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Information on EC 1.12.1.3 - hydrogen dehydrogenase (NADP+)
for references in articles please use BRENDA:EC1.12.1.3
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EC Tree 1 Oxidoreductases
1.12 Acting on hydrogen as donor
1.12.1 With NAD+ or NADP+ as acceptor
1.12.1.3 hydrogen dehydrogenase (NADP+)
IUBMB Comments
The protein from the bacterium Desulfovibrio fructosovorans is an iron-sulfur protein that exclusively functions as a hydrogen dehydrogenase , while the enzyme from the archaeon Pyrococcus furiosus is a nickel, iron, iron-sulfur protein, that is part of a heterotetrameric complex where the α and δ subunits function as a hydrogenase while the β and γ subunits function as sulfur reductase (EC 1.12.98.4, sulfhydrogenase). Different from EC 1.12.1.5, hydrogen dehydrogenase [NAD(P)+].
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
- 1.12.1.3
- hydrogenases
-
pyrococcus
- furiosus
-
desulfovibrio
- fructosovorans
-
hyperthermophilic
- synthesis
- clostridium
- ferredoxin
-
heterotetrameric
- pasteurianum
- nife-hydrogenase
- viologen
-
proton-coupled
- mononucleotide
-
midpoint
-
hydride
-
photolysis
-
binuclear
-
hydrogen-producing
- denitrificans
-
h2-dependent
-
time-resolved
-
typified
-
metalloenzyme
- taurus
-
nickel-iron
- energy production
showSynonyms
nadp-reducing hydrogenase, soluble hydrogenase i, nadp-dependent hydrogenase, nadp-linked hydrogenase, [nife]-hydrogenase i, nadph-dependent hydrogenase i, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
hydrogenase [ambiguous]
-
-
-
-
NADP-linked hydrogenase
-
-
-
-
NADP-reducing hydrogenase
-
-
-
-
NADPH-dependent [NiFe]-hydrogenase
NiFe-hydrogenase
SHI
[NiFe]-hydrogenase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
H2 + NADP+ = H+ + NADPH
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
BRENDA
MetaCyc
hydrogen oxidation III (anaerobic, NADP), hydrogen production IV
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SYSTEMATIC NAME
IUBMB Comments
hydrogen:NADP+ oxidoreductase
The protein from the bacterium Desulfovibrio fructosovorans is an iron-sulfur protein that exclusively functions as a hydrogen dehydrogenase [1], while the enzyme from the archaeon Pyrococcus furiosus is a nickel, iron, iron-sulfur protein, that is part of a heterotetrameric complex where the alpha and delta subunits function as a hydrogenase while the beta and gamma subunits function as sulfur reductase (EC 1.12.98.4, sulfhydrogenase). Different from EC 1.12.1.5, hydrogen dehydrogenase [NAD(P)+].
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CAS REGISTRY NUMBER
COMMENTARY
9027-05-8
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
H+ + NADPH
H2 + NADP+
Substrates: -
Products: -
Products: -
?
H+ + reduced benzyl viologen
H2 + oxidized benzyl viologen
Substrates: -
Products: -
Products: -
?
H2 + 2,6-dichlorophenolindophenol
H+ + ?
Substrates: -
Products: -
Products: -
r
H2 + NAD+
H+ + NADH
Substrates: specific activity with NAD+ as electron acceptor is 150times lower than that of NADP+
Products: -
Products: -
r
H2 + oxidized ferredoxin
H+ + reduced ferredoxin
-
Substrates: -
Products: -
Products: -
?
H2 + oxidized methylene blue
H+ + reduced methylene blue
Substrates: -
Products: -
Products: -
r
NADPH + H+
H2 + NADP+
Substrates: -
Products: -
Products: -
?
oxidized benzyl viologen + H2
reduced benzyl viologen + H+
-
Substrates: -
Products: -
Products: -
?
additional information
?
-
-
Substrates: the enzyme introduces corrosion of 316L stainless steel in the presence of NAD+. The enzyme induces the occurrence of cathodic and anodic microsites on the same electrode surface
Products: -
Products: -
?
H2 + NADP+
H+ + NADPH
-
Substrates: the enzyme likely functions in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration
Products: -
Products: -
r
H2 + NADP+
H+ + NADPH
-
Substrates: the hydrogen consumption activity is 1 order of magnitude higher than the hydrogen production activity
Products: -
Products: -
r
H2 + NADP+
H+ + NADPH
Substrates: -
Products: -
Products: -
?
H2 + NADP+
H+ + NADPH
Solidesulfovibrio fructosivorans
-
Substrates: enzyme does not utilize NAD+
Products: -
Products: -
?
