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Literature summary extracted from

  • 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 (2005), J. Bacteriol., 187, 3122-3132.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.12.1.2 additional information enzyme activation mechanism, overview Cupriavidus necator
1.12.1.2 NADH specific activation of the soluble oligomeric enzyme Cupriavidus necator
1.12.1.2 NADH activates hexameric enzyme form, no activation of the tetrameric enzyme form Cupriavidus necator
1.12.1.2 NADPH specific activation of the soluble oligomeric enzyme, the binding domain is located on subunit HoxI Cupriavidus necator
1.12.1.2 NADPH activates hexameric enzyme form. Subunit HoxI provides a binding domain for NADPH Cupriavidus necator

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.12.1.2 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 Cupriavidus necator

Protein Variants

EC Number Protein Variants Comment Organism
1.12.1.2 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 Cupriavidus necator

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.12.1.2 cytoplasm
-
Cupriavidus necator 5737
-
1.12.1.2 soluble
-
Cupriavidus necator
-
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.12.1.2 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 Cupriavidus necator
1.12.1.2 CO the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre Cupriavidus necator
1.12.1.2 Fe2+ the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre Cupriavidus necator
1.12.1.2 Ni2+ the active site is a (enzyme-Cys)2(CN)Ni(micro-enzyme-Cys)2Fe(CN)3(CO) centre Cupriavidus necator

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.12.1.2 H2 + NAD+ Cupriavidus necator key enzyme in H2 metabolism H+ + NADH
-
r
1.12.1.2 H2 + NAD+ Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 key enzyme in H2 metabolism H+ + NADH
-
r
1.12.1.2 additional information Cupriavidus necator the organism can grow on H2 as sole energy source in an oxic environment ?
-
?
1.12.1.2 additional information Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 the organism can grow on H2 as sole energy source in an oxic environment ?
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.12.1.2 Cupriavidus necator
-
-
-
1.12.1.2 Cupriavidus necator
-
facultative lithoautotrophic proteobacterium, formerly Alcaligenes eutrophus
-
1.12.1.2 Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
-
-
-
1.12.1.2 Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
-
facultative lithoautotrophic proteobacterium, formerly Alcaligenes eutrophus
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.12.1.2 hexameric enzyme form Cupriavidus necator
1.12.1.2 recombinant Strep-tagged HoxI-deletion mutant tetramer from Escherichia coli by 2 different procedures Cupriavidus necator

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.12.1.2 additional information
-
-
Cupriavidus necator
1.12.1.2 0.6
-
reaction with NADPH and ferrocyanide, purified wild-type enzyme complex including HoxI, no reaction with the HoxI deletion mutant Cupriavidus necator
1.12.1.2 25.2
-
reaction with H2 and benzyl viologen, purified HoxI deletion mutant enzyme Cupriavidus necator
1.12.1.2 35.1
-
reaction with H2 and benzyl viologen, purified wild-type enzyme complex including HoxI Cupriavidus necator
1.12.1.2 39
-
reaction with H2 and NAD+, purified HoxI deletion mutant enzyme Cupriavidus necator
1.12.1.2 41.9
-
reaction with H2 and NAD+, purified wild-type enzyme complex including HoxI Cupriavidus necator
1.12.1.2 61.6
-
reaction with NADH and ferrocyanide, purified HoxI deletion mutant enzyme Cupriavidus necator
1.12.1.2 64.9
-
reaction with NADH and ferrocyanide, purified wild-type enzyme complex including HoxI Cupriavidus necator
1.12.1.2 102.1
-
reaction with H2 and ferrocyanide, purified HoxI deletion mutant enzyme Cupriavidus necator
1.12.1.2 108.8
-
reaction with H2 and ferrocyanide, purified wild-type enzyme complex including HoxI Cupriavidus necator

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.12.1.2 ferrocyanide + NAD(P)H enzyme complex including HoxI Cupriavidus necator ferricyanide + NAD(P)+
-
?
1.12.1.2 H2 + ferrocyanide
-
Cupriavidus necator H+ + ferricyanide
-
r
1.12.1.2 H2 + NAD+
-
Cupriavidus necator H+ + NADH
-
r
1.12.1.2 H2 + NAD+ no activity with NADP+ Cupriavidus necator H+ + NADH
-
r
1.12.1.2 H2 + NAD+ key enzyme in H2 metabolism Cupriavidus necator H+ + NADH
-
r
1.12.1.2 H2 + NAD+
-
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 H+ + NADH
-
r
1.12.1.2 H2 + NAD+ no activity with NADP+ Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 H+ + NADH
-
r
1.12.1.2 H2 + NAD+ key enzyme in H2 metabolism Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 H+ + NADH
-
r
1.12.1.2 H2 + oxidized benzyl viologen
-
Cupriavidus necator H+ + reduced benzyl viologen
-
r
1.12.1.2 H2 + oxidized benzyl viologen
-
Cupriavidus necator reduced benzyl viologen + H+
-
?
1.12.1.2 additional information the organism can grow on H2 as sole energy source in an oxic environment Cupriavidus necator ?
-
?
1.12.1.2 additional information tetrameric or hexameric enzyme form: no H2 production from NADPH Cupriavidus necator ?
-
?
1.12.1.2 additional information the organism can grow on H2 as sole energy source in an oxic environment Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 ?
-
?
1.12.1.2 additional information tetrameric or hexameric enzyme form: no H2 production from NADPH Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 ?
-
?
1.12.1.2 NADH + K3Fe(CN)6
-
Cupriavidus necator ?
-
?
1.12.1.2 NADPH + K3Fe(CN)6 low reaction with hexameric enzyme form, no reaction with tetrameric enzyme form Cupriavidus necator ?
-
?

