Literature summary for 1.12.1.3 extracted from

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 (2010), PLoS ONE, 5, e10526 .

Cloned(Commentary)

Cloned (Comment)	Organism
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	Pyrococcus furiosus

Cloned (Comment)

Organism

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

Pyrococcus furiosus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.007	-	NADPH	80°C, pH 8.4, recombinant enzyme	Pyrococcus furiosus
0.03	-	NADPH	80°C, pH 8.4, native enzyme	Pyrococcus furiosus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
cytoplasm	-	Pyrococcus furiosus	5737	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Iron	-	Pyrococcus furiosus
Nickel	-	Pyrococcus furiosus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
150000	-	gel filtration	Pyrococcus furiosus

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus furiosus	E7FI44 AND Q8U2E5 AND Q8U2E4 AND E7FHU4	E7FI44: subunit alpha, Q8U2E5: subunit beta, Q8U2E4: subunit gamma, E7FHU4: subunit delta	-

Oxidation Stability

Oxidation Stability	Organism
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	Pyrococcus furiosus

Purification (Commentary)

Purification (Comment)	Organism
-	Pyrococcus furiosus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
H+ + NADPH	-	Pyrococcus furiosus	H2 + NADP+	-	?
H+ + reduced benzyl viologen	-	Pyrococcus furiosus	H2 + oxidized benzyl viologen	-	?

Synonyms

Synonyms	Comment	Organism
NADP-dependent hydrogenase	-	Pyrococcus furiosus
SHI	-	Pyrococcus furiosus
soluble hydrogenase I	-	Pyrococcus furiosus
[NiFe]-hydrogenase	-	Pyrococcus furiosus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
80	-	assay at	Pyrococcus furiosus

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
90	-	t1/2: 30 h for the native enzyme, t1/2: 1 h for the recombinant enzyme	Pyrococcus furiosus
100	-	t1/2: 2 h, native enzyme	Pyrococcus furiosus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8.4	-	assay at	Pyrococcus furiosus

Cofactor

Cofactor	Comment	Organism	Structure
[Ni-Fe]-center	metal content Ni:Fe is 1:25 for the native enzyme and 1:32 for the recombinant enzyme	Pyrococcus furiosus