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Information on EC 2.7.1.26 - riboflavin kinase and Organism(s) Corynebacterium ammoniagenes and UniProt Accession Q59263
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2.7.1.26 riboflavin kinaseIUBMB Comments
The cofactors FMN and FAD participate in numerous processes in all organisms, including mitochondrial electron transport, photosynthesis, fatty-acid oxidation, and metabolism of vitamin B6, vitamin B12 and folates . While monofunctional riboflavin kinase is found in eukaryotes, some bacteria have a bifunctional enzyme that exhibits both this activity and that of EC 2.7.7.2, FMN adenylyltransferase . A divalent metal cation is required for activity (with different species preferring Mg2+, Mn2+ or Zn2+). In Bacillus subtilis, ATP can be replaced by other phosphate donors but with decreasing enzyme activity in the order ATP > dATP > CTP > UTP .
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Word Map
- 2.7.1.26
- mononucleotide
- ammoniagenes
-
fadss
- isoalloxazine
-
kyphoplasty
-
flavocoenzyme
-
flavinogenic
-
ribityl
- davawensis
- roseoflavin
- famata
-
ctp-dependent
- synthesis
- drug development
The taxonomic range for the selected organisms is: Corynebacterium ammoniagenes
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
rfk, flavokinase, riboflavin kinase, fad synthetase, fmnat, cafads, fmn adenylyltransferase, hsrfk, atfmn/fhy, flavokinase/fad synthetase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
bifunctional riboflavin kinase/FMN adenylyltransferase
UniProt
FAD synthetase
FK
-
-
-
-
flavokinase
-
-
-
-
kinase, riboflavin
-
-
-
-
riboflavin kinase/FMN adenylyltransferase
-
FADS, bifunctional enzyme, the C-terminus exhibits ATP: riboflavin kinase activity
riboflavine kinase
-
-
-
-
FAD synthetase
bifunctional enzyme, the C-terminal is associated with ATP:riboflavin kinase activity and the N-terminal associated with ATP:FMN adenylyltransferase activity
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
phospho group transfer
-
-
-
-
PATHWAY SOURCE
PATHWAYS
BRENDA
-
-, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
ATP:riboflavin 5'-phosphotransferase
The cofactors FMN and FAD participate in numerous processes in all organisms, including mitochondrial electron transport, photosynthesis, fatty-acid oxidation, and metabolism of vitamin B6, vitamin B12 and folates [5]. While monofunctional riboflavin kinase is found in eukaryotes, some bacteria have a bifunctional enzyme that exhibits both this activity and that of EC 2.7.7.2, FMN adenylyltransferase [5]. A divalent metal cation is required for activity (with different species preferring Mg2+, Mn2+ or Zn2+). In Bacillus subtilis, ATP can be replaced by other phosphate donors but with decreasing enzyme activity in the order ATP > dATP > CTP > UTP [6].
CAS REGISTRY NUMBER
COMMENTARY
9032-82-0
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + 5-deazariboflavin
ADP + 5-deazariboflavin monophosphate
-
-
-
?
ATP + 7,8-dichlororiboflavin
ADP + 7,8-dichlororiboflavin monophosphate
-
-
-
?
ATP + 8-aminoriboflavin
ADP + 8-aminoriboflavin monophosphate
-
-
-
?
2'-dATP + riboflavin
2'-dADP + riboflavin 5'-phosphate
-
-
-
-
?
ATP + 3-deazariboflavin
ADP + 3-deazariboflavin 5'-phosphate
-
-
-
-
?
ATP + 5-deazariboflavin
ADP + 5-deazariboflavin 5'-phosphate
-
-
-
-
?
ATP + 5-methyl-5-deazariboflavin
ADP + 5-methyl-5-deazariboflavin 5'-phosphate
-
-
-
-
?
ATP + 6-methylriboflavin
ADP + 6-methylriboflavin 5'-phosphate
-
-
-
-
?
ATP + 9-azariboflavin
ADP + 9-azariboflavin 5'-phosphate
-
-
-
-
?
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
-
-
?
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
-
-
?
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
-
-
?
