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Information on EC 1.5.1.40 - 8-hydroxy-5-deazaflavin:NADPH oxidoreductase and Organism(s) Archaeoglobus fulgidus and UniProt Accession O29370
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1.5.1.40 8-hydroxy-5-deazaflavin:NADPH oxidoreductaseIUBMB Comments
The enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate .
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Word Map
- 1.5.1.40
-
methanogen
- archaea
-
hydride
- 8-hydroxy-5-deazaflavins
-
methanococcus
- hydrogenase
- vannielii
- thermoautotrophicum
-
methanobacterium
-
methanobrevibacter
- griseus
-
methanogenesis
-
stereochemical
- smithii
-
si-face
- methane
- fulgidus
- ch4
The taxonomic range for the selected organisms is: Archaeoglobus fulgidus
The expected taxonomic range for this enzyme is: Bacteria, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
8-hydroxy-5-deazaflavin-dependent nadp+ reductase, tfu-fno, f420-dependent nadp reductase, f420h2:nadp oxidoreductase, nadp+:f420 oxidoreductase, f420-dependent nadp oxidoreductase, 8-hydroxy-5-deazaflavin:nadph oxidoreductase, f420h2:nadp+ oxidoreductase, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
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].
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
additional information
?
-
effects of side chain length of residue Il135 on the donor-acceptor distance between NADP+ and the F420 precursor, FO, overview
-
-
?
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
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
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
-
-
-
r
INHIBITOR
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
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0023
NADPH
phase I, pH 6.5, 22°C, recombinant wild-type enzyme
0.062
NADPH
phase II, pH 6.5, 22°C, recombinant wild-type enzyme
0.0036
oxidized coenzyme F420
with F420 precursor, FO, pH 6.5, 22°C, recombinant mutant I135A
0.0036
oxidized coenzyme F420
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
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
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
5.41
NADPH
phase II, pH 6.5, 22°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
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
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
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
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
I135A
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
I135G
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
I135V
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
additional information
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
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
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
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloning and recombinant expression in Escherichia coli strain C41(DE3)
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain C41(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kunow,J.; Schwrer, B.; Stetter, K.O.; Thauer, R.K.
A F420-dependent NADP reductase in the extremely thermophilie sulfate-reducing Archaeoglobus fulgidus
Arch. Microbiol.
160
199-205
1993
Archaeoglobus fulgidus (O29370)
-
Warkentin, E.; Mamat, B.; Sordel-Klippert, M.; Wicke, M.; Thauer, R.K.; Iwata, M.; Iwata, S.; Ermler, U.; Shima, S.
Structures of F420H2:NADP+ oxidoreductase with and without its substrates bound
EMBO J.
20
6561-6569
2001
Archaeoglobus fulgidus (O29370)
Le, C.; Oyugi, M.; Joseph, E.; Nguyen, T.; Ullah, M.; Aubert, J.; Phan, T.; Tran, J.; Johnson-Winters, K.
Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F420H2 NADP+ oxidoreductase A kinetic analysis
Biochem. Biophys. Rep.
9
114-120
2017
Archaeoglobus fulgidus (O29370)
Joseph, E.; Le, C.Q.; Nguyen, T.; Oyugi, M.; Hossain, M.S.; Foss, F.W.; Johnson-Winters, K.
Evidence of negative cooperativity and half-site reactivity within an F420-dependent enzyme kinetic analysis of F420H2 NADP+ oxidoreductase
Biochemistry
55
1082-1090
2016
Archaeoglobus fulgidus (O29370)
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