Literature summary for 1.7.2.4 extracted from

Schneider, L.K.; Einsle, O.
Role of calcium in secondary structure stabilization during maturation of nitrous oxide reductase (2016), Biochemistry, 55, 1433-1440 .

Cloned(Commentary)

Cloned (Comment)	Organism
codon-optimized gene nosZ, from canonical gene cluster nosRZDFYL(X), recombinant overexpression of His6-tagged enzyme and of Strep-tagged enzyme in Escherichia coli strain BL21(DE3)	Shewanella denitrificans

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme, sitting drop vapor diffusion method, for His6-tagged Ca2+-bound apo-N2OR enzyme: mixing of 0.0003 ml of 15mg/ml protein solution with 0.0003 ml of well solution containing PEG 3350, 0.2 M ammonium sulfate, and 0.1 M Bis-Tris-HCl, pH 5.5, for Strep-tagged Ca2+-free or -bound apo-N2OR: mixing of 0.0003 ml of 15mg/ml protein solution with 0.0003 ml of well solution containing 25% w/v PEG 3350, 0.2 M lithium sulfate monohydrate, and 0.1 M Tris-HCl, pH 8.5, 7 days, 25°C, X-ray diffraction structure determination and analysis, molecular replacement and modelling using structure PDB ID 3SBQ as template. No crystals are obtained for Ca2+-free His6-tagged apo-N2OR	Shewanella denitrificans

Crystallization (Comment)

Organism

purified enzyme, sitting drop vapor diffusion method, for His6-tagged Ca2+-bound apo-N2OR enzyme: mixing of 0.0003 ml of 15mg/ml protein solution with 0.0003 ml of well solution containing PEG 3350, 0.2 M ammonium sulfate, and 0.1 M Bis-Tris-HCl, pH 5.5, for Strep-tagged Ca2+-free or -bound apo-N2OR: mixing of 0.0003 ml of 15mg/ml protein solution with 0.0003 ml of well solution containing 25% w/v PEG 3350, 0.2 M lithium sulfate monohydrate, and 0.1 M Tris-HCl, pH 8.5, 7 days, 25°C, X-ray diffraction structure determination and analysis, molecular replacement and modelling using structure PDB ID 3SBQ as template. No crystals are obtained for Ca2+-free His6-tagged apo-N2OR

Shewanella denitrificans

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
periplasm	the enzyme contains a signal peptide with the twin-arginine motif S-RR-S-F-M-G that is required for Tat-dependent translocation to the periplasm	Shewanella denitrificans	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	single Ca2+ ion binds to the Ca2+-binding loop coordinated by residues Y251, E255, M263, D269, and S316 and a water molecule, Ca2+ plays a role in secondary structure stabilization during maturation of nitrous oxide reductase, required for structural stability of the binuclear CuA site	Shewanella denitrificans
Cu2+	involved in catalysis. In the absence of Ca2+, substantial parts of the enzyme surrounding the binding sites for the copper ions show structural disorder. Reconstitution of the binuclear CuA site was possible in vitro but required the presence of Ca2+ ions for a stable insertion of the center	Shewanella denitrificans

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
130000	-	gel filtration	Shewanella denitrificans

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
nitrous oxide + 2 reduced cytochrome c	Shewanella denitrificans	-	nitrogen + H2O + 2 cytochrome c	-	?
nitrous oxide + 2 reduced cytochrome c	Shewanella denitrificans OS217	-	nitrogen + H2O + 2 cytochrome c	-	?

Organism

Organism	UniProt	Comment	Textmining
Shewanella denitrificans	Q12M27	-	-
Shewanella denitrificans OS217	Q12M27	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme and Strep-tagged enzyme from Escherichia coli strain BL21(DE3) with or without bound calcium by metal affinity and streptavidin chromatography, respectively, followed by gel filtration	Shewanella denitrificans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
nitrous oxide + 2 reduced cytochrome c	-	Shewanella denitrificans	nitrogen + H2O + 2 cytochrome c	-	?
nitrous oxide + 2 reduced cytochrome c	the copper enzyme nitrous oxide reductase catalyzes the two-electron reduction of nitrous oxide (N2O) to dinitrogen	Shewanella denitrificans	nitrogen + H2O + 2 cytochrome c	-	?
nitrous oxide + 2 reduced cytochrome c	-	Shewanella denitrificans OS217	nitrogen + H2O + 2 cytochrome c	-	?
nitrous oxide + 2 reduced cytochrome c	the copper enzyme nitrous oxide reductase catalyzes the two-electron reduction of nitrous oxide (N2O) to dinitrogen	Shewanella denitrificans OS217	nitrogen + H2O + 2 cytochrome c	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * 64810, maturated enzyme, sequence calculation, a homodimer with a distinct two-domain architecture in the protomer	Shewanella denitrificans
More	calcium plays a role in secondary structure stabilization during maturation of nitrous oxide reductase. The enzyme contains a signal peptide with the twin-arginine motif S-RR-S-F-M-G that is required for Tat-dependent translocation to the periplasm. Three-dimensional structure analysis, overview	Shewanella denitrificans

Synonyms

Synonyms	Comment	Organism
N2OR	-	Shewanella denitrificans
nitrous oxide reductase	-	Shewanella denitrificans
NosZ	-	Shewanella denitrificans
Sden_2219	-	Shewanella denitrificans
SdN2OR	-	Shewanella denitrificans
Z-type N2OR	-	Shewanella denitrificans

Cofactor

Cofactor	Comment	Organism	Structure
cytochrome c	-	Shewanella denitrificans

General Information

General Information	Comment	Organism
malfunction	involved in catalysis. In the absence of Ca2+, substantial parts of the enzyme surrounding the binding sites for the copper ions show structural disorder. Reconstitution of the binuclear CuA site was possible in vitro but required the presence of Ca2+ ions for a stable insertion of the center	Shewanella denitrificans
additional information	SdN2OR is a Z-type N2OR, structure modelling	Shewanella denitrificans