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Enzyme Nomenclature
Information on EC 1.5.1.40 - 8-hydroxy-5-deazaflavin:NADPH oxidoreductase
for references in articles please use BRENDA:EC1.5.1.40
Word Map on EC 1.5.1.40
- 1.5.1.40
-
methanogen
- archaea
-
hydride
-
methanococcus
- 8-hydroxy-5-deazaflavins
-
hydrogenase
- vannielii
- thermoautotrophicum
-
methanobacterium
- fulgidus
-
si-face
-
methanogenesis
- griseus
- methane
-
stereochemical
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The expected taxonomic range for this enzyme is: Archaea, Bacteria
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EC NUMBER 
COMMENTARY 
1.5.1.40
-
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RECOMMENDED NAME
GeneOntology No.
8-hydroxy-5-deazaflavin:NADPH oxidoreductase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
reduced coenzyme F420 + NADP+ = oxidized coenzyme F420 + NADPH + H+
-
-
-
-
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SYSTEMATIC NAME
IUBMB Comments
reduced coenzyme F420:NADP+ oxidoreductase
The enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate [1].
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
T28A mutant shows 3fold increased kinetic efficiency compared with the wild-type enzyme when NADPH is the substrate
physiological function
additional information
physiological function
-
the main function of this oxidoreductase is probably to provide cells with reduced 8-hydroxy-5-deazaflavin to be used in specific reduction reactions. The last step of the tetracycline biosynthesis in Streptomyces aureofaciens in which 5a,11a-dehydrochlortetracycline is reduced to chlortetracycline is 8-hydroxy-5-deazaflavin-dependent, and the reducing equivalents are obtained from NADPH
physiological function
-
residue I135 plays a key role in sustaining the donor-acceptor distance between the two cofactor substrates, thereby regulating the rate at which the hydride is transferred from FOH2 to NADP+. Fno is a dynamic enzyme that regulates NADPH production
physiological function
-
half-site reactivity and negative cooperativity involving the important F420 cofactor-dependent enzyme. F420H2:NADP+ oxidoreductase (Fno), an F420 cofactor-dependent enzyme that catalyzes the reversible reduction of NADP+ through the transfer of a hydride from the reduced F420 cofactor. Fno may be a functional regulatory enzyme
physiological function
-
F420-dependent NADP+ oxidoreductase (Fno) is critical to the conversion of CO2 to CH4 by methanogenic archaea, while the F420 redox moiety, FO, functions as a light-harvesting agent in DNA repair
additional information
the active site of F420-dependent enzyme Tfu-FNO is located in a hydrophobic pocket between an N-terminal dinucleotide binding domain and a smaller C-terminal domain. Residues interacting with the 2'-phosphate of NADP+, Thr28, Ser50, Arg51, and Arg55, are important for discriminating between NADP+ and NAD+. Molecular recognition of the two cofactor substrates, F420 and NAD(P)H by FNO, overview
additional information
-
the active site of F420-dependent enzyme Tfu-FNO is located in a hydrophobic pocket between an N-terminal dinucleotide binding domain and a smaller C-terminal domain. Residues interacting with the 2'-phosphate of NADP+, Thr28, Ser50, Arg51, and Arg55, are important for discriminating between NADP+ and NAD+. Molecular recognition of the two cofactor substrates, F420 and NAD(P)H by FNO, overview
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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
5'-O-methyl-7,8-didemethyl-8-hydroxyflavin + NADPH + H+
8-hydroxypyrimido[4,5-b]-2,4-(3H,10H)-dione + NADP+
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
r
5-deaza-8-hydroxyisoalloxazine + NADPH + H+
8-hydroxypyrimido[4,5-b]-2,4-(3H,10H)-dione + NADP+
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
?
7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate + NADPH + H+
1-deoxy-1-(8-hydroxy-2,4-dioxo-1,3,4,5-tetrahydropyrimido[4,5-b]quinolin-10(2H)-yl)-5-O-phospho-D-ribitol + NADP+
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
?
