Information on EC 2.7.1.26 - riboflavin kinase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
2.7.1.26
-
RECOMMENDED NAME
GeneOntology No.
riboflavin kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + riboflavin = ADP + FMN
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
5,6-dimethylbenzimidazole biosynthesis I (aerobic)
-
-
Biosynthesis of secondary metabolites
-
-
flavin biosynthesis I (bacteria and plants)
-
-
flavin biosynthesis III (fungi)
-
-
flavin biosynthesis IV (mammalian)
-
-
Metabolic pathways
-
-
Riboflavin metabolism
-
-
roseoflavin biosynthesis
-
-
flavin biosynthesis
-
-
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 hide
9032-82-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
ecotype Columbia
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
cell wall lacking mutant
-
-
Manually annotated by BRENDA team
cv. Bright Yellow 2
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2'-dATP + riboflavin
2'-dADP + riboflavin 5'-phosphate
show the reaction diagram
adenosine-5'-O-(3-thiotriphosphate) + riboflavin
?
show the reaction diagram
-
48% of the activity with ATP
-
-
?
ADP + riboflavin
AMP + FMN
show the reaction diagram
-
22% of the activity with ATP
-
-
?
ATP + 10-(D-allo)flavin
ADP + 10-(D-allo)flavin 5'-phosphate
show the reaction diagram
-
30% of the activity with riboflavin
-
-
?
ATP + 10-(L-arabo)flavin
ADP + 10-(L-arabo)flavin 5'-phosphate
show the reaction diagram
-
25% of the activity with riboflavin
-
-
?
ATP + 2'-deoxyriboflavin
ADP + 2'-deoxyriboflavin 5'-phosphate
show the reaction diagram
-
31% of the activity with riboflavin
-
-
?
ATP + 2-thioriboflavin
ADP + 2-thioriboflavin 5'-phosphate
show the reaction diagram
ATP + 3-deazariboflavin
ADP + 3-deazariboflavin 5'-phosphate
show the reaction diagram
-
-
-
-
?
ATP + 3-methylriboflavin
ADP + 3-methylriboflavin 5'-phosphate
show the reaction diagram
-
5% of the activity with riboflavin
-
-
?
ATP + 5-deazariboflavin
ADP + 5-deazaflavin monophosphate
show the reaction diagram
-
-
-
-
?
ATP + 5-deazariboflavin
ADP + 5-deazariboflavin 5'-phosphate
show the reaction diagram
ATP + 5-methyl-5-deazariboflavin
ADP + 5-methyl-5-deazariboflavin 5'-phosphate
show the reaction diagram
-
-
-
-
?
ATP + 6,7-dichloro-9-(D-1'-ribityl)isoalloxazine
ADP + 6,7-dichloro-9-(D-1'-ribityl)isoalloxazine phosphate
show the reaction diagram
ATP + 6,7-dimethyl-9-(1'-D-ribityl)-2-iminoisoalloxazine
ADP + 6,7-dimethyl-9-(1'-D-ribityl)-2-iminoisoalloxazine 5'-phosphate
show the reaction diagram
-
18% of the activity with riboflavin
-
-
?
ATP + 6-methylriboflavin
ADP + 6-methylriboflavin 5'-phosphate
show the reaction diagram
-
-
-
-
?
ATP + 7,8-dichlororiboflavin
ADP + 7,8-dichloroflavin monophosphate
show the reaction diagram
-
-
-
-
?
ATP + 7-chlororiboflavin
ADP + 7-chlororiboflavin
show the reaction diagram
-
-
-
-
?
ATP + 8-aminoriboflavin
ADP + 8-aminoflavin monophosphate
show the reaction diagram
-
-
-
-
?
ATP + 8-bromo-8-demethylriboflavin
ADP + 8-bromo-8-demethylriboflavin 5-'phosphate
show the reaction diagram
-
384% of the activity with riboflavin
-
-
?
ATP + 8-chloro-8-demethylriboflavin
ADP + 8-chloro-8-demethylriboflavin 5'-phosphate
show the reaction diagram
-
122.2% of the activity with riboflavin
-
-
?
