Any feedback?
Information on EC 2.7.7.2 - FAD synthase and Organism(s) Corynebacterium ammoniagenes and UniProt Accession Q59263
for references in articles please use BRENDA:EC2.7.7.2Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
EC Tree
2.7.7.2 FAD synthaseIUBMB Comments
Requires Mg2+ and is highly specific for ATP as phosphate donor . 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 FAD synthetase is found in eukaryotes and in some prokaryotes, most prokaryotes have a bifunctional enzyme that exhibits both this activity and that of EC 2.7.1.26, riboflavin kinase [3,5].
Specify your search results
Word Map
- 2.7.7.2
-
desaturase
-
polyunsaturated
-
arachidonic
-
docosahexaenoic
-
linoleic
-
eicosapentaenoic
-
desaturation
- elongase
-
elovl2
-
omega-6
-
delta-5
-
lcpufas
- ammoniagenes
-
srebf1
-
gene-diet
- flavokinase
- synthesis
- nutrition
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
fads1, flad1, fad synthase, fad pyrophosphorylase, flavin adenine dinucleotide synthetase, atribf1, atribf2, atp:fmn adenylyltransferase, fmn:atp adenylyltransferase, mj1179, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
adenosine triphosphate-riboflavin mononucleotide transadenylase
-
-
-
-
adenosine triphosphate-riboflavine mononucleotide transadenylase
-
-
-
-
FAD pyrophosphorylase
-
-
-
-
FMN adenylyltransferase
-
-
-
-
FMN pyrophosphorylase
-
-
-
-
lysZ
-
-
-
-
riboflavin adenine dinucleotide pyrophosphorylase
-
-
-
-
riboflavin mononucleotide adenylyltransferase
-
-
-
-
riboflavine adenine dinucleotide adenylyltransferase
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
nucleotidyl group transfer
-
-
-
-
PATHWAY SOURCE
PATHWAYS
BRENDA
-
-, -, -, -
SYSTEMATIC NAME
IUBMB Comments
ATP:FMN adenylyltransferase
Requires Mg2+ and is highly specific for ATP as phosphate donor [5]. 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 [3]. While monofunctional FAD synthetase is found in eukaryotes and in some prokaryotes, most prokaryotes have a bifunctional enzyme that exhibits both this activity and that of EC 2.7.1.26, riboflavin kinase [3,5].
CAS REGISTRY NUMBER
COMMENTARY
9026-37-3
-
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 + FMN
diphosphate + FAD
-
adenylation of FMN is reversible, FAD and diphosphate can be converted to FMN and ATP by the enzyme, under the conditions studied phosphorylation of riboflavin is irreversible
-
r
ATP + FMN
diphosphate + FAD
-
catalyzes 2 sequential steps in the biosynthesis of FAD, phosphorylation of riboflavin to produce FMN and subsequent adenylylation of FMN to form FAD
-
r
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
?
-
-
bifunctional FAD synthetase which shows FMN adenylyltransferase and flavokinase activities, producing FMN ATP:riboflavin 5'-phosphotransferase EC 2.7.1.26
-
-
?
additional information
?
-
-
bifunctional FAD synthetase which shows FMN adenylyltransferase and flavokinase activities, producing FMN ATP:riboflavin 5'-phosphotransferase EC 2.7.1.26
-
-
?
additional information
?
-
bifunctional FAD synthetase which shows FMN adenylyltransferase and flavokinase activities, producing FMN ATP:riboflavin 5'-phosphotransferase EC 2.7.1.26
-
-
?
additional information
?
-
-
bifunctional FAD synthetase which shows FMN adenylyltransferase and flavokinase activities, producing FMN ATP:riboflavin 5'-phosphotransferase EC 2.7.1.26
-
-
?
additional information
?
-
-
bifunctional FAD synthetase which shows FMN adenylyltransferase and flavokinase activities, producing FMN ATP:riboflavin 5'-phosphotransferase EC 2.7.1.26
-
-
?
additional information
?