1,5-dihydro-8-hydroxy-5-deazaflavin + NADP+
8-hydroxy-5-deazaflavin + NADPH + H+
-
the kcat value of the forward reaction is 24 times greater than that of the reverse reaction, thus the production of NADPH at pH 7.0 is more favorable than its consumption
-
-
r
1,5-dihydro-8-hydroxy-5-deazaflavin + NADP+
8-hydroxy-5-deazaflavin + NADPH + H+
-
the kcat value of the forward reaction is 24 times greater than that of the reverse reaction, thus the production of NADPH at pH 7.0 is more favorable than its consumption
-
-
r
5-deaza-8-hydroxy-10-methylisoalloxazine + NADPH + H+
? + NADP+
-
-
-
-
?
5-deaza-8-hydroxy-10-methylisoalloxazine + NADPH + H+
? + NADP+
-
-
-
-
?
coenzyme F0 + NADPH + H+
reduced coenzyme F0 + NADP+
-
i.e. 7,8-didemethyl-8-hydroxy-5-deazariboflavin. The enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
?
coenzyme F0 + NADPH + H+
reduced coenzyme F0 + NADP+
-
i.e. 7,8-didemethyl-8-hydroxy-5-deazariboflavin. The enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
?
coenzyme F0 + NADPH + H+
reduced coenzyme F0 + NADP+
-
i.e. 7,8-didemethyl-8-hydroxy-5-deazariboflavin. The enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
?
coenzyme F0 + NADPH + H+
reduced coenzyme F0 + NADP+
-
i.e. 7,8-didemethyl-8-hydroxy-5-deazariboflavin. The enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
?
coenzyme F0 + NADPH + H+
reduced coenzyme F0 + NADP+
-
i.e. 7,8-didemethyl-8-hydroxy-5-deazariboflavin. The enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
?
coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate. No activity with NAD+
-
-
?
coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate. No activity with NAD+
-
-
?
coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
-
the main function of this oxidoreductase is probably to provide cells with reduced 8-hydroxy-5-deazaflavin to be used in specific reduction reactions
-
-
?
coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate. No activity with NAD+
-
-
r
coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate. No activity with NAD+
-
-
?
coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate. No activity with NAD+
-
-
?
oxidized coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
-
-
-
?
oxidized coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
-
-
-
?
reduced coenzyme F420 + NADP+
coenzyme F420 + NADPH + H+
-
direct hydride transfer process, A side-specific, with respect to the prochiral center C5 of the dihydro-8-hydroxy-5-deazaflavin cofactor
-
-
?
reduced coenzyme F420 + NADP+
coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
of the two substrates NADP+ has to bind first, the binding being associated with an induced fit. The stereochemical analysis of the hydrode transfer leads to the conclusion that the observed orientation of the Si-face of coenzyme F420 towards the Si-face of NADP+ allows only the transfer of the proS hydrogen at C5 to the proS position at C4 and vice versa
-
-
?
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
the enzyme is Si face specific with respect to C5 of reduced coenzyme F420 and Si face specific with respect to C4 of NADP+. The enzyme is specific for both coenzyme F420 and NADP+/NADPH
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
the enzyme exhibits a sequential kinetic mechanism
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
the enzyme exhibits a sequential kinetic mechanism
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
FNO catalyzes the NADP+ reduction more efficiently compared to NADPH oxidation
-
-
r
additional information
?
-
-
F420 and the F420 redox moiety, FO, are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O2 of riboflavin-derived coenzymes (FMN and FAD), and, in general, have a more negative redox potential than NAD(P)+. A convenient synthesis of FO is achieved by improving the redox stability of synthetic intermediates containing a polar, electron-rich aminophenol fragment, Fno enzyme activity is restored with FO in the absence of F420, method optimization, overview
-
-
-
additional information
?
-
-
effects of side chain length of residue Il135 on the donor-acceptor distance between NADP+ and the F420 precursor, FO, overview
-
-
-
additional information
?
-
NADP+ binding site structure, overview. A F420-dependent enzyme
-
-
-
additional information
?
