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Literature summary for 1.7.1.17 extracted from

  • Valenzuela-Garcia, L.I.; Zapata, B.L.; Ramirez-Ramirez, N.; Huchin-Mian, J.P.; Robleto, E.A.; Ayala-Garcia, V.M.; Pedraza-Reyes, M.
    Novel biochemical properties and physiological role of the flavin mononucleotide oxidoreductase YhdA from Bacillus subtilis (2020), Appl. Environ. Microbiol., 86, e01688-20 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
cloning and sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Bacillus smithii

Crystallization (Commentary)

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

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

General Stability Organism
the recombinant enzyme is an extremely stable indigo reductase Bacillus smithii

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
48000
-
recombinant enzyme, gel filtration Bacillus smithii

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Bacillus smithii A0A0H4NXC5
-
-
Bacillus smithii DSM 4216 A0A0H4NXC5
-
-

Purification (Commentary)

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

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 and Products (Substrate)

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

Subunits Comment Organism
homodimer 2 * 23000, recombinant enzyme, SDS-PAGE Bacillus smithii

Synonyms

Synonyms Comment Organism
FMN-dependent NADH-azoreductase
-
Bacillus smithii
indigo reductase
-
Bacillus smithii
NADH-azoreductase
-
Bacillus smithii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
70
-
recombinant enzyme Bacillus smithii

Temperature Stability [°C]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
recombinant enzyme Bacillus smithii

Cofactor

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

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