Subunits

EC Number Subunits Comment Organism
1.12.1.2 hexamer subunit stoichiometry of HoxFUYI2. Subunit HoxIhas a MW of 19000 Da as determined by SDS-PAGE Cupriavidus necator
1.12.1.2 More structure analysis and subunit composition, HoxI is associated with the NADH-dehydrogenase moiety of the enzyme Cupriavidus necator
1.12.1.2 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 Cupriavidus necator

Synonyms

EC Number Synonyms Comment Organism
1.12.1.2 SH
-
Cupriavidus necator
1.12.1.2 soluble [NiFe]-hydrogenase
-
Cupriavidus necator
1.12.1.2 [NiFe]-hydrogenase
-
Cupriavidus necator

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.12.1.2 2
-
NADPH pH 8.0, wild-type enzyme complex including HoxI, with ferrocyanide Cupriavidus necator
1.12.1.2 2
-
ferrocyanide pH 8.0, wild-type enzyme complex including HoxI, with NADPH Cupriavidus necator
1.12.1.2 70
-
Oxidized benzyl viologen pH 8.0, HoxI deletion mutant enzyme, with H2 Cupriavidus necator
1.12.1.2 70
-
H2 pH 8.0, HoxI deletion mutant enzyme, with oxidized benzyl viologen Cupriavidus necator
1.12.1.2 109
-
NAD+ pH 8.0, HoxI deletion mutant enzyme, with H2 Cupriavidus necator
1.12.1.2 109
-
H2 pH 8.0, HoxI deletion mutant enzyme, with NAD+ Cupriavidus necator
1.12.1.2 120
-
Oxidized benzyl viologen pH 8.0, wild-type enzyme complex including HoxI, with H2 Cupriavidus necator
1.12.1.2 120
-
H2 pH 8.0, wild-type enzyme complex including HoxI, with oxidized benzyl viologen Cupriavidus necator
1.12.1.2 143
-
NAD+ pH 8.0, wild-type enzyme complex including HoxI, with H2 Cupriavidus necator
1.12.1.2 143
-
H2 pH 8.0, wild-type enzyme complex including HoxI, with NAD+ Cupriavidus necator
1.12.1.2 171
-
NADH pH 8.0, HoxI deletion mutant enzyme, with ferrocyanide Cupriavidus necator
1.12.1.2 171
-
ferrocyanide pH 8.0, HoxI deletion mutant enzyme, with NADH Cupriavidus necator
1.12.1.2 171
-
NADH reaction with K3Fe(CN)6, tetrameric enzyme form Cupriavidus necator
1.12.1.2 222
-
NADH pH 8.0, wild-type enzyme complex including HoxI, with ferrocyanide Cupriavidus necator
1.12.1.2 222
-
ferrocyanide pH 8.0, wild-type enzyme complex including HoxI, with NADH Cupriavidus necator
1.12.1.2 222
-
NADH reaction with K3Fe(CN)6, hexameric enzyme form Cupriavidus necator
1.12.1.2 284
-
H2 pH 8.0, HoxI deletion mutant enzyme, with ferrocyanide Cupriavidus necator
1.12.1.2 284
-
ferrocyanide pH 8.0, HoxI deletion mutant enzyme, with H2 Cupriavidus necator
1.12.1.2 372
-
H2 pH 8.0, wild-type enzyme complex including HoxI, with ferrocyanide Cupriavidus necator
1.12.1.2 372
-
ferrocyanide pH 8.0, wild-type enzyme complex including HoxI, with H2 Cupriavidus necator

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.12.1.2 8
-
assay at Cupriavidus necator

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.12.1.2 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 Cupriavidus necator

Cofactor

EC Number Cofactor Comment Organism Structure
1.12.1.2 additional information no activity with NADP+ Cupriavidus necator
1.12.1.2 NAD+
-
Cupriavidus necator