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
-
-
?
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
-
-
?
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
-
-
?
additional information
?
-
the engineered FAD synthetase from Corynebacterium ammoniagenes with deleted N-terminal adenylation domain is a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale, method evaluation, overview
-
-
?
additional information
?
-
-
the engineered FAD synthetase from Corynebacterium ammoniagenes with deleted N-terminal adenylation domain is a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale, method evaluation, overview
-
-
?
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
-
-
-
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
-
-
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
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
-
-
?
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
-
-
?
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
-
-
?
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
-
-
?
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
-
-
?
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
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ATP
the ADP/ATP-binding pocket is closed in the ligand-free structure, structure overview. The ADP molecule establishes hydrogen bonds to residues located in L1c-FlapI: Gly196, Gly198 and Gly201 interact with the alpha- and beta-phosphates of ADP, whereas Pro207 and Thr208 (both belonging to the PTAN motif) form several hydrogen bonds via side-chain and main-chain atoms
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ADP
product inhibition, the ADP product acts as a competitive inhibitor
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
riboflavin
RF, substrate inhibition, strong inhibition is observed in the RFK activity with increased substrate concentration
additional information
a steady-state study shows the different levels and potency at which substrates and products of the RFK module inhibit its activity
-
additional information
-
a steady-state study shows the different levels and potency at which substrates and products of the RFK module inhibit its activity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.01
ATP
mutant enzyme S164D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.011
ATP
mutant enzyme R161D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.011
ATP
mutant enzyme T165D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.012
ATP
mutant enzyme H28D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.012
ATP
mutant enzyme R161A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.012
ATP
mutant enzyme S164A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.012
ATP
mutant enzyme T165A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.014
ATP
mutant enzyme H28A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.014
ATP
mutant enzyme H31A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.014
ATP
wild type enzyme, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.024
ATP
mutant enzyme N125A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.035
ATP
mutant enzyme N125D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.04
ATP
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics
0.06
ATP
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics
0.00089
riboflavin
mutant enzyme R66E, at pH 7.0 and 25°C
0.0017
riboflavin
mutant enzyme N125A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0045
riboflavin
mutant enzyme H31A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0056
riboflavin
mutant enzyme S164D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0069
riboflavin
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics
0.0077
riboflavin
mutant enzyme T165D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0088
riboflavin
mutant enzyme S164A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.01
riboflavin
mutant enzyme T165A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.01
riboflavin
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics
0.0117
riboflavin
pH 7.0, 25°C, recombinant wild-type enzyme
0.012
riboflavin
mutant enzyme R161A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.013
riboflavin
mutant enzyme R161D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.013
riboflavin
wild type enzyme, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.016
riboflavin
mutant enzyme N125D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.023
riboflavin
mutant enzyme H28A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.025
riboflavin
mutant enzyme H28D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0137
ATP
-
apparent value, wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
0.0176
ATP
-
apparent value, mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
0.0453
ATP
-
apparent value, mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
0.0021
riboflavin
-
apparent value, mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
0.0041
riboflavin
-
apparent value, mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
0.013
riboflavin
-
apparent value, wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
additional information
additional information
bi-substrate enzyme kinetics of the recombinant DELTA(1-182)CaFADS module
-
additional information
additional information
-
bi-substrate enzyme kinetics of the recombinant DELTA(1-182)CaFADS module
-
additional information
additional information
Michaelis-Menten model and thermodynamic profiles of recombinant wild-type and mutant enzymes, overview
-
additional information
additional information
-
Michaelis-Menten model and thermodynamic profiles of recombinant wild-type and mutant enzymes, overview
-
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
