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Literature summary for 2.7.1.26 extracted from

  • Serrano, A.; Sebastian, M.; Arilla-Luna, S.; Baquedano, S.; Pallares, M.C.; Lostao, A.; Herguedas, B.; Velazquez-Campoy, A.; Martinez-Julvez, M.; Medina, M.
    Quaternary organization in a bifunctional prokaryotic FAD synthetase: Involvement of an arginine at its adenylyltransferase module on the riboflavin kinase activity (2015), Biochim. Biophys. Acta, 1854, 897-906.
    View publication on PubMed
Search for more literature for 2.7.1.26

Cloned(Commentary)

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 (Commentary)

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

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 [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

Metals/Ions Comment Organism Structure
Mg2+ required 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
-
 ?
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

Organism UniProt Comment Textmining
Corynebacterium ammoniagenes Q59263
-
-

Purification (Commentary)

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 and Products (Substrate)

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

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

Synonyms Comment Organism
ATP:riboflavin kinase
-
 Corynebacterium ammoniagenes
FAD synthetase
-
 Corynebacterium ammoniagenes
FADS
-
 Corynebacterium ammoniagenes
RFK
-
 Corynebacterium ammoniagenes

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25 37 assay at Corynebacterium ammoniagenes

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Corynebacterium ammoniagenes

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Corynebacterium ammoniagenes

General Information

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 [mM/s]

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