Any feedback?
Literature summary for 1.5.1.39 extracted from
- Expression, biochemical characterization, and mutation of a water forming NADH FMN oxidoreductase from Lactobacillus rhamnosus (2020), Enzyme Microb. Technol., 134, 109464 .
Application
| Application | Comment | Organism |
|---|---|---|
| biotechnology | enzyme-catalyzed cofactor regeneration is a significant approach to avoid large quantities consumption of oxidized cofactor, which is vital in a variety of bioconversion reactions. NADH: FMN oxidoreductase is an ideal regenerating enzyme because innocuous molecular oxygen is required as an oxidant. But the by-product H2O2 limits its further applications at the industrial scale, therefore, mutants with improved features are constructed | Lacticaseibacillus rhamnosus |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene LrFOR, recombinant overexpression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Lacticaseibacillus rhamnosus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| G298A | site-directed mutagenesis, inactive mutant | Lacticaseibacillus rhamnosus |
| H170A | site-directed mutagenesis, inactive mutant | Lacticaseibacillus rhamnosus |
| H319A | site-directed mutagenesis, inactive mutant | Lacticaseibacillus rhamnosus |
| M28A | site-directed mutagenesis, inactive mutant | Lacticaseibacillus rhamnosus |
| additional information | a small size or electronegative of residue in position 29 shortens the distance of NADH and FMN, promoting the electrons transfer and resulting in the increased activity | Lacticaseibacillus rhamnosus |
| N173A | site-directed mutagenesis, the mutant shows 66% reduced activity compared to wild-type enzyme | Lacticaseibacillus rhamnosus |
| T29A | site-directed mutagenesis, the mutant shows 2.91fold increased activity compared to the wild-type enzyme | Lacticaseibacillus rhamnosus |
| T29D | site-directed mutagenesis, the mutant shows 3.9fold increased activity compared to the wild-type enzyme | Lacticaseibacillus rhamnosus |
| T29G | site-directed mutagenesis, the mutant shows 3.7fold increased activity compared to the wild-type enzyme | Lacticaseibacillus rhamnosus |
| T29N | site-directed mutagenesis, the mutant shows 2.2fold increased activity compared to the wild-type enzyme | Lacticaseibacillus rhamnosus |
| T29R | site-directed mutagenesis, the mutant shows 90% reduced activity compared to wild-type enzyme | Lacticaseibacillus rhamnosus |
| T29Y | site-directed mutagenesis, the mutant shows 40% reduced activity compared to wild-type enzyme | Lacticaseibacillus rhamnosus |
| Y341A | site-directed mutagenesis, inactive mutant | Lacticaseibacillus rhamnosus |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| Co2+ | slight inhibition | Lacticaseibacillus rhamnosus |  |
| Fe3+ | slight inhibition | Lacticaseibacillus rhamnosus | |
| Mg2+ | slight inhibition | Lacticaseibacillus rhamnosus | |
| Triton X-10 | slight inhibition | Lacticaseibacillus rhamnosus | |
| Tween 80 | slight inhibition | Lacticaseibacillus rhamnosus | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten steady-state kinetics. The kinetic parameters show that the Km values of mutants T29A, T29G, and T29D are similar to that of wild-type enzyme with 0.0882 mM but the Km of mutants are increased. Also, the Vmax of mutant T29C and T29S are increased compared to wild-type, while the T29R mutant shows a significant decrease in Vmax | Lacticaseibacillus rhamnosus | |
| 0.0882 | - |
NADH | recombinant wild-type enzyme, pH 5.5, 35°C | Lacticaseibacillus rhamnosus | |
| 0.1394 | - |
NADH | recombinant mutant T29Y, pH 5.5, 35°C | Lacticaseibacillus rhamnosus | |
| 0.179 | - |
NADH | recombinant mutant T29N, pH 5.5, 35°C | Lacticaseibacillus rhamnosus | |
| 0.2579 | - |
NADH | recombinant mutant T29R, pH 5.5, 35°C | Lacticaseibacillus rhamnosus | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| additional information | Li+ and EDTA have a weak effect on enzyme activity | Lacticaseibacillus rhamnosus |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 42000 | - |
recombinant His-tagged enzyme, gel filtration | Lacticaseibacillus rhamnosus |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| FMN + NADH + H+ | Lacticaseibacillus rhamnosus | - |
FMNH2 + NAD+ | - |
? | |
| FMN + NADH + H+ | Lacticaseibacillus rhamnosus ATCC 53103 | - |
FMNH2 + NAD+ | - |
? | |
| FMN + NADPH + H+ | Lacticaseibacillus rhamnosus | - |
FMNH2 + NADP+ | - |
? | |
| FMN + NADPH + H+ | Lacticaseibacillus rhamnosus ATCC 53103 | - |
FMNH2 + NADP+ | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Lacticaseibacillus rhamnosus | - |
- |
- |
