7.4, 30°C, methyl red with FAD Escherichia coli
1.7.1.17 188.6
-
 purified recombinant enzyme, pH 7.4, 30°C, methyl red with FMN Escherichia coli
1.7.1.17 362.6
-
 purified recombinant enzyme, pH 7.4, 30°C, menadione with FMN Escherichia coli
1.7.1.17 441
-
 purified recombinant enzyme, pH 7.4, 30°C, ethyl red with FMN Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.7.1.17 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+
-
 ?
1.7.1.17 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+
-
 ?
1.7.1.17 ethyl red + 2 NADH + 2 H+
-
 Escherichia coli anthranilate + N,N-diethyl-1,4-phenylenediamine + 2 NAD+
-
 ?
1.7.1.17 menadione + 2 NADH + 2 H+
-
 Escherichia coli ? + 2 NAD+
-
 ?
1.7.1.17 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

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

Synonyms

EC Number Synonyms Comment Organism
1.7.1.17 acpD
-
 Escherichia coli
1.7.1.17 azoreductase
-
 Escherichia coli
1.7.1.17 FMN-dependent methyl red reductase
-
 Escherichia coli
1.7.1.17 FMN-dependent NADH-azoreductase
-
 Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.7.1.17 30
-
 assay at Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.7.1.17 7.4
-
 assay at Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
1.7.1.17 FAD can partially substitute for FMN Escherichia coli
1.7.1.17 FAD about 50% of the activity with FMN Escherichia coli
1.7.1.17 FMN
-
 Escherichia coli
1.7.1.17 FMN absolutely dependent on, no activity without FMN Escherichia coli
1.7.1.17 additional information no activity with NADPH Escherichia coli
1.7.1.17 additional information no cofactor: NADPH Escherichia coli
1.7.1.17 NADH
-
 Escherichia coli