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Literature summary for 1.12.1.2 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

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

Cloned(Commentary)

Cloned (Comment) Organism
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

Protein Variants Comment Organism
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

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

Metals/Ions

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

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

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

Purification (Commentary)

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

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

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

Subunits

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

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

Turnover Number [1/s]

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

pH Optimum

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

pH Stability

pH Stability pH Stability Maximum Comment Organism
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

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