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Literature summary for 1.5.1.36 extracted from

  • Groening, J.A.; Kaschabek, S.R.; Schloemann, M.; Tischler, D.
    A mechanistic study on SMOB-ADP1 an NADH flavin oxidoreductase of the two-component styrene monooxygenase of Acinetobacter baylyi ADP1 (2014), Arch. Microbiol., 196, 829-845 .
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
2-mercaptoethanol slightly activating Acinetobacter baylyi
DTT slightly activating Acinetobacter baylyi
EDTA
-
Acinetobacter baylyi
iodoacetamide slightly activating Acinetobacter baylyi
PMSF slightly activating Acinetobacter baylyi
styrene oxide slightly activating at 0.1 mM, inhibitory at 0.2 mM Acinetobacter baylyi
Thiourea slightly activating Acinetobacter baylyi

Cloned(Commentary)

Cloned (Comment) Organism
sequence comparisons and phylogenetic analysis, cloning in Escherichia coli strain DH5alpha, recombinant expression in Escherichia coli strain BL21(DE3) (pLysS) in inclusion bodies Acinetobacter baylyi

Inhibitors

Inhibitors Comment Organism Structure
Fe2+ complete inactivation at 0.1 mM, 38% activity remains in presence of 10 mM EDTA Acinetobacter baylyi
additional information poor inhibition by H2O2 Acinetobacter baylyi
Styrene
-
Acinetobacter baylyi
styrene oxide slightly activating at 0.1 mM, inhibitory at 0.2 mM Acinetobacter baylyi
thioanisole
-
Acinetobacter baylyi

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics Acinetobacter baylyi
0.0044
-
FAD pH 7.5, 25°C, recombinant enzyme, with NADH Acinetobacter baylyi
0.0055
-
FMN pH 7.5, 25°C, recombinant enzyme, with NADH Acinetobacter baylyi
0.011
-
riboflavin pH 7.5, 25°C, recombinant enzyme, with NADH Acinetobacter baylyi
0.024
-
NADH pH 7.5, 25°C, recombinant enzyme, with FAD Acinetobacter baylyi

Metals/Ions

Metals/Ions Comment Organism Structure
additional information no effect on enzyme activity by Cl- and NaN3 Acinetobacter baylyi

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
FAD + NADH Acinetobacter baylyi
-
FADH2 + NAD+
-
r

Organism

Organism UniProt Comment Textmining
Acinetobacter baylyi Q6F936
-
-

Purification (Commentary)

Purification (Comment) Organism
refolded recombinant enzyme by anion exchange chromatography and gel filtration, or by nickel affinity chromatography and gel filtration, when a His10-tagged enzyme is expressed Acinetobacter baylyi

Reaction

Reaction Comment Organism Reaction ID
reduced flavin + NAD+ = flavin + NADH + H+ FAD reduction by recombinant His10-tagged SMOB-ADP1 follows a random sequential mechanism Acinetobacter baylyi

Renatured (Commentary)

Renatured (Comment) Organism
refolding of recombinant enzyme from Escherichia coli strain BL21(DE3) (pLysS) inclusion bodies, with Triton X100 used in the washing buffer, refolding from solubilization buffer containing 8 M urea, 10 mM DTT, 50 mM Tris-HCl, pH 8.0, per mg pellet at 50°C for 30 min, refolding in a 20fold volume of refolding buffer.containing 50 mM Tris–HCl, pH 8.0, 240 mM NaCl, 10 mM KCl, 1 mM Na2EDTA, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, and 0.05 % w/v PEG 4000, followed by ultrafiltration Acinetobacter baylyi

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
FAD + NADH
-
Acinetobacter baylyi FADH2 + NAD+
-
r
FMN + NADH
-
Acinetobacter baylyi FMNH2 + NAD+
-
r
riboflavin + NADH
-
Acinetobacter baylyi reduced riboflavin + NAD+
-
r

Subunits

Subunits Comment Organism
homodimer 2 * 22665, His10-tagged enzyme, sequence calculation, 2 * 20000-25000, recombinant His10-tagged enzyme, SDS-PAGE, 2 * 18679, native enzyme, sequence calculation Acinetobacter baylyi

Synonyms

Synonyms Comment Organism
NAD(P)H:flavin oxidoreductase
-
Acinetobacter baylyi
NADH:flavin oxidoreductase
-
Acinetobacter baylyi
SMOB-ADP1
-
Acinetobacter baylyi

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25 26 assay at Acinetobacter baylyi

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
7.8
-
FMN pH 7.5, 25°C, recombinant enzyme, with NADH Acinetobacter baylyi
21
-
riboflavin pH 7.5, 25°C, recombinant enzyme, with NADH Acinetobacter baylyi
56
-
FAD pH 7.5, 25°C, recombinant enzyme, with NADH Acinetobacter baylyi
64
-
NADH pH 7.5, 25°C, recombinant enzyme, with FAD Acinetobacter baylyi

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Acinetobacter baylyi

pH Range

pH Minimum pH Maximum Comment Organism
6.3 7.5 activity irreversibly drops to under 5% at pH 5.3 indicating denaturation Acinetobacter baylyi

Cofactor

Cofactor Comment Organism Structure
additional information no activity with NADPH Acinetobacter baylyi
NADH
-
Acinetobacter baylyi

General Information

General Information Comment Organism
evolution SMOB-ADP1 belongs to the flavin reductases of the HpaC-like subfamily. NAD(P)H:flavin oxidoreductase structure comparisons, overview Acinetobacter baylyi

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0013
-
FAD pH 7.5, 25°C, recombinant enzyme, with NADH Acinetobacter baylyi
0.0014
-
FMN pH 7.5, 25°C, recombinant enzyme, with NADH Acinetobacter baylyi
0.0019
-
riboflavin pH 7.5, 25°C, recombinant enzyme, with NADH Acinetobacter baylyi
0.0027
-
NADH pH 7.5, 25°C, recombinant enzyme, with FAD Acinetobacter baylyi