-
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
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.75
ATP
mutant enzyme H28A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.75
ATP
mutant enzyme T165D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.77
ATP
mutant enzyme N125A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.92
ATP
mutant enzyme H28D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.92
ATP
mutant enzyme S164D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.97
ATP
mutant enzyme H31A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.98
ATP
mutant enzyme S164A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
1.08
ATP
mutant enzyme R161A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
1.1
ATP
mutant enzyme T165A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
1.13
ATP
mutant enzyme R161D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
1.13
ATP
wild type enzyme, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
1.33
ATP
mutant enzyme N125D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
2.17
ATP
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics
2.67
ATP
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics
1.85
riboflavin
mutant enzyme N125A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
2.82
riboflavin
mutant enzyme H31A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
2.85
riboflavin
mutant enzyme T165A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
3
riboflavin
mutant enzyme T165D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
3.08
riboflavin
mutant enzyme S164D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
4.32
riboflavin
mutant enzyme S164A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
4.78
riboflavin
mutant enzyme H28D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
5
riboflavin
mutant enzyme R161A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
5
riboflavin
mutant enzyme R161D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
5.02
riboflavin
wild type enzyme, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
5.33
riboflavin
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics
6.8
riboflavin
pH 7.0, 25°C, recombinant wild-type enzyme
6.92
riboflavin
mutant enzyme N125D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
7.12
riboflavin
mutant enzyme H28A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
7.33
riboflavin
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics
0.042
ATP
-
apparent value, mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
0.07
ATP
-
apparent value, mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
1.13
ATP
-
apparent value, wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
0.045
riboflavin
-
apparent value, mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
0.085
riboflavin
-
apparent value, mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
5
riboflavin
-
apparent value, wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
31.7
ATP
mutant enzyme N125A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
38.3
ATP
mutant enzyme N125D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
44.5
ATP
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics
51.7
ATP
mutant enzyme H28A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
54.25
ATP
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics
68.3
ATP
mutant enzyme T165D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
71.7
ATP
mutant enzyme H31A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
78.3
ATP
mutant enzyme H28D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
81.7
ATP
wild type enzyme, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
83.3
ATP
mutant enzyme S164A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
88.3
ATP
mutant enzyme S164D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
90
ATP
mutant enzyme T165A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
93.3
ATP
mutant enzyme R161A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
101.7
ATP
mutant enzyme R161D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
200
riboflavin
mutant enzyme H28D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
283
riboflavin
mutant enzyme T165A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
300
riboflavin
mutant enzyme H28A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
383
riboflavin
mutant enzyme R161D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
383
riboflavin
mutant enzyme T165D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
383
riboflavin
wild type enzyme, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
417
riboflavin
mutant enzyme R161A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
433
riboflavin
mutant enzyme N125D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
483
riboflavin
mutant enzyme S164A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
550
riboflavin
mutant enzyme S164D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
581.7
riboflavin
pH 7.0, 25°C, recombinant wild-type enzyme
617
riboflavin
mutant enzyme H31A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
733
riboflavin
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics
772.5
riboflavin
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics
1083
riboflavin
mutant enzyme N125A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.83
ATP
-
apparent value, mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
3.83
ATP
-
apparent value, mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
82.17
ATP
-
apparent value, wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
10.83
riboflavin
-
apparent value, mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
40.3
riboflavin
-
apparent value, mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