-
-
NADP+ binding site structure, overview. A F420-dependent enzyme
-
-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
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
coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
-
the main function of this oxidoreductase is probably to provide cells with reduced 8-hydroxy-5-deazaflavin to be used in specific reduction reactions
-
-
?
reduced coenzyme F420 + NADP+
coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
O29370
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
-
r
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
Q47RA9
-
-
-
r
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
NaCl
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
the enzyme is not inhibited by iodoacetamide (20 mM), chloromercuribenzoate (20 mM) or N-ethylmaleimide (7 mM) when preincubated for 30 min in the presence of these reagents. The enzyme is not inactivated by dioxygen
-
additional information
inhibitor prediction studies reveal that lovastatin and compactin (mevastatin) produced more affinity for model structure of NADP oxidoreducatse as compared to F420. It indicates that the lovastatin and compactin (mevastatin) compounds (negative regulator) may act as potential inhibitor of F420 dependent NADP oxidoreducatse protein
-
additional information
-
inhibitor prediction studies reveal that lovastatin and compactin (mevastatin) produced more affinity for model structure of NADP oxidoreducatse as compared to F420. It indicates that the lovastatin and compactin (mevastatin) compounds (negative regulator) may act as potential inhibitor of F420 dependent NADP oxidoreducatse protein
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0155
7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate
-
pH 6.0, 22°C
0.0625
coenzyme F420
-
pH and temperature not specified in the publication
0.07
NADP+
pH 8.0, temperature not specified in the publication
0.05
NADPH
pH 6.0, temperature not specified in the publication
10
NADPH
pH 6.0, 25°C, recombinant mutant R51E/R55A; pH 6.0, 25°C, recombinant mutant R51V/R55V
12
NADPH
pH 6.0, 25°C, recombinant mutant T28A/R51V; pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V
180
NADPH
above, pH 6.0, 25°C, recombinant mutant R51A; above, pH 6.0, 25°C, recombinant mutant R51V
500
NADPH
above, pH 6.0, 25°C, recombinant mutant R51E/R55A; above, pH 6.0, 25°C, recombinant mutant R51E/R55N; above, pH 6.0, 25°C, recombinant mutant R51E/R55S; above, pH 6.0, 25°C, recombinant mutant R51V/R55V; above, pH 6.0, 25°C, recombinant mutant R55N; above, pH 6.0, 25°C, recombinant mutant S50E; above, pH 6.0, 25°C, recombinant mutant S50E/R55A; above, pH 6.0, 25°C, recombinant mutant S50E/R55V; above, pH 6.0, 25°C, recombinant mutant S50Q; above, pH 6.0, 25°C, recombinant mutant T28A/R51V; above, pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V
0.0036
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135A; with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135G
0.0037
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135V
0.004
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant wild-type enzyme
0.3
oxidized coenzyme F420
pH 6.0, temperature not specified in the publication
0.0077
reduced coenzyme F420
-
pH and temperature not specified in the publication
0.15
reduced coenzyme F420
pH 8.0, temperature not specified in the publication
additional information
additional information
-
substrate binding studies, steady-state and pre steady-state kinetic analysis with wild-type enzyme Fno and Ile135 Fno mutant variants, I135A, I135V, and I135G, overview. Steady-state kinetic analysis of wild-type Fno and the variants show classical Michaelis-Menten kinetics with varying FO concentrations. The data reveal a decreased kcat as side chain length decreased, with varying FO concentrations. The steady-state plots reveal non-Michaelis-Menten kinetic behavior when NADPH is varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs420 versus time trace reveals biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displays an increased rate constant as side chain length decreases. The rate constant for the second phase, remained about 2/s for each variant. Pre-steady-state data with F420 cofactor and NADPH for the enzyme Fno mutant variants reveal biphasic kinetics with a fast and slow phase, similar with wild-type Fno, overview
-
additional information
additional information
-
the enzyme shows half-site reactivity and negative cooperativity (Koshland-Nemethy-Filmer model) in the reversible reduction of NADP+ through the transfer of a hydride from the reduced F420 cofactor, steady-state kinetic analysis revealing classical Michaelis-Menten kinetics with varying concentrations of the F420 redox moiety, and non-Michaelis-Menten kinetic behavior when NADPH is varied. Pre-steady-state, stopped flow, Single-turnover, and steady-state kinetics, detailed overview