-
-
bifunctional FAD synthetase which shows FMN adenylyltransferase and flavokinase activities, producing FMN ATP:riboflavin 5'-phosphotransferase EC 2.7.1.26
-
-
?
additional information
?
-
-
FAD synthetase presents two catalytic modules, a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide adenylyltransferase activity
-
-
?
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
ATP + FMN
diphosphate + FAD
-
catalyzes 2 sequential steps in the biosynthesis of FAD, phosphorylation of riboflavin to produce FMN and subsequent adenylylation of FMN to form FAD
-
r
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
flavin
presence of a flavin binding site for the adenylylation activity, independent from that related with the phosphorylation actiity
ATP
-
nucleoside triphosphates other than ATP do not act as substrates or inhibitors
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+
Mg2+
additional information
-
not basically effected by Mg2+, FAD production slightly inhibited at high concentrations
Mg2+
Mg2+ and the concerted fit of substrates are required to achieve a catalytically competent geometry
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(1R,2S,4aS,4bS,7R,8aR,10aS)-7-hydroxy-2,4b-dimethyl-1-[(3-oxo-3H-indol-2-yl)methyl]tetradecahydrophenanthrene-2-carboxylic acid
inhibitor of the activity of the flavin adenylyl transferase module of the FADS
2-([1-[4-(trifluoromethyl)pyrimidin-2-yl]piperidin-4-yl]carbamoyl)benzoic acid
inhibitor of the activity of the flavin adenylyl transferase module of the FADS
2-([[(9H-fluoren-9-yl)methoxy]carbonyl]amino)benzoic acid
inhibitor of the activity of the flavin adenylyl transferase module of the FADS, able to inhibit growth of Corynebacterium ammoniagenes and Mycobacterium tuberculosis
methyl 2-[(4-chlorophenyl)carbamoyl]-N-(3,5-dimethyl-1,2-oxazole-4-sulfonyl)hydrazine-1-carboximidothioate
56% resiudal activity at 0.25 mM, inhibitor of the activity of the flavin adenylyl transferase module of the FADS
N-(5-nitrothiophene-3-carbonyl)-2-[[4-(propan-2-yl)phenyl]carbamothioyl]hydrazine-1-carboxamide
inhibitor of the activity of the flavin adenylyl transferase module of the FADS, able to inhibit growth of Corynebacterium ammoniagenes and Mycobacterium tuberculosis
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.03568
ATP
-
wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.0382
ATP
-
mutant enzyme T208D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.0387
ATP
-
mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.0435
ATP
-
FADS trimer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
0.04537
ATP
-
mutant enzyme T208A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.04647
ATP
-
mutant enzyme E268A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.04704
ATP
-
mutant enzyme N210A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.07667
ATP
-
mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.079
ATP
-
FADS monomer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
0.00088
FMN
-
mutant enzyme E268A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.00088
FMN
-
mutant enzyme T208D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.00095
FMN
-
mutant enzyme T208A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.00117
FMN
-
mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.00119
FMN
-
wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.0054
FMN
-
FADS monomer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
0.00823
FMN
-
mutant enzyme N210A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.01501
FMN
-
mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.0311
FMN
-
FADS trimer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
additional information
additional information
MichaelisMenten model
-
additional information
additional information
-
MichaelisMenten model
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.047
FMN
-
FADS trimer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
0.33
FMN
-
FADS monomer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.1
ATP
-
FADS trimer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
3.8
ATP
-
mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
4.2
ATP
-
FADS monomer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
4.3
ATP
-
mutant enzyme N210A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
7.3
ATP
-
mutant enzyme E268A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
7.7
ATP
-
mutant enzyme T208A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
8
ATP
-
wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
8.2
ATP
-
mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
8.5
