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Literature summary for 3.4.15.6 extracted from

  • Law, A.M.; Lai, S.W.; Tavares, J.; Kimber, M.S.
    The structural basis of beta-peptide-specific cleavage by the serine protease cyanophycinase (2009), J. Mol. Biol., 392, 393-404.
    View publication on PubMed
Search for more literature for 3.4.15.6

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Synechocystis sp. PCC 6803

Crystallization (Commentary)

Crystallization (Comment) Organism
12 mg/ml protein, 1.6 M ammonium sulfate, 0.1 M MES, 12% v/v dioxane, pH 6.6, vapor diffusion, sitting drop, temperature 293 K, space group C2221, resolution 1.5 A, the native crystal is grown in the presence of 5 mM cyanophycinase product, but no electron density for the product is interpretable in the final map Synechocystis sp. PCC 6803

Protein Variants

Protein Variants Comment Organism
additional information Gly101, Asp172, Gly173, Arg178, Arg180 and Arg183 form a conserved pocket adjacent to the catalytic Ser132, these are functionally critical residues. The cyanophycinase active site contains three absolutely conserved Arg residues (Arg178, Arg180 and Arg183), which line up along one side of the pocket Synechocystis sp. PCC 6803
N100A site-directed mutagenesis, 40% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
N158A site-directed mutagenesis, 41% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
N172A site-directed mutagenesis, 0.02% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
N17A site-directed mutagenesis, 60% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
N202A site-directed mutagenesis, 8% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
Q101A site-directed mutagenesis, 0.02% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
Q173A site-directed mutagenesis, 0.5% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
R178A site-directed mutagenesis, 1% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
R180A site-directed mutagenesis, 0.01% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
R183a site-directed mutagenesis, 0.01% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803
S132A site-directed mutagenesis, 0.002% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C Synechocystis sp. PCC 6803

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0022
-
 [L-Asp(4-Arg)]n 30°C in 50 mM N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine, pH 8.0 Synechocystis sp. PCC 6803

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
29700
-
 2 * 29700, SDS-PAGE Synechocystis sp. PCC 6803
56400
-
 gel filtration Synechocystis sp. PCC 6803

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
[L-Asp(4-Arg)]n + H2O Synechocystis sp. PCC 6803
-
 [L-Asp(4-Arg)]n-1 + [L-Asp(4-Arg)]
-
 ?

Organism

Organism UniProt Comment Textmining
Synechocystis sp. PCC 6803 P73832
-
-

Purification (Commentary)

Purification (Comment) Organism
several steps Synechocystis sp. PCC 6803

Source Tissue

Source Tissue Comment Organism Textmining
additional information cyanophycinase is amplified from Synechocystis sp. PCC 6803 genomic DNA using iProof polymerase and is ligated into the BamHI and NdeI sites of pET-28a, plasmids are transformed into Rosetta cells Synechocystis sp. PCC 6803
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
[L-Asp(4-Arg)]n + H2O
-
 Synechocystis sp. PCC 6803 [L-Asp(4-Arg)]n-1 + [L-Asp(4-Arg)]
-
 ?

Subunits

Subunits Comment Organism
dimer 2 * 29700, SDS-PAGE Synechocystis sp. PCC 6803

Synonyms

Synonyms Comment Organism
cyanophycinase
-
 Synechocystis sp. PCC 6803

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
16.5
-
 [L-Asp(4-Arg)]n 30°C in 50 mM N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine, pH 8.0, maximum at pH 9.0 Synechocystis sp. PCC 6803

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
7500
-
 [L-Asp(4-Arg)]n 30°C in 50 mM N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine, maximum at pH 8.0 Synechocystis sp. PCC 6803