ATP + 8-demethylriboflavin
ADP + 8-demethylriboflavin 5'-phosphate
show the reaction diagram
ATP + 8-dimethylamino-8-demethylriboflavin
ADP + 8-dimethylamino-8-demethylriboflavin 5'-phosphate
show the reaction diagram
ATP + 8-ethoxy-8-demethylriboflavin
ADP + 8-ethoxy-8-demethylriboflavin 5'-phosphate
show the reaction diagram
-
210% of the activity with riboflavin
-
-
?
ATP + 8-fluoro-8-demethylriboflavin
ADP + 8-fluoro-8-demethylriboflavin 5'-phosphate
show the reaction diagram
-
132.2% of the activity with riboflavin
-
-
?
ATP + 8-iodo-8-demethylriboflavin
ATP + 8-iodo-8-demethylriboflavin 5'-phosphate
show the reaction diagram
-
334.7% of the activity with riboflavin
-
-
?
ATP + 8-methoxy-8-demethylriboflavin
ADP + 8-methoxy-8-demethylriboflavin 5'-phosphate
show the reaction diagram
-
114.5% of the activity with riboflavin
-
-
?
ATP + 8-methylamino-8-demethylriboflavin
ATP + 8-methylamino-8-demethylriboflavin 5'-phosphate
show the reaction diagram
-
237.3% of the activity with riboflavin
-
-
?
ATP + 9-azariboflavin
ADP + 9-azariboflavin 5'-phosphate
show the reaction diagram
-
-
-
-
?
ATP + alloflavin
ADP + alloflavin 5'-phosphate
show the reaction diagram
-
-
-
-
?
ATP + arabitylflavin
ADP + arabitylflavin phosphate
show the reaction diagram
ATP + D-erythroflavin
ADP + D-erythroflavin 5'-phosphate
show the reaction diagram
ATP + riboflavin
ADP + FMN
show the reaction diagram
ATP + roseoflavin
ADP + roseoflavin 5'-phosphate
show the reaction diagram
CTP + riboflavin
CTP + riboflavin 5'-phosphate
show the reaction diagram
dATP + riboflavin
dADP + FMN
show the reaction diagram
GTP + riboflavin
GTP + riboflavin 5'-phosphate
show the reaction diagram
riboflavin + ATP
FMN + ADP
show the reaction diagram
-
-
-
-
?
UTP + riboflavin
UDP + FMN
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + riboflavin
ADP + FMN
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
can substitute for Mg2+
Cd2+
-
38% of the activation with Zn2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1'-DL-glyceryl-6,7-dimethylisoalloxazine
-
competitive
1-Deazariboflavin
-
-
10-(2'-Hydroxyethyl)-isoalloxazine
-
0.01 mM, 34% inhibition
10-(4'-Carboxybutyl)-isoalloxazine
-
0.001 mM, 16% inhibition
10-(5'-Hydroxypentyl)-isoalloxazine
-
0.01 mM, 38% inhibition
10-(5'-Hydroxypentyl)flavin
-
-
10-(Hydroxyethyl)flavin
-
-
2'-Thioriboflavin
-
0.01 mM, 59% inhibition
3'-hydroxypropyl-6,7-dimethylisoalloxazine
-
competitive
3-Deazariboflavin
-
-
3-methylriboflavin
-
0.01 mM, 5% inhibition
4'-hydroxybutyl-6,7-dimethylisoalloxazine
-
competitive
5'-hydroxypentyl-6,7-dimethylisoalloxazine
-
competitive
5-Deazariboflavin
-
-
7,8-dimethyl-10-(2'-hydroxyethyl)-isoalloxazine
-
0.5 mM, 10% inhibition
7,8-dimethyl-10-(O-methylacetoxime)-isoaloxazine
-
0.5 mM, 34% inhibition
7alpha-Methylriboflavin
-
0.01 mM, 95% inhibition
8-Aminoriboflavin
-
-
8-Diethylaminoriboflavin
-
-
8-Ethoxyriboflavin
-
-
8-Ethylaminoriboflavin
-
-
8-hydroxyriboflavin
-
-
8-Methoxyriboflavin
-
-
8-Methylaminoriboflavin
-
-
8-Methylethylaminoriboflavin
-
-
9-(6'-hydroxyhexyl)-6,7-dimethylisoalloxazine
-
competitive
FAD
product inhibition, wild-type enzyme FMNAT is strongly inhibited by FAD, whereas D181A mutant enzyme has an attenuated product inhibition
FMN
-
product inhibition
lumichrome
-
-
Lumiflavin
riboflavin
-
substrate inhibition
riboflavin 5'-phosphate
-
-
roseoflavin
-
0.4 mM, 8% inhibition
ZnADP-
-
product inhibition
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Carbonate
-
50 mM, activates
NO3-
-
50 mM, activates
phosphate
-
50 mM, activates
SO42-
-
50 mM, activates
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.025
adenosine-5'-O-(3-thiotriphosphate)
-
-
0.0002 - 4.55
ATP
0.0015
D-alloflavin
-
-
0.0011
D-erythroflavin
-
-
0.0012
D-riboflavin
-
-
0.000021 - 0.005
MgATP2-
0.0000103 - 0.18
riboflavin
0.03
roseoflavin