-
additional information
additional information
steady-state kinetics
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
35.57
7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate
-
pH 6.0, 22°C
1.6
NADPH
above, pH 6.0, 25°C, recombinant mutant R51A; pH 6.0, 25°C, recombinant mutant R51E/R55A
2.7
NADPH
pH 6.0, 25°C, recombinant mutant R51E/R55N; pH 6.0, 25°C, recombinant mutant S50E; pH 6.0, 25°C, recombinant mutant T28A/R51V
2.8
NADPH
pH 6.0, 25°C, recombinant mutant R51V/R55V; pH 6.0, 25°C, recombinant mutant R55N
3.2
NADPH
pH 6.0, 25°C, recombinant mutant R51A; pH 6.0, 25°C, recombinant mutant R55V
3.3
NADPH
pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V; pH 6.0, 25°C, recombinant mutant T28A/R55A; pH 6.0, 25°C, recombinant wild-type enzyme
0.7
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135G
1.6
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135A
1.8
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135V
5.3
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant wild-type enzyme
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2295
7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate
-
pH 6.0, 22°C
0.16
NADPH
pH 6.0, 25°C, recombinant mutant R51E/R55A; pH 6.0, 25°C, recombinant wild-type enzyme
0.28
NADPH
pH 6.0, 25°C, recombinant mutant R51V/R55V; pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V
0.42
NADPH
pH 6.0, 25°C, recombinant mutant R51E/R55N; pH 6.0, 25°C, recombinant mutant R55A
194.4
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135G
444.4
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135A
486.5
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135V
1325
oxidized coenzyme F420
-
with F420 precursor, FO, pH 6.5, 22°C, recombinant wild-type enzyme
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3.5
reduction of oxidized coenzyme F420 with NADPH
5.5
7.4
7.9
9.2
pH optimum for reduction of NADP+ with reduced coenzyme F420
5.5
pH optimum for reduction of oxidized coenzyme F420 with NADPH
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.3 - 6.7
-
pH 4.3: about 50% of maximal activity, pH 6.7: about 50% of maximal activity
4.5 - 6.5
-
pH 4.5: about 45% of maximal activity, pH 6.5: about 35% of maximal activity, reduction of oxidized coenzyme F420 with NADPH
7 - 9
-
pH 7.0: about 60% of maximal activity, pH 9.0: about 50% of maximal activity, reduction of NADP+ with reduced coenzyme F420
7.5 - 10
pH 7.5: about 50% of maximal activity, pH 10.0: about 90% of maximal activity, reduction of NADP+ with reduced coenzyme F420
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
22
37
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
15 - 50
-
15°C: about 45% of maximal activity, 50°C: about 45% of maximal activity
25 - 90
the enzyme displays highest activity between 60°C and 70°C. The activity at 65°C is almost 4fold higher than that at 25°C. The apparent melting temperature of Tfu-FNO is 75°C, about 50% of maximal activity at 40°C and 90°C
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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PDB
SCOP
CATH
ORGANISM
UNIPROT
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
28000
45000
-
1 * 60000 (alpha) + 1 * 50000 (beta) + 1 * 45000 (gamma), SDS-PAGE
50000
-
1 * 60000 (alpha) + 1 * 50000 (beta) + 1 * 45000 (gamma), SDS-PAGE
60000
-
1 * 60000 (alpha) + 1 * 50000 (beta) + 1 * 45000 (gamma), SDS-PAGE
90000
gradient gel electrophoresis under nondenaturating conditions
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homotetramer
trimer
homotetramer
4 * 23500, calculated from sequence; 4 * 28000, SDS-PAGE
homotetramer
-
4 * 23500, calculated from sequence; 4 * 28000, SDS-PAGE
-
trimer
-
1 * 60000 (alpha) + 1 * 50000 (beta) + 1 * 45000 (gamma), SDS-PAGE
trimer
-
1 * 60000 (alpha) + 1 * 50000 (beta) + 1 * 45000 (gamma), SDS-PAGE
-
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour diffusion method, crystal structure of the enzyme bound with coenzyme F420. The structure resolved to 1.65 A contains two domains: an N-terminal domain characteristic of a dinucleotide-binding Rossmann fold and a smaller C-terminal domain. The nicotinamide and the deazaflavin part of the two coenzymes are bound in the cleft between the domains such that the Si-faces of both face each other at a distance of 3.1 A, which is optimal for hydride transfer
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25 - 90
the enzyme displays highest activity between 60°C and 70°C. The activity at 65°C is almost 4fold higher than that at 25°C. The apparent melting temperature of Tfu-FNO is 75°C, about 50% of maximal activity at 40°C and 90°C