| Lacticaseibacillus rhamnosus ATCC 53103 | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Lacticaseibacillus rhamnosus |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| FMNH2 + NAD(P)+ = FMN + NAD(P)H + H+ | the enzyme FOR can catalyze the oxidation of NADH to NAD+ with the flavin mononucleotide (FMN) functions as the prosthetic group. In the first step, a semiquinone intermediate (FMNH) is formed by the transfer of the hydride from the nicotinamide group of NADH to the N5 in the isoalloxazine moiety of the oxidized FMN. Then, a proton transfer to the N atom near the ribitol moiety of FMNH which may result in the formation of FMNH2. Eventually, the reduced FMNH2 is oxidized to FMN by O2 molecules | Lacticaseibacillus rhamnosus |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 39.8 | - |
purified recombinant His-tagged enzyme, pH 5.5, 35°C | Lacticaseibacillus rhamnosus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| FMN + NADH + H+ | - |
Lacticaseibacillus rhamnosus | FMNH2 + NAD+ | - |
? | |
| FMN + NADH + H+ | - |
Lacticaseibacillus rhamnosus ATCC 53103 | FMNH2 + NAD+ | - |
? | |
| FMN + NADPH + H+ | - |
Lacticaseibacillus rhamnosus | FMNH2 + NADP+ | - |
? | |
| FMN + NADPH + H+ | - |
Lacticaseibacillus rhamnosus ATCC 53103 | FMNH2 + NADP+ | - |
? | |
| additional information | the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. The optimum FMN concentration for the LrFOR is 0.015 mM. With increasing of FMN concentration from 0.001 to 0.015 mM, the relative activity of LrFOR is also increased from 42% to 100%. When the concentration of FMN is increased to 0.030 mM, LrFOR displays more than 90% of the optimal activity. And the activity retains more than 80% of the optimal value when the FMN concentration is 0.070 mM | Lacticaseibacillus rhamnosus | ? | - |
- |
|
| additional information | the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. The optimum FMN concentration for the LrFOR is 0.015 mM. With increasing of FMN concentration from 0.001 to 0.015 mM, the relative activity of LrFOR is also increased from 42% to 100%. When the concentration of FMN is increased to 0.030 mM, LrFOR displays more than 90% of the optimal activity. And the activity retains more than 80% of the optimal value when the FMN concentration is 0.070 mM | Lacticaseibacillus rhamnosus ATCC 53103 | ? | - |
- |
|
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| monomer | 1 * 42000, recombinant His-tagged enzyme, SDS-PAGE | Lacticaseibacillus rhamnosus |
| More | the conserved protein fold of LrFOR is comprised of about eight alpha-helices and eight parallel beta-strands that alternate along the peptide backbones (A (beta/alpha) 8 barrel) | Lacticaseibacillus rhamnosus |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| H2O-forming FOR | - |
Lacticaseibacillus rhamnosus |
| LrFOR | - |
Lacticaseibacillus rhamnosus |
| NADH: FMN oxidoreductase | - |
Lacticaseibacillus rhamnosus |
| water forming NADH: FMN oxidoreductase | - |
Lacticaseibacillus rhamnosus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 35 | - |
recombinant enzyme | Lacticaseibacillus rhamnosus |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | 70 | recombinant enzyme, activity range | Lacticaseibacillus rhamnosus |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | 55 | the enzyme is stable at 25°C and 35°C with no obvious decline in activity after 2.5 h. 70.0% residual activity after 2.5 h at 45°C, 5.4% residual activity after 2.5 h at 55°C. The half-lives at 45°C and 55°C are 273.9 min and 17.3 min, respectively | Lacticaseibacillus rhamnosus |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 5.5 | - |
recombinant enzyme | Lacticaseibacillus rhamnosus |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 5.5 | 8 | optimum pH for the oxidation of NADH is pH 5.5, whereas the enzyme shows 87.5% and 86.7% of maximum activity at pH 6.0 and 6.5, respectively. 69.3% residual activity is measured at pH 7.5, 36.8% at pH 8.0 | Lacticaseibacillus rhamnosus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| additional information | the purified recombinant LrFOR has both the NADPH and NADH oxidation activity | Lacticaseibacillus rhamnosus | |
| NADH | NADH is docked into docking into the substrate-binding site of wild-type LrFOR, preferred cofactor | Lacticaseibacillus rhamnosus | |
| NADPH | less active than NADH | Lacticaseibacillus rhamnosus | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | NADH: FMN oxidoreductases (FOR) are old yellow enzyme members with a (beta/alpha) 8-barrel structure and can catalyze the oxidation of NADH to NAD+ with the flavin mononucleotide (FMN) functions as the prosthetic group | Lacticaseibacillus rhamnosus |
| additional information | enzyme structure homology modeling and docking using the structure of Thermoanaerobacter pseudethanolicus strain E39 enzyme (PDB ID 3KRZ) as the template, overview. Molecular dynamic simulation | Lacticaseibacillus rhamnosus |
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
