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Literature summary for 2.7.1.26 extracted from
- Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes (2017), Sci. Rep., 7, 7281 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene ribF, recombinant expression of the isolated RF module | Corynebacterium ammoniagenes |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | isolation of the RFK module of CaFADS (DELTA(1-182)CaFADS). This truncated form of the enzyme has shown to perform the RFK activity with ligand binding profiles and strong substrate inhibition that are similar to those observed in the full-length bifunctional enzyme | Corynebacterium ammoniagenes |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| ADP | product inhibition, the ADP product acts as a competitive inhibitor | Corynebacterium ammoniagenes |  |
| ATP | substrate inhibition | Corynebacterium ammoniagenes | |
| FMN | product inhibition. FMN is not able to bind quickly enough to inhibit the free RFK module, but it behaves as an uncompetitive inhibitor that binds to the preformed ATP-enzyme complex and forms a highly stable dead-end complex. The phosphate group at the ribityl end in the FMN product might prevent the placement and enclosure of this cofactor. The FMN product acts as an uncompetitive inhibitor | Corynebacterium ammoniagenes | |
| additional information | a steady-state study shows the different levels and potency at which substrates and products of the RFK module inhibit its activity | Corynebacterium ammoniagenes | |
| riboflavin | RF, substrate inhibition, strong inhibition is observed in the RFK activity with increased substrate concentration | Corynebacterium ammoniagenes | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten steady-state kinetic study. While Km ATP values increase with the ADP concentration, kcat values remain constant. The high affinity for the ADP product inhibitor considerably increases the estimated error for KmATP. Association and dissociation kinetics of flavin ligands to the RFK module are measured by flavin fluorescence changes, stopped-flow kinetics. Pre-steady-state kinetic analysis of the binding of flavins to the RFK module of CaFADS, thermodynamic diagram for the RFK-ligand interactions. Adenine and flavin nucleotide ligands cooperate in their binding to the RFK module. Detailed kinetic analysis, overview | Corynebacterium ammoniagenes | |
| 0.0069 | - |
riboflavin | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics | Corynebacterium ammoniagenes | |
| 0.01 | - |
riboflavin | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics | Corynebacterium ammoniagenes | |
| 0.04 | - |
ATP | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics | Corynebacterium ammoniagenes | |
| 0.06 | - |
ATP | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics | Corynebacterium ammoniagenes | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | dependent on | Corynebacterium ammoniagenes | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + riboflavin | Corynebacterium ammoniagenes | - |
ADP + FMN | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Corynebacterium ammoniagenes | Q59263 | - |
- |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + riboflavin | - |
Corynebacterium ammoniagenes | ADP + FMN | - |
? | |
| additional information | riboflavin kinase activity of the FAD synthetase (EC 2.7.7.2) from Corynebacterium ammoniagenes. Adenine and flavin nucleotide ligands cooperate in their binding to the RFK module | Corynebacterium ammoniagenes | ? | - |
- |
|
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| More | the RFK module is formed by residues 187-338 and shows a globular shape with a beta-barrel formed by six antiparallel strands, a terminal alpha-helix that is perpendicular to the barrel and seven loops connecting them | Corynebacterium ammoniagenes |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| bifunctional riboflavin kinase/FMN adenylyltransferase | UniProt | Corynebacterium ammoniagenes |
| CaFADS | - |
Corynebacterium ammoniagenes |
| FADS | - |
Corynebacterium ammoniagenes |
| More | cf. EC 2.7.7.2 | Corynebacterium ammoniagenes |
| ribF | - |
Corynebacterium ammoniagenes |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | - |
assay at | Corynebacterium ammoniagenes |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 2.17 | - |
ATP | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics | Corynebacterium ammoniagenes | |
| 2.67 | - |
ATP | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics | Corynebacterium ammoniagenes | |
| 5.33 | - |
riboflavin | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics | Corynebacterium ammoniagenes | |
| 7.33 | - |
riboflavin | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics | Corynebacterium ammoniagenes | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7 | - |
assay at | Corynebacterium ammoniagenes |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | inhibition kinetics, dead-end inhibition by substrate excess | Corynebacterium ammoniagenes | |
| 0.0014 | - |
FMN | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics | Corynebacterium ammoniagenes | |
| 0.0019 | - |
riboflavin | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics | Corynebacterium ammoniagenes | |
| 0.017 | - |
ADP | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics | Corynebacterium ammoniagenes | |
| 2.67 | - |
ATP | pH 7.0, 25°C, recombinant RFK activity of the RFK module | Corynebacterium ammoniagenes | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| metabolism | biosynthesis of FMN and FAD from riboflavin (RF) involves two reactions: RF is first phosphorylated to FMN in an ATP-Mg2+-dependent reaction carried out by an ATP:riboflavin kinase (RFK), and then an FMN:ATP adenylyltransferase (FMNAT) transfers the adenylyl group from a second ATP molecule to FMN to yield FAD. In eukaryotes, these reactions are preferentially performed by two independent monofunctional enzymes, but in most prokaryotes, the two reactions are sequentially catalyzed by a bifunctional enzyme known as prokaryotic type I FAD synthetase (FADS). These bifunctional proteins are organized in two nearly independent modules with each one catalyzing one of the two activities | Corynebacterium ammoniagenes |
| additional information | adenine and flavin nucleotide ligands cooperate in their binding to the RFK module | Corynebacterium ammoniagenes |
| physiological function | Enzymes known as bifunctional and bimodular prokaryotic type-I FAD synthetase (FADS) exhibit ATP:riboflavin kinase (RFK) and FMN:ATP adenylyltransferase (FMNAT) activities in their C-terminal and N-terminal modules, respectively, and produce flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These act as cofactors of a plethora of flavoproteins in all organisms. Kinetics and thermodynamics of the protein-ligand interactions of riboflavin kinase activity of FAD synthetase from Corynebacterium ammoniagenes, overview. FMN synthesis is a key process that requires tight regulation. FMN production by CaFADS is highly regulated by substrate and product inhibition of its RFK activity to avoid overproduction even though the RF substrate might transiently increases in the media, where usually the amount of RF is considerably lower than that of FMN and particularly of FAD (either free or as part of flavoproteins) | Corynebacterium ammoniagenes |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 44.5 | - |
ATP | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics | Corynebacterium ammoniagenes | |
| 54.25 | - |
ATP | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics | Corynebacterium ammoniagenes | |
| 733 | - |
riboflavin | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics | Corynebacterium ammoniagenes | |
| 772.5 | - |
riboflavin | pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics | Corynebacterium ammoniagenes | |
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