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.042 - 6.8
ATP
0.1
MgATP2-
Neurospora crassa
-
pH 8.5, 30C
0.045 - 7.12
riboflavin
0.4 - 0.5
roseoflavin
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.83 - 788.3
ATP
4
0.0758 - 1083
riboflavin
351
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0065
1'-DL-glyceryl-6,7-dimethylisoalloxazine
-
competitive
0.021
1-Deazariboflavin
-
pH 8, 37C
0.008
10-(5'-Hydroxypentyl)flavin
-
pH 8, 37C
0.007
10-(Hydroxyethyl)flavin
-
pH 8, 37C
0.0071
3'-hydroxypropyl-6,7-dimethylisoalloxazine
-
competitive
0.41
3-Deazariboflavin
-
pH 8, 37C
0.0076
4'-hydroxybutyl-6,7-dimethylisoalloxazine
-
competitive
0.0078
5'-hydroxypentyl-6,7-dimethylisoalloxazine
-
competitive
0.275
5-Deazariboflavin
-
pH 8, 37C
0.25
8-Aminoriboflavin
-
pH 8, 37C
0.02
8-Diethylaminoriboflavin
-
pH 8, 37C
0.016
8-Ethoxyriboflavin
-
pH 8, 37C
0.175
8-Ethylaminoriboflavin
-
pH 8, 37C
0.5
8-hydroxyriboflavin
-
pH 8, 37C
0.015
8-Methoxyriboflavin
-
pH 8, 37C
0.47
8-Methylaminoriboflavin
-
pH 8, 37C
0.03
8-Methylethylaminoriboflavin
-
pH 8, 37C
0.0079
9-(6'-hydroxyhexyl)-6,7-dimethylisoalloxazine
-
competitive
0.006
FMN
-
pH 8.0, 37C, against riboflavin
0.01
lumichrome
-
pH 8, 37C
0.007
Lumiflavin
-
pH 8, 37C
0.0018 - 0.0063
riboflavin
0.018
riboflavin 5'-phosphate
-
pH 8, 37C
0.023 - 0.12
ZnADP-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0083
0.716
-
enzyme from brain
additional information
-
bioluminescent assay
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5 - 9
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
-
pH 5.0: about 30% of maximal activity, pH 9.0: about 65% of maximal activity
5 - 8.5
-
enzyme is active between pH 5 and 8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 45
-
15C: about 60% of maximal activity, 45C: about 75% of maximal activity
20 - 45
-
the relative activities at pH 7.2 and 20C, 25C, 37C and 45C are 0.22, 0.46, 1, and 1.35, respectively
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
13500
-
gel filtration
16600
-
recombinant truncated C-terminal RF kinase domain, gel filtration
27000
-
gel filtration
27250
-
gel filtration
28000
-
1 * 28000, SDS-PAGE
30000
-
1 * 30000, SDS-PAGE
34200
gel filtration
35000
-
gel filtration
35500
-
gel filtration
36000
1 * 36000, SDS-PAGE
40000
-
gel filtration
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
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
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
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
-
purified recombinant enzyme with bound products FMN and MgADP, hanging drop vapour diffusion method, 34 mg/ml protein in 50 mM Tris, pH 7.4, 0.3 M NaCl, 1 mM DTT, 20C, mixing with equal volume of reservoir solution containing 0.1 M sodium acetate, pH 4.7, 30% PEG monomethyl ether 5000, and 0.2 M ammonium sulfate, followed by microseeding in reservoir solution containing 0.1 M sodium acetate, pH 4.4, 22.5% PEG monomethyl ether 5000, and 0.2 M ammonium sulfate, cryoprotection in 30% glycerol in reservoir solution, storage in liquid propane, X-ray diffraction structure determination and analysis at 2.4 A resolution
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
enzyme immobilized by amide linkage to omega aminoalkyl-agarose-beads has a half-life of three weeks
100
-
purified recombinant RFK module of enzyme FADS, DELTA(1-182)CaFADS, pH 7.0, 5 min, inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
50% of the activity is lost on freezing, but the stability of the enzyme is not greatly affected by the period of freezing
-
enzyme immobilized by amide linkage to omega aminoalkyl-agarose-beads has a half-life of three weeks at 25C
-
enzyme is inactivated by freezing and thawing unless both riboflavin and 20% glycerol are added
-
loss of activity during repeated freezing and thawing
