Cloned (Comment) | Organism |
---|---|
cloning and sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) | Bacillus smithii |
Crystallization (Comment) | Organism |
---|---|
purified recombinant His-tagged wild-type enzyme in complex with FMN/N-cyclohexyl-2-aminoethanesulfonate (CHES) and Y151F enzyme mutant in complex with FMN, sitting drop vapor diffusion method, mixing of 0.002 ml of 42 mg/ml protein solution with an equal volume of reservoir solution containing 40% PEG 600 and 100 mM CHES, pH 9.5 for the wild-type, and 36% PEG 600 and 100 mM phosphate-citrate buffer, pH 4.2 for the mutant, equilibration against 0.1 m reservoir resolution, 20°C, X-ray diffraction structure determination and analysis at 1.97 and 1.95 A resolution respectively, molecular replacement method. Diffraction-quality crystals for the Y127F mutant cannot be obtained | Bacillus smithii |
Protein Variants | Comment | Organism |
---|---|---|
Y127F | site-directed mutagenesis, Diffraction-quality crystals for the Y127F mutant cannot be obtained | Bacillus smithii |
Y151F | site-directed mutagenesis, structure comparison with the wild-type enzyme | Bacillus smithii |
General Stability | Organism |
---|---|
the recombinant enzyme is an extremely stable indigo reductase | Bacillus smithii |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
48000 | - |
recombinant enzyme, gel filtration | Bacillus smithii |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
acid red 88 + NADH + H+ | Bacillus smithii | - |
? + NAD+ | - |
r | |
acid red 88 + NADH + H+ | Bacillus smithii DSM 4216 | - |
? + NAD+ | - |
r | |
Congo red + NADH + H+ | Bacillus smithii | - |
? + NAD+ | - |
r | |
Congo red + NADH + H+ | Bacillus smithii DSM 4216 | - |
? + NAD+ | - |
r | |
methyl orange + NADH + H+ | Bacillus smithii | - |
? + NAD+ | - |
r | |
methyl orange + NADH + H+ | Bacillus smithii DSM 4216 | - |
? + NAD+ | - |
r | |
methyl red + NADH + H+ | Bacillus smithii | - |
? + NAD+ | - |
r | |
methyl red + NADH + H+ | Bacillus smithii DSM 4216 | - |
? + NAD+ | - |
r | |
orange II + NADH + H+ | Bacillus smithii | - |
? + NAD+ | - |
r | |
orange II + NADH + H+ | Bacillus smithii DSM 4216 | - |
? + NAD+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus smithii | A0A0H4NXC5 | - |
- |
Bacillus smithii DSM 4216 | A0A0H4NXC5 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzyme from Escherichia coli strain BL21(DE3) by cobalt affinity chromatography, dialysis, and ultrafiltration | Bacillus smithii |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
additional information | Bacillus smithii strain DSM 4216 grows over a wide temperature range of 25-65°C | Bacillus smithii | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
acid red 88 + NADH + H+ | - |
Bacillus smithii | ? + NAD+ | - |
r | |
acid red 88 + NADH + H+ | - |
Bacillus smithii DSM 4216 | ? + NAD+ | - |
r | |
Congo red + NADH + H+ | - |
Bacillus smithii | ? + NAD+ | - |
r | |
Congo red + NADH + H+ | - |
Bacillus smithii DSM 4216 | ? + NAD+ | - |
r | |
methyl orange + NADH + H+ | - |
Bacillus smithii | ? + NAD+ | - |
r | |
methyl orange + NADH + H+ | - |
Bacillus smithii DSM 4216 | ? + NAD+ | - |
r | |
methyl red + NADH + H+ | - |
Bacillus smithii | ? + NAD+ | - |
r | |
methyl red + NADH + H+ | - |
Bacillus smithii DSM 4216 | ? + NAD+ | - |
r | |
orange II + NADH + H+ | - |
Bacillus smithii | ? + NAD+ | - |
r | |
orange II + NADH + H+ | - |
Bacillus smithii DSM 4216 | ? + NAD+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
homodimer | 2 * 23000, recombinant enzyme, SDS-PAGE | Bacillus smithii |
Synonyms | Comment | Organism |
---|---|---|
FMN-dependent NADH-azoreductase | - |
Bacillus smithii |
indigo reductase | - |
Bacillus smithii |
NADH-azoreductase | - |
Bacillus smithii |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
70 | - |
recombinant enzyme | Bacillus smithii |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
the recombinant enzyme is an extremely stable indigo reductase, analysis of the structure of this highly thermostable indigo reductase and identification of the key amino acid residues responsible for the thermostability | Bacillus smithii |
20 | 100 | purified recombinant wild-type enzyme, completely stable, while the mutant shows loss of 50% activity at about 55°C and inactivation at 90°C | Bacillus smithii |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
recombinant enzyme | Bacillus smithii |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FMN | FMN is present in the enzyme as a coenzyme and is not covalently bound to the enzyme | Bacillus smithii | |
NAD+ | - |
Bacillus smithii | |
NADH | - |
Bacillus smithii |
General Information | Comment | Organism |
---|---|---|
additional information | structure homology modeling. An indigo carmine molecule is modeled into the active site using the molecular docking simulation, and the binding mode of indigo carmine is elucidated. Residue F105 plays a major role in the intersubunit aromatic interaction. Crystal structure analysis, overview | Bacillus smithii |