ATP
-
mutant enzyme T208D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
0.0311
FMN
-
FADS trimer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
19.7
FMN
-
mutant enzyme N210D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
24.8
FMN
-
mutant enzyme N210A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
61.7
FMN
-
FADS monomer, 10 mM MgCl2 in 50 mM TrisHCl (pH 8.0), at 37°C
238.3
FMN
-
wild type enzyme, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
270.2
FMN
-
mutant enzyme E268D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
365.7
FMN
-
mutant enzyme T208D, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
369.2
FMN
-
mutant enzyme T208A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
386
FMN
-
mutant enzyme E268A, at 37°C in 20 mM PIPES pH 7.0, 10 mM MgCl2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.238
(1R,2S,4aS,4bS,7R,8aR,10aS)-7-hydroxy-2,4b-dimethyl-1-[(3-oxo-3H-indol-2-yl)methyl]tetradecahydrophenanthrene-2-carboxylic acid
Corynebacterium ammoniagenes
pH 7.0, 25°C
0.1543
2-([1-[4-(trifluoromethyl)pyrimidin-2-yl]piperidin-4-yl]carbamoyl)benzoic acid
Corynebacterium ammoniagenes
pH 7.0, 25°C
0.053
2-([[(9H-fluoren-9-yl)methoxy]carbonyl]amino)benzoic acid
Corynebacterium ammoniagenes
pH 7.0, 25°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
bifunctional enzyme, displays activities of EC 2.7.7.2 and EC 2.7.1.26
SwissProt
bifunctional riboflavin kinase/FMN adenylyltransferase, cf. EC 2.7.1.26
SwissProt
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
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
distortion of aromaticity at the FMNAT isoalloxazine binding cavity prevents FMN and FAD from correct accommodation in their binding cavity and decreases the efficiency of the FMNAT activity. The side-chains of F62, Y106 and F128 are relevant in the formation of the catalytic competent complex during FMNAT catalysis in bifunctional FAD synthase
physiological function
in a truncated FADS variant consisting in the isolated C-terminal ATP:riboflavin kinase RFK module, RFK activity is similar to that of the full-length enzyme. Inhibition of the RFK activity by the RF substrate is independent of the FMN:ATP adenylyltransferase module, and FMN production, in addition to being inhibited by an excess of riboflavin, is also inhibited by both of the reaction products. Mg2+ and the concerted fit of substrates are required to achieve a catalytically competent geometry
physiological function
molecular docking and molecular dynamics simulations with ATP/Mg2+ and FMN in both the monomeric and dimer-of-trimers assemblies. For the dimer-of-trimers conformation, the RFK module negatively influences FMN binding at the interacting FMNAT module. FMN binds to the monomer but not to the dimer-of-trimers. The presence of the RFK module (residues E268, D298, and V300) considerably impairs FMN binding at the FMNAT active site and decreases the FMNAT catalytic efficiency
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
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
37120
-
electrospray mass spectrometry, Se-Met-enzyme, 36843.5 is the theoretical value for the native protein
38000
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
hexamer
dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other
monomer
x * 16600, recombinant truncated C-terminal RF kinase domain, SDS-PAGE
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 the monomeric enzyme
additional information
additional information
prokaryotic FAD synthetases (FADSs) are bifunctional enzymes composed of two modules, the C-terminal module with ATP:riboflavin kinase (RFK) activity, and the N-terminus with ATP:FMN adenylyltransferase (FMNAT) activity
additional information
-
prokaryotic FAD synthetases (FADSs) are bifunctional enzymes composed of two modules, the C-terminal module with ATP:riboflavin kinase (RFK) activity, and the N-terminus with ATP:FMN adenylyltransferase (FMNAT) activity
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
3D structural model for based on the structure of Thermotoga maritima FADS
crystal structure predicts a dimer of trimers organization
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
D298A
mutation at the macromolecular interface between two protomers within the trimer
D298E
mutation at the macromolecular interface between two protomers within the trimer
E203A
mutation at the macromolecular interface between two protomers within the trimer
E301A
mutation at the macromolecular interface between two protomers within the trimer
E301K
mutation at the macromolecular interface between two protomers within the trimer
