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Information on EC 1.12.1.2 - hydrogen dehydrogenase and Organism(s) Cupriavidus necator and UniProt Accession P22319
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1.12.1.2 hydrogen dehydrogenaseIUBMB Comments
An iron-sulfur flavoprotein (FMN or FAD). Some forms of this enzyme contain nickel.
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Word Map
- 1.12.1.2
- alcaligenes
- eutrophus
- hydrogenases
-
ralstonia
-
hydrogen-oxidizing
-
h2-dependent
-
lithoautotrophic
-
thiocapsa
- roseopersicina
-
h2-oxidizing
-
oxygen-tolerant
- synthesis
The taxonomic range for the selected organisms is: Cupriavidus necator
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
soluble hydrogenase, hoxefuyh, hox hydrogenase, nad-dependent hydrogenase, nad-reducing hydrogenase, soluble [nife]-hydrogenase, bidirectional [nife] hydrogenase, fehyd, iron-hydrogenase, bidirectional nife-hydrogenase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
dehydrogenase, hydrogen
-
-
-
-
H2:NAD+ oxidoreductase
-
-
-
-
hydrogenase
-
-
-
-
SH
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
H2 + NAD+ = H+ + NADH
catalytic activity may be regulated by the reduction of a regulatory center followed by a series of structural changes
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
hydrogen:NAD+ oxidoreductase
An iron-sulfur flavoprotein (FMN or FAD). Some forms of this enzyme contain nickel.
CAS REGISTRY NUMBER
COMMENTARY
37256-54-5
deleted
9027-05-8
-
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
ferrocyanide + NAD(P)H
ferricyanide + NAD(P)+
-
enzyme complex including HoxI
-
-
?
H+ + NADH
H2 + NAD+
module HoxFU shows clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple
-
-
r
NAD(P)H + H+ + oxidized 2,6-dichlorophenolindophenol
NAD(P)+ + reduced 2,6-dichlorophenolindophenol
-
-
-
?
NADH + oxidized 2,6-dichlorophenol indophenol
NAD+ + reduced 2,6-dichlorophenol indophenol
-
-
-
-
?
NADH + reduced 3-acetylpyridine adenine dinucleotide
NAD+ + oxidized 3-acetylpyridine adenine dinucleotide
-
-
-
-
?
NADPH + K3Fe(CN)6
?
-
low reaction with hexameric enzyme form, no reaction with tetrameric enzyme form
-
-
?
oxidized 2,6-dichlorophenol indophenol + NADH + H+
reduced 2,6-dichlorophenolindophenol + NAD+
-
-
-
-
r
oxidized cytochrome c + NAD(P)H
reduced cytchrome c + NAD(P)+
-
-
-
?
oxidized methylene blue + NAD(P)H
reduced methylene blue + NAD(P)+
-
-
-
?
oxidized methylene blue + NADH + H+
reduced methylene blue + NAD+
-
-
-
-
r
H2 + ferricyanide
H+ + ferrocyanide
-
708% activity compared to electron acceptor NAD+
-
?
H2 + NAD+
H+ + NADH
-
artificial electron acceptors: Janus green, 2,6-dichlorophenolindophenol, phenazine methosulfate, menaquinone, ubiquinone, cytochrome c
-
r
H2 + NAD+
H+ + NADH
-
hydrogenase produces superoxide free radical anions, which are responsible for enzyme inactivation
-
?
H2 + NAD+
H+ + NADH
-
artificial electron acceptor benzyl viologen
-
r
H2 + NAD+
H+ + NADH
-
artificial electron acceptor methyl viologen
-
r
H2 + NAD+
H+ + NADH
-
enzyme also has diaphorase and NAD(P)H oxidase activity
-
r
H2 + NAD+
H+ + NADH
-
artificial electron acceptor ferricyanide
-
r
H2 + NAD+
H+ + NADH
-
artificial electron acceptor methylene blue
-
r
H2 + NAD+
H+ + NADH
-
utilization of H2 as energy source during autotrophic growth on hydrogen and CO2
-
r
H2 + NAD+
H+ + NADH
-
utilization of H2 as energy source during autotrophic growth on hydrogen and CO2
-
r
H2 + NAD+
H+ + NADH
-
enzyme provides reducing equivalents for CO2 fixation
-
-
r
H2 + NAD+
H+ + NADH
-
the enzyme catalyzes electron transfer from molecular hydrogen to NAD+, thereby producing reducing equivalents for CO2 fixation in the form of NADH
-
-
?