H2 + NADP+
H+ + NADPH
Solidesulfovibrio fructosivorans
-
Substrates: heteromeric complex in which HndA and HndC constitute the NADP-reducing unit and HndD constitutes the hydrogenase unit
Products: -
Products: -
?
H2 + NADP+
H+ + NADPH
Solidesulfovibrio fructosivorans
-
Substrates: biological function probably involves production of reduced pyridine nucleotides
Products: -
Products: -
?
H2 + NADP+
H+ + NADPH
-
Substrates: in the presence of ferredoxin and a ferredoxin-NADP reductase
Products: -
Products: -
?
H2 + NADP+
H+ + NADPH
Substrates: -
Products: -
Products: -
r
H2 + oxidized benzyl viologen
H+ + reduced benzyl viologen
-
Substrates: -
Products: -
Products: -
?
H2 + oxidized benzyl viologen
H+ + reduced benzyl viologen
Substrates: -
Products: -
Products: -
r
H2 + oxidized methyl viologen
H+ + reduced methyl viologen
-
Substrates: -
Products: -
Products: -
?
H2 + oxidized methyl viologen
H+ + reduced methyl viologen
Solidesulfovibrio fructosivorans
-
Substrates: reduction requires all four subunits, a form lacking HndD shows no activity towards methyl viologen
Products: -
Products: -
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
H2 + oxidized ferredoxin
H+ + reduced ferredoxin
-
Substrates: -
Products: -
Products: -
?
additional information
?
-
-
Substrates: the enzyme introduces corrosion of 316L stainless steel in the presence of NAD+. The enzyme induces the occurrence of cathodic and anodic microsites on the same electrode surface
Products: -
Products: -
?
H2 + NADP+
H+ + NADPH
-
Substrates: the enzyme likely functions in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration
Products: -
Products: -
r
H2 + NADP+
H+ + NADPH
Substrates: -
Products: -
Products: -
?
H2 + NADP+
H+ + NADPH
Solidesulfovibrio fructosivorans
-
Substrates: biological function probably involves production of reduced pyridine nucleotides
Products: -
Products: -
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NAD+
specific activity as electron acceptor is 150times lower than that of NADP+
[Ni-Fe]-center
metal content Ni:Fe is 1:25 for the native enzyme and 1:32 for the recombinant enzyme
-
FAD
the alphabetagammadelta subunit contains 1.01 mol of FAD
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe
Iron
Nickel
Fe
Solidesulfovibrio fructosivorans
-
the HndA subunit belongs to the [2Fe-2S] ferredoxin family. HndB is a FeS subunit
Iron
NiFe-protein, the alphabetagammadelta subunit contains 0.8 mol of Ni
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.007
NADPH
80°C, pH 8.4, recombinant enzyme
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
8.4
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
80
90
H2 production with methyl viologen
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
60 - 90
-
60°C: about 40% of maximal activity, 90°C: about 80% of maximal activity
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
E7FHU4: sulfhydrogenase 1 subunit delta, E7FI44: sulfhydrogenase 1 subunit alpha
SwissProt
brenda
Solidesulfovibrio fructosivorans
E7FHU4: sulfhydrogenase 1 subunit delta, E7FI44: sulfhydrogenase 1 subunit alpha
SwissProt
brenda
E7FI44: subunit alpha, Q8U2E5: subunit beta, Q8U2E4: subunit gamma, E7FHU4: subunit delta
SwissProt
brenda
Q8U2E5: gene PF0891, Q8U2E4: gene PF0892, E7FHU4: gene PF0893, E7FI44: gene PF0894
SwissProt
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
Highest Expressing Human Cell Lines
Cell Line Links Show AA Sequence and Transmembrane Helices (2367 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
150000
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
(alphabetagammadelta)n, n is 3 or more
heterotetramer
tetramer
heterotetramer
a maturation pathway is proposed where, in the terminal step, the NiFe-containing catalytic subunit assembles with NADPH-oxidizing trimeric form to give the active holoenzym
tetramer
Solidesulfovibrio fructosivorans
-
four different subunits with the following molecular weights: HndA 19000, HndB 14000, HndC 52000, HndD 64000, SDS-PAGE
tetramer
Solidesulfovibrio fructosivorans
-
four different subunits with the following molecular weights: HndA 18800, HndB 13800, HndC 52000, HndD 63400, SDS-PAGE
tetramer
Solidesulfovibrio fructosivorans
-
the HndC subunit (52 kDa) corresponds to the NADP-reducing unit, and the HndD subunit (63.5 kDa) is homologous to Clostridium pasteurianum hydrogenase. The role of HndA and HndB subunits (18.8 kDa and 13.8 kDa, respectively) in the complex remains unknown. HndAc and HndB can form a heterodimeric intermediate in the electron transfer between the hydrogenase (HndD) active site and the NADP reduction site in HndC
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
