Literature summary for 4.2.1.104 extracted from

Butryn, A.; Stoehr, G.; Linke-Winnebeck, C.; Hopfner, K.P.
Serendipitous crystallization and structure determination of cyanase (CynS) from Serratia proteamaculans (2015), Acta Crystallogr. Sect. F, 71, 471-476 .

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Cloned(Commentary)

Cloned (Comment)	Organism
gene cynS, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain Rosetta (DE3) or Trichoplusia ni High Five cells	Serratia proteamaculans

Crystallization (Commentary)

Crystallization (Comment)	Organism
enzyme SpCynS as DNA-protein complex is crystallized as an impurity and its identity is determined using mass-spectrometric analysis. The crystals seems to have a ligand bound in the active site, glycerol molecules are bound at the entry to the active site of the enzyme. Mixing of 500 nl of 5.5 mg/ml protein-DNA complex in 20 mM MES potassium, pH 6.5, 60 mM KCl, 5 mM MgCl2, 2 mM DTT, with 500 nl of reservoir solution containing 0.1 M HEPES, pH 7.5, 2% v/v 2-propanol, 0.1 M sodium acetate, 18% w/v PEG 8000, and equilibration against 0.055 ml of reservoir solution, four months at 20°C, X-ray diffraction structure determination and analysis at 2.1 A resolution, the cryoprotecting solution consists of mother liquor containing 25% v/v glycerol, the crystals belong to space group P1, molecular replacement solution using the target DNA-protein complex or its components as a search model and the EcCynS crystal structure (PDB ID 1dw9) as a search model, modeling without ligand	Serratia proteamaculans

Crystallization (Comment)

Organism

enzyme SpCynS as DNA-protein complex is crystallized as an impurity and its identity is determined using mass-spectrometric analysis. The crystals seems to have a ligand bound in the active site, glycerol molecules are bound at the entry to the active site of the enzyme. Mixing of 500 nl of 5.5 mg/ml protein-DNA complex in 20 mM MES potassium, pH 6.5, 60 mM KCl, 5 mM MgCl2, 2 mM DTT, with 500 nl of reservoir solution containing 0.1 M HEPES, pH 7.5, 2% v/v 2-propanol, 0.1 M sodium acetate, 18% w/v PEG 8000, and equilibration against 0.055 ml of reservoir solution, four months at 20°C, X-ray diffraction structure determination and analysis at 2.1 A resolution, the cryoprotecting solution consists of mother liquor containing 25% v/v glycerol, the crystals belong to space group P1, molecular replacement solution using the target DNA-protein complex or its components as a search model and the EcCynS crystal structure (PDB ID 1dw9) as a search model, modeling without ligand

Serratia proteamaculans

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
172000	-	homodecamer, gel filtration	Serratia proteamaculans
220000	-	protein-DNA complex, gel filtration	Serratia proteamaculans

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
cyanate + HCO3- + 2 H+	Serratia proteamaculans	-	NH3 + 2 CO2	-	?
cyanate + HCO3- + 2 H+	Serratia proteamaculans 568	-	NH3 + 2 CO2	-	?

Organism

Organism	UniProt	Comment	Textmining
Serratia proteamaculans	A8GBZ7	-	-
Serratia proteamaculans 568	A8GBZ7	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain Rosetta (DE3) or Trichoplusia ni High Five cells by nickel affinity chromatography, anion and/or cation ion exchange chromatography, and gel filtration to over 90% purity	Serratia proteamaculans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
cyanate + HCO3- + 2 H+	-	Serratia proteamaculans	NH3 + 2 CO2	-	?
cyanate + HCO3- + 2 H+	-	Serratia proteamaculans 568	NH3 + 2 CO2	-	?

Subunits

Subunits	Comment	Organism
homodecamer	-	Serratia proteamaculans
homodimer	-	Serratia proteamaculans
More	CynS protomers form dimers through intricate interactions of their C-terminal domains. Both protomers contribute two beta-strands each to one continuous antiparallel beta-sheet, and the two beta-strands of one protomer are packed against a long alpha-helix from the other protomers within the dimer. Like EcCynS, SpCynS assembles into a ring-like decamer with 5/2 symmetry	Serratia proteamaculans

Synonyms

Synonyms	Comment	Organism
CynS	-	Serratia proteamaculans
SpCynS	-	Serratia proteamaculans
Spro_1533	-	Serratia proteamaculans

General Information

General Information	Comment	Organism
additional information	glycerol molecules bound at the entry to the active site of the enzyme during crystallization indicate conserved residues that might be important for the trafficking of substrates and products. The enzyme binds specifically to DNA	Serratia proteamaculans
physiological function	cyanate hydratase (CynS) catalyzes the decomposition of cyanate and bicarbonate into ammonia and carbon dioxide	Serratia proteamaculans