H2 + NAD+
H+ + NADH
module HoxFU shows clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple
-
-
r
H2 + oxidized benzyl viologen
H+ + reduced benzyl viologen
-
-
-
r
H2 + oxidized benzyl viologen
H+ + reduced benzyl viologen
-
-
-
-
r
H2 + oxidized benzyl viologen
H+ + reduced benzyl viologen
-
hydrogenase activity
-
-
r
H2 + oxidized methyl viologen
H+ + reduced methyl viologen
-
-
-
?
H2 + oxidized methyl viologen
H+ + reduced methyl viologen
-
-
-
r
additional information
?
-
-
activity is equally high in atmosperes of 20% O2, 20% N2, or 100% H2, the latter being slightly preferred
-
-
?
additional information
?
-
-
enzyme regulation involves a regulatory hydrogenase RH which acts as a sensor for H2 content, interaction via signal cascade
-
-
?
additional information
?
-
-
the organism can grow on H2 as sole energy source in an oxic environment
-
-
?
additional information
?
-
-
addition of NADH prolonged the lag phase before H2 consumption
-
-
?
additional information
?
-
-
deuterium-hydrogen proton exchange activity of wild-type and mutantenzymes, overview
-
-
?
additional information
?
-
-
enzyme shows both hydrogenase and diaphorase activities, proton channeling
-
-
?
additional information
?
-
-
FMN release induces reduction with NADH, enzyme shows both hydrogenase and diaphorase activities, proton channeling
-
-
?
additional information
?
-
-
tetrameric or hexameric enzyme form: no H2 production from NADPH
-
-
?
additional information
?
-
in protein film electrochemical experiments the electrocatalytic current is unaffected by O2. In aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein is observed, meaning that the formation of reactive oxygen species observed for the native enzyme can be attributed mainly to module HoxFU
-
-
?
additional information
?
-
in protein film electrochemical experiments the electrocatalytic current is unaffected by O2. In aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein is observed, meaning that the formation of reactive oxygen species observed for the native enzyme can be attributed mainly to module HoxFU
-
-
?
additional information
?
-
-
in protein film electrochemical experiments the electrocatalytic current is unaffected by O2. In aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein is observed, meaning that the formation of reactive oxygen species observed for the native enzyme can be attributed mainly to module HoxFU
-
-
?
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
H2 + NAD+
H+ + NADH
-
utilization of H2 as energy source during autotrophic growth on hydrogen and CO2
-
r
H2 + NAD+
H+ + NADH
-
utilization of H2 as energy source during autotrophic growth on hydrogen and CO2
-
r
H2 + NAD+
H+ + NADH
-
enzyme provides reducing equivalents for CO2 fixation
-
-
r
H2 + NAD+
H+ + NADH
-
the enzyme catalyzes electron transfer from molecular hydrogen to NAD+, thereby producing reducing equivalents for CO2 fixation in the form of NADH
-
-
?
additional information
?
-
-
activity is equally high in atmosperes of 20% O2, 20% N2, or 100% H2, the latter being slightly preferred
-
-
?
additional information
?
-
-
enzyme regulation involves a regulatory hydrogenase RH which acts as a sensor for H2 content, interaction via signal cascade
-
-
?
additional information
?