80
90
80
203 min, 50% loss of activity of evolution reaction
90
t1/2: 30 h for the native enzyme, t1/2: 1 h for the recombinant enzyme
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
50% loss of enzyme activity when extracts are exposed for 15 min to air
Solidesulfovibrio fructosivorans
-
spontaneous proteolysis of the HndA subunit occurs during desalting preparation performed in a Centricon
Solidesulfovibrio fructosivorans
-
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
t1/2 for the native enzyme is 25 h at 23°C in presence of air, t1/2 for for the recombinant enzyme is 20 h at 23°C in presence of air
746235
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
an active, purified 12-His tagged recombinant SHI (rSHI) is obtained by one-step affinity adsorption on nickel-charged resin
-
C-terminal domain of subunit HndA and N-terminal domain of subunit HndD recombinantly expressed in Escherichia coli
Solidesulfovibrio fructosivorans
-
one-step purification of the recombinant enzyme including a polyhistidine affinity tag results in a 50fold increase in yield compared to the four-step purification procedure for the native enzyme
strategies for reliable and improved large-scale production of Pyrococcus furiosus with integrated purification of hydrogenase I
-
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
construction of a strain of Pyrococcus furiosus that contains a second set of the eight genes involved in the maturation of the catalytic subunit and insertion of the [NiFe]-site, along with a second set of the four genes encoding the NADPH-dependent cytoplasmic [NiFe]-hydrogenase (SHI) structural subunits. This results in a 40% higher yield of the purified affinity-tagged enzyme and the content of the inactive trimeric form decreases to 5% of the total protein
expression in Escherichia coli. The native Escherichia coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from Pyrococcus furiosus. The expression is induced by anaerobic conditions, whereby Escherichia coli is grown aerobically and production of recombinant hydrogenase is achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme is purified and shown to be functionally similar to the native enzyme purified from Pyrococcus furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins
expression of C-terminal domain of subunit HndA and N-terminal domain of subunit HndD in Escherichia coli
Solidesulfovibrio fructosivorans
-
overexpressed in Thermococcus kodakarensis KOD1 results in more than 1200-fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector
-
the four gene operon encoding the enzyme is overexpressed in Pyrococcus furiosus and includes a polyhistidine affinity tag
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
energy production
-
application in biofuel cells, to generate an electric current
synthesis
synthesis
-
biohydrogen production. Photochemical hydrogen production system using zinc porphyrin and hydrogenase in a micellar system of cetyltrimethylammonium bromide. Cetyltrimethylammonium bromide acts as a cationic surfactant to effectively separate the charges
synthesis
-
biohydrogen production. The enzyme is immobilized between two layers of montmorillonite clay and poly(butylviologen) mixture. The amount of hydrogen produced relates closely to the applied potential, buffer pH and temperature
synthesis
biohydrogen production from sugars using a mixture of enzymes in an in vitro cell-free synthetic pathway. Development of this process at an industrial scale is limited by the availability of the H2-producing enzyme
synthesis
-
the study provides a cost-efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production. It has great potential in biocatalysis, bioelectrochemistry, and clean energy production
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Casalot, L.; Valette, O.; De Luca, G.; Dermoun, Z.; Rousset, M.; de Philip, P.
Construction and physiological studies of hydrogenase depleted mutants of Desulfovibrio fructosovorans
FEMS Microbiol. Lett.
214
107-112
2002
Solidesulfovibrio fructosivorans
brenda
De Luca, G.; Asso, M.; Belaiech, J.P.; Dermoun, Z.
Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli
Biochemistry
37
2660-2665
1998
Solidesulfovibrio fructosivorans
brenda
De Luca, G.; De Philip, P.; Rousset, M.; Belaich, J.P.; Dermoun, Z.
The NADP-reducing hydrogenase of Desulfovibrio fructosovorans: evidence for a native complex with hydrogen-dependent methyl-viologen-reducing activity
Biochem. Biophys. Res. Commun.
248
591-596
1998
Solidesulfovibrio fructosivorans
brenda
Dermoun, Z.; De Luca, G.; Asso, M.; Bertrand, P.; Guerlesquin, F.; Guigliarelli, B.
