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Literature summary extracted from

  • Campbell, Z.T.; Baldwin, T.O.
    Fre Is the Major Flavin Reductase Supporting Bioluminescence from Vibrio harveyi Luciferase in Escherichia coli (2009), J. Biol. Chem., 284, 8322-8328.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.5.1.30 NADH
-
 Escherichia coli
1.5.1.30 NADPH
-
 Escherichia coli

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.30 PCR-amplification, expression in Escherichia coli Bl21 (deltaDE3) Escherichia coli
1.14.14.3 expressed in Escherichia coli from pJHD500, ligated into a pET21b vector. Luciferase subcloned from pZCH2 into a pASKIBA-3c vector with the restriction sites XbaI and XhoI. The resulting luciferase containing a strep-II tag on the C terminus of the beta-subunit (pZCB4) expressed Vibrio harveyi

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.30 additional information Fre oxidoreductase deletion strain shows 99% reduced bioluminescence in coupled assay with luciferase Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.30 0.0009
-
 FMN coupled oxidoreductase-luciferase assay, kcat/Km = 73/(M*min), in 100 mM Na+/K+ phosphate buffer with 100 mM NaCl, pH 7.0, 1 microM Fre oxidoreductase, 10 microM decanal, 10 microM NADPH, 5 microM luciferase Escherichia coli
1.5.1.30 0.0013
-
 riboflavin coupled oxidoreductase-luciferase assay, kcat/Km = 1257/(M*min), in 100 mM Na+/K+ phosphate buffer with 100 mM NaCl, pH 7.0, 1 microM Fre oxidoreductase, 10 microM decanal, 10 microM NADPH, 5 microM luciferase, riboflavin is favoured by Fre oxidoreductase but a poor substrate for bacterial luciferase Escherichia coli
1.14.14.3 0.0009
-
 FMN in the presence of different flavin concentrations, 0.001 mM Fre oxidoreductase, 10 mM decanal, and 0.01 mM NADPH and 0.005 mM luciferase Vibrio harveyi
1.14.14.3 0.0013
-
 riboflavin in the presence of different flavin concentrations, 0.001 mM Fre oxidoreductase, 10 mM decanal, and 0.01 mM NADPH and 0.005 mM luciferase Vibrio harveyi

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.14.3 K+
-
 Vibrio harveyi
1.14.14.3 Na+
-
 Vibrio harveyi

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.5.1.30 26000
-
 SDS-PAGE Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.30 FMN + NADH + H+ Escherichia coli
-
 FMNH2 + NAD+
-
 ?
1.5.1.30 FMN + NADPH Escherichia coli
-
 FMNH2 + NADP+ + H+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.30 Escherichia coli
-
-
-
1.14.14.3 Vibrio harveyi
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.30 crude lysate assay with briefly centrifuged cells, resuspended in B-PER bacterial protein extraction reagent, recombinant pZCFRE1 expression product is clarified by centrifugation and applied to a custom nickel affinity column, dialyzed into buffer containing 100 mM Na+/K+ phosphate and 100 mM NaCl, pH 7.0 Escherichia coli
1.14.14.3 nickel affinity column Vibrio harveyi

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.30 FMN + NADH + H+
-
 Escherichia coli FMNH2 + NAD+
-
 ?
1.5.1.30 FMN + NADPH
-
 Escherichia coli FMNH2 + NADP+ + H+
-
 ?
1.5.1.30 FMN + NADPH + H+
-
 Escherichia coli FMNH2 + NADP+
-
 ?
1.5.1.30 riboflavin + NADPH + H+
-
 Escherichia coli reduced riboflavin + NADP+
-
 ?
1.14.14.3 aldehyde + FMNH2 + O2
-
 Vibrio harveyi ?
-
 ?
1.14.14.3 decanal + FMNH + O2
-
 Vibrio harveyi decanoic acid + FMN + H2O + light
-
 ?
1.14.14.3 decanal + riboflavin + O2 riboflavin is a very poor substrate for bacterial luciferase Vibrio harveyi ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.5.1.30 monomer 1 * 26000 Escherichia coli

Synonyms

EC Number Synonyms Comment Organism
1.5.1.30 flavin oxidoreductase
-
 Escherichia coli
1.5.1.30 flavin reductase
-
 Escherichia coli
1.5.1.30 Fre oxidoreductase
-
 Escherichia coli
1.14.14.3 bacterial luciferase
-
 Vibrio harveyi

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.14.3 FMNH
-
 Vibrio harveyi
1.14.14.3 FMNH2
-
 Vibrio harveyi

General Information

EC Number General Information Comment Organism
1.5.1.30 physiological function provides reducing equivalents required to drive the luciferase genes in Escherichia coli but is not coupled to luciferase Escherichia coli
1.14.14.3 physiological function bacterial luciferase requires an exogenous source of reduced flavin mononucleotide for bioluminescence activity. There is no stable complex formation between luciferase and oxidoreductases Fre/NfsA from Escherichia coli or Frp from Vibrio harveyi, which are believed to provide reduced flavin for luciferase activity. No difference in the levels of luciferase expression in either the DELTAfre or DELTANsfA strains Vibrio harveyi

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.5.1.30 0.0012
-
 FMN coupled oxidoreductase-luciferase assay, in 100 mM Na+/K+ phosphate buffer with 100 mM NaCl, pH 7.0, 1 microM Fre oxidoreductase, 10 microM decanal, 10 microM NADPH, 5 microM luciferase Escherichia coli
1.5.1.30 0.021
-
 riboflavin coupled oxidoreductase-luciferase assay, in 100 mM Na+/K+ phosphate buffer with 100 mM NaCl, pH 7.0, 1 microM Fre oxidoreductase, 10 microM decanal, 10 microM NADPH, 5 microM luciferase, riboflavin is favoured by Fre oxidoreductase but a poor substrate for bacterial luciferase Escherichia coli