-
-
the organism can grow on H2 as sole energy source in an oxic environment
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4Fe-4S-center
presence of a 4Fe-4S-center in addition to the active site iron
[2Fe-2S]-center
HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters
[4Fe-4S]-center
HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters
FMN
-
2 molecules per enzyme molecule, one of 2 can be reversibly released upon reduction of the enzyme
FMN
-
required, 2 molecules per tetrameric enzyme complex, reduced enzyme reversibly releases half of the FMN bound, kinetics, FMN release induces reduction with NADH, overview
FMN
module HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters
additional information
-
Ni-Fe cofactor, analysis of the non-standard structure of the Ni-Fe cofactor bound at the active site, interaction mechanism with H2, structural changes during reaction, reduction, and inactivation, the cofactor structure is (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO), overview
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CN-
CO
cyanide
-
enzyme contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen
Fe
Fe2+
Iron
Ni
Ni2+
additional information
-
no increase of activity by addition of Co2+, Mn2+, Ni2+ or Fe2+
CN-
-
enzyme contains four cyanides in its active site, one is bound to the Ni2+, the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre, the CN- bound to the nickel ion can be irreversibly removed inducing enzyme inhibition by oxygen
CN-
-
enzyme contains four cyanides in its active site, the Ni2+ bound one is responsible for the insensitivity towards oxygen, the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre, the CN- bound to the nickel ion can be irreversibly removed inducing enzyme inhibition by oxygen
CO
-
bound to the active site, the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre
CO
-
the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre
Fe
-
Ni-Fe enzyme. Analysis of the Ni-Fe cofactor revealed a nonstandard structure, (CN)(O)3NiII(mu-CysS)2FeII(CN)3(CO)
Fe
-
Ni-Fe hydrogenase. Monitoring of the structure and oxidation state of its metal centers during H2 turnover
Fe2+
-
the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre
Iron
-
11.5 iron atoms per enzyme molecule, enzyme contains 2 [4Fe-4S] and 2 [2Fe-2S] clusters
Iron
-
the diaphorase contains 3 [2Fe-2S] cluster, the hydrogenase subunit HoxY contains one [2Fe-2S] cluster coordinated by 9 Cys residues
Iron
the active site iron atom has a standard ligation, i.e., one CO and two cyanide ligands
Ni
-
Ni-Fe enzyme. Analysis of the Ni-Fe cofactor revealed a nonstandard structure, (CN)(O)3NiII(mu-CysS)2FeII(CN)3(CO). The unusual ligation of the Ni by only two thiols plus further (C,O) ligands seems to be a prerequisite of the exceptionally rapid activation of the SH by NADH, involving the loss of an oxygen ligand from the Ni. Evidence for the binding of hydrogen to the open coordination site at Ni has been obtained. The hydrogen cleavage reaction seems not to involve a Ni-C state (Ni(III)-H-). The CN ligand at the Ni may be involved in establishing both rapid activation and oxygen-insensitive catalytic behavior in the SH. Possibly, one important function of the CN is stabilization of the Ni(II) oxidation state throughout the catalytic cycle of hydrogen cleavage
Ni
-
Ni-Fe hydrogenase. Monitoring of the structure and oxidation state of its metal centers during H2 turnover
Ni2+
-
nickel is essential for the catalytic activity of the enzyme
Ni2+
-
the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre
Ni2+
-
the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre, H2 activation solely takes place on Ni2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
diethyl dicarbonate
-
chemical modification of active site His residues results in reduced enzymatic hydrogenase and diaphorase activities, pH-dependence and kinetics of modifications/inactivation
DTNB
-
modification of thiol groups of activated enzyme results in rapid inactivation of both activities, modification of the residues of nonactivated enzyme has small effects on the enzyme activities, pH-dependence and kinetics of modifications/inactivation
iodoacetic acid
-
modification of thiol groups of activated enzyme results in rapid inactivation of both activities, modification of the residues of nonactivated enzyme has small effects on the enzyme activities, pH-dependence and kinetics of modifications/inactivation
O2
-
irreversible enzyme inhibition by oxygen occurs if the CN- bound to Ni2+ is irreversibly removed or if the enzyme is reduced by NADH
O2
incubation of subunits HoxHY with O2 at high potentials causes slow inactivation, but activity is recovered within seconds at potentials below -170 mV at 30°C, even in the presence of 2% O2