additional information
additional information
F420-dependent enzyme Tfu-FNO is highly thermostable
additional information
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F420-dependent enzyme Tfu-FNO is highly thermostable
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
in 0.08 M NaCl a half-life of 9 h is found which increased to greater than 6000 h in 1 M NaCl (both in the presence of 2% ethylene glycol)
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neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ethylene glycol
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enhanced the stability: half-life increased from 9 to 750 h in 18% ethylene glycol (in 0.08 M NaCl)
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-75°C, 2 months, at low concentration (0.066 mg/ml), in the presence of ethylene glycol, 20% (v/v), no loss of activity
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4°C, at low concentration (0.066 mg/ml), in the presence of ethylene glycol, 20% (v/v), 22% of the activity is lost
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4°C, at low concentration (0.066 mg/ml), the purified enzyme loses 84% of its activity in 2 days
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4°C, purified enzyme at a protein concentration of 20 mg/ml is completely stable when stored at 4°C in 50 mM Tricine/KOH pH 8.0 containing 0.2 M KCl
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme from cell-free extracts of Escherichia coli strain C41(DE3) by heat treatment at 90°C for 30 min, ammonium sulfate fractionation with addition of 0.05% polyethylenimine, followed by an ion exchange chromatography, ultrafiltration, and gel filtration
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recombinant enzyme from Escherichia coli by ammonium sulfate fractionation followed by anion exchange chromatography, DNase I treatment during protein purification is essential to remove residual DNA
recombinant wild-type and mutant enzymes from cell-free extracts of Escherichia coli strain C41(DE3) by heat treatment at 90°C for 30 min, ammonium sulfate fractionation with addition of 0.05% polyethylenimine, followed by an ion exchange chromatography, ultrafiltration, and gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and recombinant expression in Escherichia coli strain C41(DE3)
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gene Tfu-fno, DNA and amino acid sequence determination and analysis, recombinant expression in Escherichia coli
overexpression in Escherichia coli
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain C41(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
I135A
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site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
I135G
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site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
I135V
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site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
R51A
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R51E/R55A
site-directed mutagenesis, the mutant shows similar catalytic efficiency compared to the wild-type enzyme
R51E/R55N
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R51E/R55S
site-directed mutagenesis, the mutant shows similar catalytic efficiency compared to the wild-type enzyme
R51V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R51V/R55V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R55A
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R55N
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R55S
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R55V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
S50E
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
S50E/R55A
site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type enzyme
S50E/R55V
site-directed mutagenesis, the mutant shows slightly increased catalytic efficiency compared to the wild-type enzyme
S50Q
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
T28A
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
T28A/R51V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
T28A/R51V/R55V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
T28A/R55A
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
additional information
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pre-steady-state data with F420 cofactor and NADPH for the enzyme Fno mutant variants reveal biphasic kinetics with a fast and slow phase, similar with wild-type Fno, overview
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LINKS TO OTHER DATABASES (specific for EC-Number 1.5.1.40)
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