386
riboflavin
-
apparent value, wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 0.8 mM MgCl2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.017
ADP
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics
2.67
ATP
pH 7.0, 25°C, recombinant RFK activity of the RFK module
0.0014
FMN
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Lineweaver-Burk equation kinetics
0.0018
riboflavin
mutant enzyme H28A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0019
riboflavin
mutant enzyme H28D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0019
riboflavin
pH 7.0, 25°C, recombinant RFK activity of the RFK module, Michaelis-Menten kinetics
0.0032
riboflavin
mutant enzyme T165A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0033
riboflavin
mutant enzyme N125D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.004
riboflavin
wild type enzyme, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0041
riboflavin
mutant enzyme T165D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0044
riboflavin
mutant enzyme S164A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0046
riboflavin
mutant enzyme N125A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0051
riboflavin
mutant enzyme R161A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0051
riboflavin
mutant enzyme R161D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0058
riboflavin
mutant enzyme S164D, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
0.0063
riboflavin
mutant enzyme H31A, in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0, at 37°C
additional information
additional information
inhibition kinetics, dead-end inhibition by substrate excess
-
additional information
additional information
-
inhibition kinetics, dead-end inhibition by substrate excess
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
whereas the N-terminal module of FADS lacks structural homology to eukaryotic FMNATs, the kinase module is homologous to monofunctional RFKs
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
physiological function
additional information
physiological function
the essential cofactors of flavoproteins and flavoenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are synthesized from riboflavin in two sequential reactions: riboflavin phosphorylation is catalysed by an ATP-riboflavin kinase (RFK, EC 2.7.1.26) to produce FMN, which can be then converted to FAD by an FMN:ATP adenylyltranferase (FMNAT, EC 2.7.7.2). Bacteria contain a single bifunctional polypeptide called FAD synthetase (FADS)
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)
additional information
molecular dynamics simulations of riboflavin kinase domain bound to FMN, ADP, and Mg2+, structure-function analysis, flavin-binding site structure in the RFK module of CaFADS, overview
additional information
-
molecular dynamics simulations of riboflavin kinase domain bound to FMN, ADP, and Mg2+, structure-function analysis, flavin-binding site structure in the RFK module of CaFADS, overview
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
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
additional information
adenine and flavin nucleotide ligands cooperate in their binding to the RFK module
additional information
-
adenine and flavin nucleotide ligands cooperate in their binding to the RFK module
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
16600
recombinant truncated C-terminal RF kinase domain, gel filtration
additional information
additional information
the elution profile of full-length FADS shows two characteristic peaks at molecular weights corresponding to its monomeric and trimeric forms, while the tcRFK elutes as a single peak at an estimated molecular weight of 16.6 kDa, consistent with monomeric enzyme
additional information
-
the elution profile of full-length FADS shows two characteristic peaks at molecular weights corresponding to its monomeric and trimeric forms, while the tcRFK elutes as a single peak at an estimated molecular weight of 16.6 kDa, consistent with monomeric enzyme
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
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
additional information
monomer
x * 16600, recombinant truncated C-terminal RF kinase domain, SDS-PAGE
additional information
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
additional information
-
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
additional information
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
additional information
-
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
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant DELTA(1-182)CaFADS module in binary complex with ADP-Ca2+ and in ternary complex with FMN-ADP-Mg2+, mixing of 0.002 ml of 7.5-10 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1mM FMN and/or 1 mM ADP, with 0.002 ml of reservoir solution containing 10-14% PEG 8000, 20% glycerol, 0.1 M MES-NaOH pH 6.5, 200 mM CaCl2 for the binary complex, or with 0.002 ml of reservoir solution containing 26-30% PEG 4000, 200 mM Li2SO4, 100 mM sodium acetate, pH 5.0, as well as 0.002 ml of 1 M NaI solution, for the ternary complex, X-ray diffraction structure determination and analysis at 1.65-2.15 A resolution, modelling
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
H28A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
H28D
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
H31A
the mutant shows increased catalytic efficiency compared to the wild type enzyme
N125A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
N125D
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
R161A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
R66A
R66E
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
S164A