F206A
mutation at the macromolecular interface between two protomers within the trimer
F206K
mutation at the macromolecular interface between two protomers within the trimer
F206W
mutation at the macromolecular interface between two protomers within the trimer
H28A
loss of both riboflavin kinase and FAD synthetase activities
H28D
loss of both riboflavin kinase and FAD synthetase activities
H31D
residual activity, involved in the stabilisation of the phosphate groups and the adenine moiety of ATP and the phosophate of FMN
K202A
mutation at the macromolecular interface between two protomers within the trimer
L304K
mutation at the macromolecular interface between two protomers within the trimer
L98A
mutation totally prevents the binding of FMN and/or FAD. Residues P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis
L98K
mutation totally prevents the binding of FMN and/or FAD. Residues P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis
L98W
residues P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis
P56A/P58A
variant exhibits lower KdATP values and altered thermodynamic profile for ATP binding. Residues P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis
P56W
variant exhibits lower KdATP values and altered thermodynamic profile for ATP binding. Residues P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis
P58W
residues P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis
R161A
active, residue R161 does not play a critical role in catalysis
R161D
active, residue R161 does not play a critical role in catalysis
R66A
site-directed mutagenesis, R66A CaFADS shows a considerable increase in the amount of oligomeric species
R66E
site-directed mutagenesis, R66E CaFADS shows a considerable increase in the amount of oligomeric species
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
residual activity, involved in the stabilisation of the phosphate groups and the adenine moiety of ATP and the phosophate of FMN
S164D
residual activity, involved in the stabilisation of the phosphate groups and the adenine moiety of ATP and the phosophate of FMN
T165A
residual activity, involved in the stabilisation of the phosphate groups and the adenine moiety of ATP and the phosophate of FMN
T165D
residual activity, involved in the stabilisation of the phosphate groups and the adenine moiety of ATP and the phosophate of FMN
V300A
mutation at the macromolecular interface between two protomers within the trimer
V300K
mutation at the macromolecular interface between two protomers within the trimer
E268A
-
the mutant shows increased catalytic efficiency for FMN and reduced catalytic efficiency for ATP compared to the wild type enzyme
E268D
-
the mutant shows about wild type catalytic efficiencies for ATP and FMN
N210A
-
the mutant shows strongly reduced catalytic efficiencies for FMN and ATP compared to the wild type enzyme
N210D
-
the mutant shows strongly reduced catalytic efficiencies for FMN and ATP compared to the wild type enzyme
T208A
-
the mutant shows increased catalytic efficiency for FMN and reduced catalytic efficiency for ATP compared to the wild type enzyme
T208D
-
the mutant shows increased catalytic efficiency for FMN and increased catalytic efficiency for ATP compared to the wild type enzyme
additional information
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DEAE-cellulose column chromatography, phenyl Sepharose column chromatography, and Sephacryl S-200 gel filtration
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
ammonium sulfate fractionation, Phenyl-Sepharose chromatography, DEAE-Cellulose chromatography
-
phenyl Sepharose column chromatography and DEAE-cellulose column chromatography
-
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 wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
recombinant expression of C-terminally poly-His-tagged N-terminally truncated mutant in Escherichia coli
expressed in Escherichia coli BL21 (DE3), a SeMet-version is generated
-
FAD synthetase gene cloned and overexpressed in Escherichia coli JM105
FAD synthetase overproducing recombinant Corynebacterium ammoniagenes KY13315 constructed from ATCC6872, gene cannot be expressed in Escherichia coli
-
vector pET-23a(+) cloned and overexpressed in Escherichia coli JM109(DE-3)
-
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
nutrition
nutrition
-
industrial production of FAD and FMN as nutrient additives, pharmaceuticals and biochemical agents
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Hagihara, T.; Fujio, T.; Aisaka, K.
Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production
Appl. Microbiol. Biotechnol.
