Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL21(DE3) cells | Corynebacterium ammoniagenes |
gene ribF, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Corynebacterium ammoniagenes |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme mutant R66A and R66E, mixing of equal volumes of 10 mg/ml protein in 20mMTris/HCl, pH 8.0, and 1 mM DTT, with reservoir solution containing 1.5 M Li2SO4, 0.1 M HEPES/NaOH, pH 7.5, X-ray diffraction structure determination and analysis, molecular replacment and modelling using the native CaFADS structure, PDB ID 2X0K, as search model | Corynebacterium ammoniagenes |
Protein Variants | Comment | Organism |
---|---|---|
R66A | inactive | Corynebacterium ammoniagenes |
R66A | site-directed mutagenesis, R66A CaFADS shows a considerable increase in the amount of oligomeric species | Corynebacterium ammoniagenes |
R66E | the mutant shows increased activity compared to the wild type enzyme | Corynebacterium ammoniagenes |
R66E | site-directed mutagenesis, R66E CaFADS shows a considerable increase in the amount of oligomeric species | Corynebacterium ammoniagenes |
R66X | point mutations at R66 have only mild effects on ligand binding and kinetic properties of the FMNAT-module (where R66 is located), but considerably impair the RFK activity turnover. Substitutions of R66 also modulate the ratio between monomeric and oligomeric species and modify the quaternary arrangement observed by single-molecule methods | Corynebacterium ammoniagenes |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten model and thermodynamic profiles of recombinant wild-type and mutant enzymes, overview | Corynebacterium ammoniagenes | |
0.00089 | - |
riboflavin | mutant enzyme R66E, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
0.00089 | - |
riboflavin | pH 7.0, 25°C, recombinant mutant R66E | Corynebacterium ammoniagenes | |
0.0117 | - |
riboflavin | pH 7.0, 25°C, recombinant wild-type enzyme | Corynebacterium ammoniagenes | |
0.0117 | - |
riboflavin | wild type enzyme, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
0.0215 | - |
ATP | mutant enzyme R66E, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
0.0215 | - |
ATP | pH 7.0, 25°C, recombinant mutant R66E | Corynebacterium ammoniagenes | |
0.0282 | - |
ATP | pH 7.0, 25°C, recombinant wild-type enzyme | Corynebacterium ammoniagenes | |
0.0282 | - |
ATP | wild type enzyme, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
0.087 | - |
ATP | pH 7.0, 25°C, recombinant mutant R66A | Corynebacterium ammoniagenes |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Corynebacterium ammoniagenes |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + riboflavin | Corynebacterium ammoniagenes | - |
ADP + FMN | - |
? | |
additional information | Corynebacterium ammoniagenes | bifunctional FAD synthetase exhibiting both the activities of FAD synthetase, EC 2.7.7.2, and riboflavin kinase, EC 2.7.1.26 | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Corynebacterium ammoniagenes | Q59263 | - |
- |
Purification (Comment) | Organism |
---|---|
ammonium sulfate precipitation, phenyl Sepharose column chromatography, and DEAE-cellulose column chromatography | Corynebacterium ammoniagenes |
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, hydrophobic interaction and ion exchange chromatography, followed by dialysis | Corynebacterium ammoniagenes |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + riboflavin | - |
Corynebacterium ammoniagenes | ADP + FMN | - |
? | |
additional information | bifunctional FAD synthetase exhibiting both the activities of FAD synthetase, EC 2.7.7.2, and riboflavin kinase, EC 2.7.1.26 | Corynebacterium ammoniagenes | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | - |
Corynebacterium ammoniagenes |
More | prokaryotic FAD synthetases (FADSs) are bifunctional enzymes composed of two modules, the C-terminal module with RFK activity, and the N-terminus with FMNAT activity | Corynebacterium ammoniagenes |
oligomer | the enzyme forms transient oligomers during catalysis stabilized by several interactions between the RFK and FMNAT sites from neighboring protomers, which otherwise are separated in themonomeric enzyme | Corynebacterium ammoniagenes |
Synonyms | Comment | Organism |
---|---|---|
ATP:riboflavin kinase | - |
Corynebacterium ammoniagenes |
FAD synthetase | - |
Corynebacterium ammoniagenes |
FADS | - |
Corynebacterium ammoniagenes |
RFK | - |
Corynebacterium ammoniagenes |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | 37 | assay at | Corynebacterium ammoniagenes |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.4 | - |
ATP | mutant enzyme R66E, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
0.5 | - |
ATP | pH 7.0, 25°C, recombinant mutant R66A | Corynebacterium ammoniagenes | |
0.5 | - |
riboflavin | pH 7.0, 25°C, recombinant mutant R66A | Corynebacterium ammoniagenes | |
0.7 | - |
riboflavin | mutant enzyme R66E, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
0.7 | - |
ATP | pH 7.0, 25°C, recombinant mutant R66E | Corynebacterium ammoniagenes | |
0.7 | - |
riboflavin | pH 7.0, 25°C, recombinant mutant R66E | Corynebacterium ammoniagenes | |
2.6 | - |
ATP | wild type enzyme, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
6.8 | - |
ATP | pH 7.0, 25°C, recombinant wild-type enzyme | Corynebacterium ammoniagenes | |
6.8 | - |
riboflavin | pH 7.0, 25°C, recombinant wild-type enzyme | Corynebacterium ammoniagenes | |
6.8 | - |
riboflavin | wild type enzyme, at pH 7.0 and 25°C | Corynebacterium ammoniagenes |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Corynebacterium ammoniagenes |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Corynebacterium ammoniagenes |
General Information | Comment | Organism |
---|---|---|
additional information | residue E268 is the catalytic base of the kinase reaction. The salt bridge between E268 at the RFK site and R66 at the FMNAT-module is important for the riboflavinkinase activity. Cross-talk between the RFK- and FMNAT-modules of neighboring protomers in the CaFADS enzyme, and participation of R66 in the modulation of the geometry of the RFK active site during catalysis | Corynebacterium ammoniagenes |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0758 | - |
riboflavin | pH 7.0, 25°C, recombinant mutant R66E | Corynebacterium ammoniagenes | |
0.142 | - |
riboflavin | pH 7.0, 25°C, recombinant mutant R66A | Corynebacterium ammoniagenes | |
18.3 | - |
ATP | mutant enzyme R66E, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
91.6 | - |
ATP | pH 7.0, 25°C, recombinant wild-type enzyme | Corynebacterium ammoniagenes | |
91.7 | - |
riboflavin | wild type enzyme, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
581.7 | - |
riboflavin | pH 7.0, 25°C, recombinant wild-type enzyme | Corynebacterium ammoniagenes | |
582 | - |
ATP | wild type enzyme, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
788 | - |
riboflavin | mutant enzyme R66E, at pH 7.0 and 25°C | Corynebacterium ammoniagenes | |
788.3 | - |
ATP | pH 7.0, 25°C, recombinant mutant R66E | Corynebacterium ammoniagenes |