the mutant shows increased catalytic efficiency compared to the wild type enzyme
S164D
the mutant shows increased catalytic efficiency compared to the wild type enzyme
T165A
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
E268D
-
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
N210D
-
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
additional information
R66A
site-directed mutagenesis, R66A CaFADS shows a considerable increase in the amount of oligomeric species
R66E
the mutant shows increased activity compared to the wild type enzyme
R66E
site-directed mutagenesis, R66E CaFADS shows a considerable increase in the amount of oligomeric species
additional information
engineering of the FAD synthetase from Corynebacterium ammoniagenes by deleting its N-terminal adenylation domain leads to a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale. Deletion of the N-terminal adenosyl transfer domain in the truncated C-terminal RF kinase domain, tcRFK, variants results in a drop in the TM value from 40°C (parental CaFADS) to 35°C for tcRFK. Addition of the C-terminal poly-His tag further reduces the TM to 30°C, presumably due to the conformationally flexible tail formed by the extra amino acids
additional information
-
engineering of the FAD synthetase from Corynebacterium ammoniagenes by deleting its N-terminal adenylation domain leads to a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale. Deletion of the N-terminal adenosyl transfer domain in the truncated C-terminal RF kinase domain, tcRFK, variants results in a drop in the TM value from 40°C (parental CaFADS) to 35°C for tcRFK. Addition of the C-terminal poly-His tag further reduces the TM to 30°C, presumably due to the conformationally flexible tail formed by the extra amino acids
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
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
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
100
purified recombinant RFK module of enzyme FADS, DELTA(1-182)CaFADS, pH 7.0, 5 min, inactivation
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, phenyl Sepharose column chromatography, and DEAE-cellulose column chromatography
recombinant C-terminally poly-His-tagged N-terminally truncated mutant_187-338 from Escherichia coli by ion exchange chromatography and gel filtration
recombinant RFK module of enzyme FADS, DELTA(1-182)CaFADS, from Escherichia coli strain BL21(DE3)
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
presence of ATP:riboflavin 5'-phosphotransferase and ATP:FMN adenylyltransferase on a single polypeptide
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene ribF, individual expression of the riboflavin kinase, RFK, module of enzyme FAD synthetase, FADS, i.e. DELTA(1-182)CaFADS, in Escherichia coli strain BL21(DE3)
gene ribF, recombinant expression of C-terminally poly-His-tagged N-terminally truncated mutant in Escherichia coli
gene ribF, recombinant expression of the isolated RF module
gene ribF, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
the enzyme is used for the preparation of flavin mononucleotide (FMN) and FMN analogues from their corresponding riboflavin precursors, which is performed in a two-step procedure. After initial enzymatic conversion of riboflavin to FAD by the bifunctional FAD synthetase, the adenyl moiety of FAD is hydrolyzed with snake venom phosphodiesterase to yield FMN. The engineered FAD synthetase from Corynebacterium ammoniagenes with deleted N-terminal adenylation domain is a biocatalyst that is stable and efficient for direct and quantitative phosphorylation of riboflavin and riboflavin analogues to their corresponding FMN cofactors at preparative-scale
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Walsh, C.; Fisher, J.; Spencer, R.; Graham, D.W.; Ashton, W.T.; Brown, J.E.; Brown, R.D.; Rogers, E.F.
Chemical and enzymatic properties of riboflavin analogues
Biochemistry
17
1942-1951
1978
Corynebacterium ammoniagenes
Manstein, D.J.; Pai, E.F.
Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes
J. Biol. Chem.
261
16169-16173
1986
Corynebacterium ammoniagenes
Serrano, A.; Frago, S.; Herguedas, B.; Martinez-Julvez, M.; Velazquez-Campoy, A.; Medina, M.
Key residues at the riboflavin kinase catalytic site of the bifunctional riboflavin kinase/FMN adenylyltransferase from Corynebacterium ammoniagenes
Cell Biochem. Biophys.
65
57-68
2013
Corynebacterium ammoniagenes
Serrano, A.; Frago, S.; Velazquez-Campoy, A.; Medina, M.
Role of key residues at the flavin mononucleotide (FMN):adenylyltransferase catalytic site of the bifunctional riboflavin kinase/flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes
Int. J. Mol. Sci.
13
14492-14517
2012
Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes
Herguedas, B.; Lans, I.; Sebastian, M.; Hermoso, J.A.; Martinez-Julvez, M.; Medina, M.
Structural insights into the synthesis of FMN in prokaryotic organisms
Acta Crystallogr. Sect. D
71
2526-2542
2015
Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes
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
Biochim. Biophys. Acta
1854
897-906
2015
Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes
Iamurri, S.M.; Daugherty, A.B.; Edmondson, D.E.; Lutz, S.
Truncated FAD synthetase for direct biocatalytic conversion of riboflavin and analogs to their corresponding flavin mononucleotides
Protein Eng. Des. Sel.
26
791-795
2013
Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes
Sebastian, M.; Serrano, A.; Velazquez-Campoy, A.; Medina, M.
Kinetics and thermodynamics of the protein-ligand interactions in the riboflavin kinase activity of the FAD synthetase from Corynebacterium ammoniagenes
Sci. Rep.
7
7281
2017
Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes
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