42
724-729
1995
Corynebacterium ammoniagenes
Nakagawa, S.; Igarashi, A.; Ohta, T.; Hagihara, T.; Fujio, T.; Aisaka, K.
Nucleotide sequence of the FAD synthetase gene from Corynebacterium ammoniagenes and its expression in Escherichia coli
Biosci. Biotechnol. Biochem.
59
694-702
1995
Corynebacterium ammoniagenes, Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes ATCC 6872
McCormick, D.B.; Oka, M.; Bowers-Komro, D.M.; Yamada, Y.; Hartman, H.A.
Purification and properties of FAD synthetase from liver
Methods Enzymol.
280
407-413
1997
Bos taurus, Corynebacterium ammoniagenes, Rattus norvegicus
Efimov, I.; Kuusk, V.; Zhang, X.; McIntire, W.S.
Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli
Biochemistry
37
9716-9723
1998
Corynebacterium ammoniagenes
Krupa, A.; Sandhya, K.; Srinivasan, N.; Jonnalagadda, S.
A conserved domain in prokaryotic bifunctional FAD synthetases can potentially catalyze nucleotide transfer
Trends Biochem. Sci.
28
9-12
2003
Corynebacterium ammoniagenes
Frago, S.; Martinez-Julvez, M.; Serrano, A.; Medina, M.
Structural analysis of FAD synthetase from Corynebacterium ammoniagenes
BMC Microbiol.
8
160
2008
Corynebacterium ammoniagenes (Q59263)
Herguedas, B.; Martinez-Julvez, M.; Frago, S.; Medina, M.; Hermoso, J.A.
Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes
Acta Crystallogr. Sect. F
65
1285-1288
2009
Corynebacterium ammoniagenes
Frago, S.; Velazquez-Campoy, A.; Medina, M.
The puzzle of ligand binding to Corynebacterium ammoniagenes FAD synthetase
J. Biol. Chem.
284
6610-6619
2009
Corynebacterium ammoniagenes
Marcuello, C.; Arilla-Luna, S.; Medina, M.; Lostao, A.
Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level
Biochim. Biophys. Acta
1834
665-676
2013
Corynebacterium ammoniagenes (Q59263)
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
Herguedas, B.; Martinez-Julvez, M.; Frago, S.; Medina, M.; Hermoso, J.A.
Oligomeric state in the crystal structure of modular FAD synthetase provides insights into its sequential catalysis in prokaryotes
J. Mol. Biol.
400
218-230
2010
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)
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)
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
Lans, I.; Seco, J.; Serrano, A.; Burbano, R.; Cossio, P.; Daza, M.C.; Medina, M.
The dimer-of-trimers assembly prevents catalysis at the transferase site of prokaryotic FAD synthase
Biophys. J.
115
988-995
2018
Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes
Arilla-Luna, S.; Serrano, A.; Medina, M.
Specific features for the competent binding of substrates at the FMN adenylyltransferase site of FAD synthase from Corynebacterium ammoniagenes
Int. J. Mol. Sci.
20
5083
2019
Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes
Serrano, A.; Arilla-Luna, S.; Medina, M.
Insights into the fmnat active site of FAD synthase Aromaticity is essential for flavin binding and catalysis
Int. J. Mol. Sci.
21
3738
2020
Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes
Lans, I.; Anoz-Carbonell, E.; Palacio-Rodriguez, K.; Ainsa, J.A.; Medina, M.; Cossio, P.
In silico discovery and biological validation of ligands of FAD synthase, a promising new antimicrobial target
PLoS Comput. Biol.
16
e1007898
2020
Corynebacterium ammoniagenes (Q59263), Corynebacterium ammoniagenes
Serrano, A.; Sebastian, M.; Arilla-Luna, S.; Baquedano, S.; Herguedas, B.; Velazquez-Campoy, A.; Martinez-Julvez, M.; Medina, M.
The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes
Sci. Rep.
7
404
2017
Corynebacterium ammoniagenes (Q59263)
EXTERNAL LINKS (specific for EC-Number 2.7.7.2)
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