additional information
-
release of one FMN reduces the NAD+ reduction by 90%, but is reversible by addition of excess FMN, insensitivity towards oxygen, with all 4 CN- groups bound to the enzyme, and towards CO
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Na2S2O4
-
1 mM, activity of initially inactive enzyme increases within 1 h to a stable level in the presence of 101 kPa H2
FMN
H2 oxidation by subunits HoxHY is enhanced on addition of excess FMN
NADH
-
activates hexameric enzyme form, no activation of the tetrameric enzyme form
NADPH
-
specific activation of the soluble oligomeric enzyme, the binding domain is located on subunit HoxI
NADPH
-
activates hexameric enzyme form. Subunit HoxI provides a binding domain for NADPH
additional information
-
at a redox potential of approx. -100 mV the inactive form of the enzyme is transformed into an active one
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.197
NAD+
pH 8.0, temperature not specified in the publication
0.056
NADH
pH 8.0, temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2
NADPH
-
pH 8.0, wild-type enzyme complex including HoxI, with ferrocyanide
2
ferrocyanide
-
pH 8.0, wild-type enzyme complex including HoxI, with NADPH
222
ferrocyanide
-
pH 8.0, wild-type enzyme complex including HoxI, with NADH
372
ferrocyanide
-
pH 8.0, wild-type enzyme complex including HoxI, with H2
70
H2
-
pH 8.0, HoxI deletion mutant enzyme, with oxidized benzyl viologen
120
H2
-
pH 8.0, wild-type enzyme complex including HoxI, with oxidized benzyl viologen
372
H2
-
pH 8.0, wild-type enzyme complex including HoxI, with ferrocyanide
222
NADH
-
pH 8.0, wild-type enzyme complex including HoxI, with ferrocyanide
70
Oxidized benzyl viologen
-
pH 8.0, HoxI deletion mutant enzyme, with H2
120
Oxidized benzyl viologen
-
pH 8.0, wild-type enzyme complex including HoxI, with H2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.2
NAD+
pH 8.0, temperature not specified in the publication
0.2
NADH
pH 8.0, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.6
-
reaction with NADPH and ferrocyanide, purified wild-type enzyme complex including HoxI, no reaction with the HoxI deletion mutant
102.1
-
reaction with H2 and ferrocyanide, purified HoxI deletion mutant enzyme
108.8
-
reaction with H2 and ferrocyanide, purified wild-type enzyme complex including HoxI
230
-
recombinant protein, pH 8.0, temperature not specified in the publication
25.2
-
reaction with H2 and benzyl viologen, purified HoxI deletion mutant enzyme
30 - 100
-
purified enzyme, forward reaction under aerobic conditions with NAD+
35.1
-
reaction with H2 and benzyl viologen, purified wild-type enzyme complex including HoxI
41.9
-
reaction with H2 and NAD+, purified wild-type enzyme complex including HoxI
61.6
-
reaction with NADH and ferrocyanide, purified HoxI deletion mutant enzyme
64.9
-
reaction with NADH and ferrocyanide, purified wild-type enzyme complex including HoxI
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
-
cells are grown heterotrophically in a fructose-glycerol minimal medium
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
-
-
439694, 439695, 439699, 439700, 439706, 439707, 439708, 439709, 439710, 654562, 655890, 656298, 674924
-
located as inclusion bodies in the DNA region of the cell
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
26000
30000
-
x * 63000 + x * 30000 + x * 26000 + x * 56000, SDS-PAGE, deduced from nucleotide sequence
56000
-
x * 63000 + x * 30000 + x * 26000 + x * 56000, SDS-PAGE, deduced from nucleotide sequence
63000
-
x * 63000 + x * 30000 + x * 26000 + x * 56000, SDS-PAGE, deduced from nucleotide sequence
26000
-
x * 63000 + x * 30000 + x * 26000 + x * 56000, SDS-PAGE, deduced from nucleotide sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
-
x * 63000 + x * 30000 + x * 26000 + x * 56000, SDS-PAGE, deduced from nucleotide sequence
hexamer
-
subunit stoichiometry of HoxFUYI2. Subunit HoxIhas a MW of 19000 Da as determined by SDS-PAGE
oligomer
tetramer
additional information
oligomer
-
enzyme is composed as a dimer of pentamers, the latter consisting of subunit pairs HoxU with HoxF, and HoxY with HoxH, and a fifth 19 kDa subunit HoxI, i.e. B protein, the HoxFU dimer is the NADH-dehydrogenase moiety, the HoxHY dimer is the hydrogenase moiety, overview
oligomer
-
heterotetrameric multifunctional enzyme showing both hydrogenase and diaphorase activities, subunits HoxHY form a heterodimer which is responsible for the hydrogenase activity with 56 kDa for HoxH and 26 kDa for HoxY, subunits HoxFU form a heterodimer which is responsible for the NADH-dehydrogenase, i.e. diaphorase activity
tetramer
-
enzyme is composed of 4 Hox subunits, HoxF, HoxH, HoxU, and HoxY, with MWs of 67 kDa, 55 kDa, 26 kDa, and 23 kDa