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
3C, MOPS buffer, activity decreases to approximately 35, 30 and 13% of the initial level after 24, 48, and 96 h, respectively
-
4C, 50% loss of activity after 2 d when the enzyme is stored in buffer alone, complete protection by 0.01 mM riboflavin
-
purified enzyme is unstable in dilute solution and can not be stored at -20C
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography
-
ammonium sulfate precipitation, phenyl Sepharose column chromatography, and DEAE-cellulose column chromatography; 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
-
enzyme from brain
-
partial, affinity chromatography
-
presence of ATP:riboflavin 5'-phosphotransferase and ATP:FMN adenylyltransferase on a single polypeptide
-
recombinant C-terminally poly-His-tagged N-terminally truncated mutant_187-338 from Escherichia coli by ion exchange chromatography and gel filtration
-
recombinant enzyme
recombinant RFK module of enzyme FADS, DELTA(1-182)CaFADS, from Escherichia coli strain BL21(DE3)
-
recombinant, His-tagged enzyme by nickel affinity chromatography, removal of His-tag by TEV-protease treatment
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amplification, cloning and expression of ribR gene in Escherichia coli; expression of ribC gene in Escherichia coli
-
expressed in Escherichia coli
-
expressed in Escherichia coli BL21(DE3) cells; gene ribF, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
-
expressed in Escherichis coli BL-21pLysS (DE3), C-terminal glutathione S-transferase fusionprotein
-
expression of ribC gene in Escherichia coli
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
-
overproduced in Escherichia coli
the functional overexpression of the individual domains in Escherichia coli establishes that the riboflavin kinase and FMN hydrolase activities reside, respectively, in the C-terminal (AtFMN) and N-terminal (AtFHy) domains of AtFMN/FHy
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D168A
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
D181A
site-directed mutagenesis, the mutant shows reduced sensitivity to inhibition by FAD compared to the wild-type enzyme and has a much faster turnover rate than the wild-type enzyme
D66A
site-directed mutagenesis, inactive mutant
N62A
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
N62S
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
R297A
site-directed mutagenesis, the mutant shows a 2fold increased activity compared to the wild-type enzyme
R297A/R300A
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
R300A
site-directed mutagenesis, the mutant shows 93% reduced activity compared to the wild-type enzyme
W184A
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
E268D
-
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
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
N210D
-
the mutant shows strongly reduced catalytic efficiency compared to the wild type enzyme
R161A
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
R161D
-
the mutant shows wild type catalytic efficiency
R66A
-
inactive; 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; the mutant shows increased activity compared to the wild type enzyme
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
T165D
-
the mutant shows wild type catalytic efficiency
E86Q
-
destroying the kinase domain, purified as C-terminal glutathione S-transferase fusionprotein
N36D
-
purified as C-terminal glutathione S-transferase fusionprotein
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 40C (parental CaFADS) to 35C for tcRFK. Addition of the C-terminal poly-His tag further reduces the TM to 30C, presumably due to the conformationally flexible tail formed by the extra amino acids
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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