The NADP-reducing hydrogenase from Desulfovibrio fructosovorans: functional interaction between the C-terminal region of HndA and the N-terminal region of HndD subunits
Biochim. Biophys. Acta
1556
217-225
2002
Solidesulfovibrio fructosivorans
brenda
Malki, S.; De Luca, G.; Fardeau, M.L.; Rousset, M.; Belaich, J.P.; Dermoun, Z.
Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans
Arch. Microbiol.
167
38-45
1997
Solidesulfovibrio fructosivorans
brenda
Malki, S.; Saimmaime, I.; De Luca, G.; Rousset, M.; Dermoun, Z.; Belaich, J.P.
Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibrio fructosovorans
J. Bacteriol.
177
2628-2636
1995
Solidesulfovibrio fructosivorans
brenda
Wunschiers, R.; Senger, H.; Schulz, R.
Electron pathways involved in H2-metabolism in the green alga Scenedesmus obliquus
Biochim. Biophys. Acta
1503
271-278
2001
Tetradesmus obliquus
brenda
Mertens, R.; Liese, A.
Biotechnological applications of hydrogenases
Curr. Opin. Biotechnol.
15
343-348
2004
Clostridium paraputrificum, Cupriavidus necator, Desulfovibrio vulgaris, Pyrococcus furiosus, Thiocapsa roseopersicina
brenda
Kanai, T.; Ito, S.; Imanaka, T.
Characterization of a cytosolic NiFe-hydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1
J. Bacteriol.
185
1705-1711
2003
Thermococcus kodakarensis (Q8NKS2), Thermococcus kodakarensis (Q8NKS3), Thermococcus kodakarensis (Q8NKS4), Thermococcus kodakarensis (Q8NKS5)
brenda
Nouailler, M.; Morelli, X.; Bornet, O.; Chetrit, B.; Dermoun, Z.; Guerlesquin, F.
Solution structure of HndAc: a thioredoxin-like domain involved in the NADP-reducing hydrogenase complex
Protein Sci.
15
1369-1378
2006
Solidesulfovibrio fructosivorans
brenda
van Haaster, D.J.; Silva, P.J.; Hagedoorn, P.L.; Jongejan, J.A.; Hagen, W.R.
Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus
J. Bacteriol.
190
1584-1587
2008
Pyrococcus furiosus
brenda
Germer, F.; Zebger, I.; Saggu, M.; Lendzian, F.; Schulz, R.; Appel, J.
Overexpression, isolation and spectroscopic characterization of the bidirectional [NiFe]-hydrogenase from Synechocystis sp. PCC 6803
J. Biol. Chem.
284
36462-36472
2009
Synechocystis sp.
brenda
Rieckenberg, F.; Gtz, K.; Hilterhaus, L.; Liese, A.; Zeng, A.P.
Strategies for reliable and improved large-scale production of Pyrococcus furiosus with integrated purification of hydrogenase I
Bioprocess Biosyst. Eng.
37
2475-2482
2014
Pyrococcus furiosus
brenda
Chandrayan, S.K.; Wu, C.H.; McTernan, P.M.; Adams, M.W.
High yield purification of a tagged cytoplasmic [NiFe]-hydrogenase and a catalytically-active nickel-free intermediate form
Protein Expr. Purif.
107
90-94
2015
Pyrococcus furiosus (Q8U2E5 and Q8U2E4 and E7FHU4 and E7FI44), Pyrococcus furiosus
brenda
Sun, J.; Hopkins, R.; Jenney Jr., F.; McTernan, P.; Adams, M.
Heterologous expression and maturation of an NADP-Dependent [NiFe]-Hydrogenase A key enzyme in biofuel production
PLoS ONE
5
e10526
2010
Pyrococcus furiosus (E7FI44 AND Q8U2E5 AND Q8U2E4 AND E7FHU4), Pyrococcus furiosus
brenda
Song, Y.; Liu, M.; Xie, L.; You, C.; Sun, J.; Zhang, Y.P.J.
A recombinant 12-His tagged Pyrococcus furiosus soluble [NiFe]-hydrogenase I overexpressed in Thermococcus kodakarensis KOD1 facilitates hydrogen-powered in vitro NADH regeneration
Biotechnol. J.
14
e1800301
2019
Pyrococcus furiosus
brenda
Wu, C.H.; Ponir, C.A.; Haja, D.K.; Adams, M.W.W.
Improved production of the NiFe-hydrogenase from Pyrococcus furiosus by increased expression of maturation genes
Protein Eng. Des. Sel.
31
337-344
2018
Pyrococcus furiosus (E7FHU4 AND E7FI44), Pyrococcus furiosus ATCC 43587 (E7FHU4 AND E7FI44)
brenda
EXTERNAL LINKS (specific for EC-Number 1.12.1.3)
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