additional information
-
structure analysis and subunit composition, HoxI is associated with the NADH-dehydrogenase moiety of the enzyme
additional information
-
subunits HoxHY form a heterodimer which is responsible for the hydrogenase activity, subunits HoxFU form a heterodimer which is responsible for the NADH-dehydrogenase or diaphorase activity
additional information
-
use of a reverse micelle system for study of oligomeric structure
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C461A
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, inactive
D456N
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, nearly inactive
D456S
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, nearly inactive
E14Q
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, inactive
E14V
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, nearly inactive
G15A
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, nearly inactive
H16L
H396L
-
site-directed mutagenesis, catalytic subunit HoxH allele, highly reduced activity compared to the wild-type strain
H396Q
-
site-directed mutagenesis, catalytic subunit HoxH allele, about 3.5fold reduced activity compared to the wild-type strain
H69Q
-
site-directed mutagenesis, catalytic subunit HoxH allele, about 5fold reduced activity compared to the wild-type strain
H69Q/P390A
-
site-directed mutagenesis, catalytic subunit HoxH alleles, reduced activity compared to the wild-type strain
I64A
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, highly reduced activity compared to the wild-type strain
L118A
-
site-directed mutagenesis, catalytic subunit HoxH allele, reduced activity compared to the wild-type strain
L118F
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow at O2 concentrations above 5%, about 4fold reduced activity compared to the wild-type strain
L118I
-
site-directed mutagenesis, catalytic subunit HoxH allele, reduced activity compared to the wild-type strain
L394A
-
site-directed mutagenesis, catalytic subunit HoxH allele, about 5fold reduced activity compared to the wild-type strain
L394N
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, nearly inactive
P390A
-
site-directed mutagenesis, catalytic subunit HoxH allele, about 5fold reduced activity compared to the wild-type strain
R12L
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, highly reduced activity compared to the wild-type strain
R12Q
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, highly reduced activity compared to the wild-type strain
R391L
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, inactive
R391Q
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, inactive
R60Q
-
site-directed mutagenesis, catalytic subunit HoxH allele, highly reduced activity compared to the wild-type strain
S68V
-
site-directed mutagenesis, catalytic subunit HoxH allele, highly reduced activity compared to the wild-type strain
T415S
-
site-directed mutagenesis, catalytic subunit HoxH allele, about 5fold reduced activity compared to the wild-type strain
T415V
-
site-directed mutagenesis, catalytic subunit HoxH allele, highly reduced activity compared to the wild-type strain
T415V/N415H
-
site-directed mutagenesis, catalytic subunit HoxH alleles, O2-sensitive growth, highly reduced activity compared to the wild-type strain
additional information
H16L
-
site-directed mutagenesis, catalytic subunit HoxH allele, mutant strain does not grow, highly reduced activity compared to the wild-type strain
H16L
-
in the HoxH-mutant protein H16L, H2 oxidation is impaired, but H2 production occurrs via a stable Ni-C state (N(III)-(H-)-Fe(II))
additional information
-
construction of a HoxI deletion mutant enzyme, which shows different activation behaviour than the wild-type, it is not activated by and does not react with NADPH
additional information
-
construction of an inactive null mutant strain, mutant strain growth characteristics, overview
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.2 - 7
-
stability of the enzyme complex including HoxI in potassium phosphate buffer at pH 6.2 and pH 7.0, not at pH 8.0 or in Tris-HCl buffer at pH 7.2 and pH 8.0, or in Tris-nitrate buffer at pH 7.4
655890
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50
-
thermoinactivation by dissociation of the native enzyme tetramer into constituent heterodimers
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
highly diluted enzyme preparations are significantly inactivated even at 20°C
-
oxidized inactive form of the enzyme is the most stable against thermodenaturation, proteolysis, and inactivation in urea
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
air the presence of NADH leads to rapid destruction of the hexameric enzyme
-
674924
