Literature summary for 6.3.2.30 extracted from

Steinle, A.; Witthoff, S.; Krause, J.P.; Steinbuechel, A.
Establishment of cyanophycin biosynthesis in Pichia pastoris and optimization by use of engineered cyanophycin synthetases (2010), Appl. Environ. Microbiol., 76, 1062-1070.

Application

Application	Comment	Organism
synthesis	establishment of cyanophycin, i.e. multi-L-arginylpoly-L-aspartic acid or CGP, synthesis and applicability of this industrially widely used microorganism for the production of this polyamide, overview	Komagataella pastoris

Cloned(Commentary)

Cloned (Comment)	Organism
gene cphA6308, expression in Pichia pastoris and Escherichia coli from expression vectors pPICHOLI-C with the copper-inducible CUP1 promoter and pPICHOLI-3 with the methanol-inducible AOX1 promoter. Stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae is introduced and the expression is made in the His auxotrophic Pichia pastoris strain GS115. Recombinant polymer production by wild-type and mutant enzymes and product compositions, overview	Synechocystis sp.

Cloned (Comment)

Organism

gene cphA6308, expression in Pichia pastoris and Escherichia coli from expression vectors pPICHOLI-C with the copper-inducible CUP1 promoter and pPICHOLI-3 with the methanol-inducible AOX1 promoter. Stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae is introduced and the expression is made in the His auxotrophic Pichia pastoris strain GS115. Recombinant polymer production by wild-type and mutant enzymes and product compositions, overview

Synechocystis sp.

Protein Variants

Protein Variants	Comment	Organism
C595S	site-directed mutagenesis, the mutant shows a 2.5fold increased catalytic activity compared to the wild-type enzyme	Synechocystis sp.
additional information	construction of deletion mutants CphA6308DELTA1, CphA6308DELTA2, and CphA6308DELTA3, which are truncated by one, two, or three amino acid(s), respectively, at the C-terminus, and a combination of the deletion mutation with point mutation C595S, mutants CphA6308DELTA1 and CphA6308DELTA1/C595S show increased catalytic activity compared to the wild-type enzyme, while mutants CphA6308DELTA2 and CphA6308DELTA3 show similar or reduced CphA enzyme activity in comparison to wild-type CphA6308	Synechocystis sp.

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	-	Komagataella pastoris
Mg2+	-	Synechocystis sp.

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
98230	-	x * 100000, recombinant His6-tagged wild-type enzyme, SDS-PAGE, x * 98230, His6-tagged wild-type enzyme, sequence calcualtion	Synechocystis sp.
100000	-	x * 100000, recombinant His6-tagged wild-type enzyme, SDS-PAGE, x * 98230, His6-tagged wild-type enzyme, sequence calcualtion	Synechocystis sp.

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + [L-Asp(4-L-Arg)]n-L-Asp + L-Arg	Komagataella pastoris	-	ADP + phosphate + [L-Asp(4-L-Arg)]n+1	-	?
ATP + [L-Asp(4-L-Arg)]n-L-Asp + L-Arg	Synechocystis sp.	-	ADP + phosphate + [L-Asp(4-L-Arg)]n+1	-	?

Organism

Organism	UniProt	Comment	Textmining
Komagataella pastoris	-	-	-
Synechocystis sp.	-	strain PCC 6308, gene cphA6308	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged wild-type and mutant enzymes by His affinity chromatography	Synechocystis sp.

Source Tissue

Source Tissue	Comment	Organism	Textmining
additional information	pH 6.0 and 26°C are optimal for recombinant for CGP synthesis	Synechocystis sp.	-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
2.01	-	wild-type enzyme	Synechocystis sp.
3.2	-	mutant CphA6308DELTA1	Synechocystis sp.
4.17	-	mutant CphA6308DELTA1/C595S	Synechocystis sp.
4.95	-	mutant C595S	Synechocystis sp.

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + [L-Asp(4-L-Arg)]n-L-Asp + L-Arg	-	Komagataella pastoris	ADP + phosphate + [L-Asp(4-L-Arg)]n+1	-	?
ATP + [L-Asp(4-L-Arg)]n-L-Asp + L-Arg	-	Synechocystis sp.	ADP + phosphate + [L-Asp(4-L-Arg)]n+1	-	?

Subunits

Subunits	Comment	Organism
?	x * 100000, recombinant His6-tagged wild-type enzyme, SDS-PAGE, x * 98230, His6-tagged wild-type enzyme, sequence calcualtion	Synechocystis sp.

Synonyms

Synonyms	Comment	Organism
CGP synthetase	-	Komagataella pastoris
CGP synthetase	-	Synechocystis sp.
cyanophycin synthetase	-	Komagataella pastoris
cyanophycin synthetase	-	Synechocystis sp.

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
26	-	recombinant enzyme in Pichia pastoris strain GS115	Synechocystis sp.

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	recombinant enzyme in Pichia pastoris strain GS115	Synechocystis sp.

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Komagataella pastoris
ATP	-	Synechocystis sp.