insensitivity towards oxygen, irreversible enzyme inhibition by oxygen occurs if the CN- bound to Ni2+ is irreversibly removed or if the enzyme is reduced by NADH
-
656298
oxidation with O2 and ferricyanide in absence of reducing components stabilizes, simultaneous presence of H2, NADH and O2 causes irreversible inactivation, half-lives in H2/O2 mixtures: 5% O2, 60 min, 20% O2, 30 min, 50% O2, 12 min
-
439708
oxidized hydrogenase purified under aerobic conditions is highly stable but not reactive, reductive activation of the enzyme by H2 in the presence of catalytic amounts of NADH or by reducing agents destabilizes
-
439699
rapid and irreversible inactivation under aerobic conditiones in the presence of reductants e.g. 1-10 mM NADH
-
439704
redox-dependent inactivation and activation, proposed inactivation mechanism
-
439704, 439705
the hexameric SH preparation is both active and stable in the presence of NADPH and air
-
674924
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant protein. The as-isolated module HoxHY is initially catalytically inactive, but after reductive activation at low potentials, exhibits both H2 oxidation and H+ reduction
recombinant Strep-tagged HoxI-deletion mutant tetramer from Escherichia coli by 2 different procedures
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sp. H16, cetavlon, ammonium sulfate, DEAE-cellulose, Sephadex G-200, hydroxyapatite
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene cluster HoxFUYHI, cloning and expression of the Strep-tagged HoxI-deletion mutant, a tetramer composed of HoxFU and HoxHY dimers forming a tetramer in Escherichia coli strains JM109 and XL-1 Blue
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over-expression of a plasmid carrying the hoxFUYWI-hypA2B2F2CDEXA genes in Cupriavidus necator
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
synthesis
-
coupling of enzyme reaction to carbonyl reductase from Candida parapsilosis leads to total turnover numbers up to 143666 with maximum activity for NAD+ reduction at 35°C and pH 8.0. Half-life of enzyme is 5.3 h under these conditions. Presence of ammonium and potassium ions increases the enzyme stability
synthesis
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immobilization of enzyme onto the anionic resin AmberliteTMFPA54 with immobilisation yield of 93.4% for adsorptive and 100% forcovalent attachment, corresponding activities of 48.9 and 39.3%, respectively, leading to stabilisation of enzyme. At elevated temperature and under agitation, stabilisation is further increased by modification of the covalently bound SH with methoxy-poly(ethylene) glycol(mPEG). In stationary aqueous solution, half-lives of up to 161 h at 25°C and 32 h at 35°C are obtained.In presence of the DMSO, DMF, 2-propanol and [EMIM][EtSO4] half-lives of 14-29 h can be achieved
synthesis
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system for cloning and expression of multiple genes in Escherichia coli BL21 demonstrate tby production and maturation of the NAD+reducing soluble [NiFe]-hydrogenase from Cupriavidus necator H16. The enzyme encoded in hoxFUYHI is successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in enzyme maturation reduces maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease, considerably increases maturation efficiency in Escherichia coli
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Burgdorf, T.; De Lacey, A.L.; Friedrich, B.
Functional analysis by site-directed mutagenesis of the NAD+-reducing hydrogenase from Ralstonia eutropha
J. Bacteriol.
184
6280-6288
2002
Cupriavidus necator
Tran-Betcke, A.; Warnecke, U.; Bcker, C.; Zaborosch, C.; Friedrich, B.
Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16
J. Bacteriol.
172
2920-2929
1990
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
Rohde, M.; Johannssen, W.; Mayer, F.
Immunocytochemical localization of the soluble NAD-dependent hydrogenase in cells of alcaligenes eutrophus
FEMS Microbiol. Lett.
36
83-86
1986
Cupriavidus necator
-
Hyman, M.R.; Arp, D.J.
Reversible and irreversible effects of nitric oxide on the soluble hydrogenase from Alcaligenes eutrophus H16
Biochem. J.
254
469-475
1988
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
Hyman, M.R.; Fox, C.A.; Arp, D.J.
Role of hydrogen in the activation and regulation of hydrogen oxidation by the soluble hydrogenase from Alcaligenes eutrophus H16
Biochem. J.
254
463-468
1988
Cupriavidus necator
Popov, V.O.; Ovchinnikov, A.N.; Utkin, I.B.; Gazaryan, I.G.; Egorov, A.M.; Berezin, I.V.
Inactivation of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z1: Thermoinactivation mechanism
Biochim. Biophys. Acta
831
297-301
1985
Cupriavidus necator, Cupriavidus necator Z1
-
Popov, V.O.; Gazaryan, I.G.; Egorov, A.M.; Berezin, I.V.
NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Alcaligenes eutrophum Z1. Kinetic studies of the NADH-dehydrogenase activity
Biochim. Biophys. Acta
827
466-471
1985
Cupriavidus necator, Cupriavidus necator Z1
-
Schneider, K.; Schlegel, H.G.
Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16
Biochim. Biophys. Acta
452
66-80
1976
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
Schneider, K.; Schlegel, H.G.
Localization and stability of hydrogenases from aerobic hydrogen bacteria
Arch. Microbiol.
112
229-238
1977
Cupriavidus necator, Cupriavidus necator b19, Cupriavidus necator G27, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1, Cupriavidus necator N9A, no activity in Alcaligenes paradoxus, no activity in Aquaspirillum autotrophicum, no activity in Corynebacterium autotrophicum, no activity in Hydrogenophaga palleronii, no activity in Paracoccus denitrificans, no activity in Pseudomonas facilis
Petrov, R.R.; Utkin, I.B.; Popov, V.O.
Redox-dependent inactivation of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1
Arch. Biochem. Biophys.
268
298-305
1989
Cupriavidus necator, Cupriavidus necator Z1
Petrov, R.R.; Utkin, I.B.; Popov, V.O.
Effect of redox potential on the activation of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1
Arch. Biochem. Biophys.
268
287-297
1989
Cupriavidus necator, Cupriavidus necator Z1
Friedrich, C.G.; Suetin, S.; Lohmeyer, M.
Nickel and iron incoorporation into soluble hydrogenase of alcaligenes eutrophus
Arch. Microbiol.
140
206-211
1984
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
-
Friedrich, C.G.; Schneider, K.; Friedrich, B.
Nickel in the catalytically active hydrogenase of Alcaligenes eutrophus
J. Bacteriol.
152
42-48
1982
Cupriavidus necator
Schneider, K.; Schlegel, H.G.
Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16
Biochem. J.
193
99-107
1981
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
Schneider, K.; Cammack, R.; Schlegel, H.G.; Hall, D.O.
The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus
Biochim. Biophys. Acta
578
445-461
1979
Cupriavidus necator
Schneider, K.; Schlegel, H.G.
Identification and quantitative determination of the flavin component of soluble hydrogenase from Alcaligenes eutrophus
Biochem. Biophys. Res. Commun.
84
564-571
1978
Cupriavidus necator
Grzeszik, C.; Ross, K.; Schneider, K.; Reh, M.; Schlegel, H.G.
Location, catalytic activity, and subunit composition of NAD-reducing hydrogenases of some Alcaligenes strains and Rhodococcus opacus MR22
Arch. Microbiol.
167
172-176
1997
Achromobacter denitrificans, Achromobacter denitrificans 4a-2, Achromobacter ruhlandii, Cupriavidus necator, Cupriavidus necator Cd2/01, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1, Rhodococcus opacus
Happe, R.P.; Roseboom, W.; Egert, G.; Friedrich, C.G.; Massanz, C.; Friedrich, B.; Albracht, S.P.
Unusual FTIR and EPR properties of the H2-activating site of the cytoplasmic NAD-reducing hydrogenase from Ralstonia eutropha
FEBS Lett.
466
259-263
2000
Cupriavidus necator
Loescher, S.; Burgdorf, T.; Buhrke, T.; Friedrich, B.; Dau, H.; Haumann, M.
Non-standard structures of the Ni-Fe cofactor in the regulatory and the NAD-reducing hydrogenases from Ralstonia eutropha
Biochem. Soc. Trans.
33
25-27
2005
Cupriavidus necator
Tikhonova, T.V.; Savel'eva, N.D.; Popov, V.O.
Chemical modification of catalytically essential functional groups of NAD-dependent hydrogenase from Ralstonia eutropha H16
Biochemistry
68
994-1001
2003
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
Burgdorf, T.; van der Linden, E.; Bernhard, M.; Yin, Q.Y.; Back, J.W.; Hartog, A.F.; Muijsers, A.O.; de Koster, C.G.; Albracht, S.P.; Friedrich, B.
The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH
J. Bacteriol.
187
3122-3132
2005
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
Van der Linden, E.; Burgdorf, T.; Bernhard, M.; Bleijlevens, B.; Friedrich, B.; Albracht, S.P.
The soluble [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen
J. Biol. Inorg. Chem.
9
616-626
2004
Cupriavidus necator
Loescher, S.; Burgdorf, T.; Zebger, I.; Hildebrandt, P.; Dau, H.; Friedrich, B.; Haumann, M.
Bias from H2 cleavage to production and coordination changes at the Ni-Fe active site in the NAD+-reducing hydrogenase from Ralstonia eutropha
Biochemistry
45
11658-11665
2006
Cupriavidus necator
Tikhonova, T.V.; Kurkin, S.A.; Klyachko, N.L.; Popov, V.O.
Use of a reverse micelle system for study of oligomeric structure of NAD+-reducing hydrogenase from Ralstonia eutropha H16
Biochemistry
70
645-651
2005
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
Vignais, P.M.
H/D exchange reactions and mechanistic aspects of the hydrogenases
Coord. Chem. Rev.
249
1677-1690
2005
Cupriavidus necator
-
Burgdorf, T.; Loescher, S.; Liebisch, P.; Van der Linden, E.; Galander, M.; Lendzian, F.; Meyer-Klaucke, W.; Albracht, S.P.; Friedrich, B.; Dau, H.; Haumann, M.
Structural and oxidation-state changes at its nonstandard Ni-Fe site during activation of the NAD-reducing hydrogenase from Ralstonia eutropha detected by X-ray absorption, EPR, and FTIR spectroscopy
J. Am. Chem. Soc.
127
576-592
2005
Cupriavidus necator
van der Linden, E.; Burgdorf, T.; de Lacey, A.L.; Buhrke, T.; Scholte, M.; Fernandez, V.M.; Friedrich, B.; Albracht, S.P.
An improved purification procedure for the soluble [NiFe]-hydrogenase of Ralstonia eutropha: new insights into its (in)stability and spectroscopic properties
J. Biol. Inorg. Chem.
11
247-260
2006
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
Burgdorf, T.; Lenz, O.; Buhrke, T.; van der Linden, E.; Jones, A.K.; Albracht, S.P.; Friedrich, B.
[NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation
J. Mol. Microbiol. Biotechnol.
10
181-196
2005
Cupriavidus necator, Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
Ratzka, J.; Lauterbach, L.; Lenz, O.; Ansorge-Schumacher, M.
Systematic evaluation of the dihydrogen-oxidising and NAD +-reducing soluble [NiFe]-hydrogenase from Ralstonia eutropha H16 as a cofactor regeneration catalyst
Biocatal. Biotransform.
29
246-252
2011
Cupriavidus necator, Cupriavidus necator DSM 428
-
Lauterbach, L.; Liu, J.; Horch, M.; Hummel, P.; Schwarze, A.; Haumann, M.; Vincent, K.; Lenz, O.; Zebger, I.
The hydrogenase subcomplex of the NAD+-reducing [NiFe] hydrogenase from Ralstonia eutropha - Insights into catalysis and redox interconversions
Eur. J. Inorg. Chem.
2011
1067-1079
2011
Cupriavidus necator (P22319), Cupriavidus necator (P22320), Cupriavidus necator DSM 428 (P22319), Cupriavidus necator DSM 428 (P22320)
-
Herr, N.; Ratzka, J.; Lauterbach, L.; Lenz, O.; Ansorge-Schumacher, M.
Stability enhancement of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase by a combination of immobilisation and chemical modification
J. Mol. Catal. B
97
169-174
2013
Cupriavidus necator, Cupriavidus necator DSM 428
-
Lauterbach, L.; Idris, Z.; Vincent, K.; Lenz, O.
Catalytic properties of the isolated diaphorase fragment of the NAD +-reducing [NiFe]-hydrogenase from Ralstonia eutropha
PLoS ONE
6
e25939
2011
Cupriavidus necator (P22317), Cupriavidus necator (P22318), Cupriavidus necator, Cupriavidus necator DSM 428 (P22317), Cupriavidus necator DSM 428 (P22318)
Schiffels, J.; Pinkenburg, O.; Schelden, M.; Aboulnaga, e.l.-.H.A.; Baumann, M.E.; Selmer, T.
An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli
PLoS ONE
8
e68812
2013
Cupriavidus necator
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