Literature summary for 2.7.7.3 extracted from

Chatterjee, R.; Mondal, A.; Basu, A.; Datta, S.
Transition of phosphopantetheine adenylyltransferase from catalytic to allosteric state is characterized by ternary complex formation in Pseudomonas aeruginosa (2016), Biochim. Biophys. Acta, 1864, 773-786.

Cloned(Commentary)

Cloned (Comment)	Organism
gene coaD, recombinant His-tagged enzyme expression in Escherichia coli strain BL21(DE3)	Pseudomonas aeruginosa

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His-tagged enzyme in complex with diphosphate, substrate analogue AMPPNP, and inhibitors acetyl-CoA and CoA, sitting drop vapor diffusion method, mixing of 0.002 ml of protein in 25 mM HEPES, pH 7.0, 300 mM NaCl, and 2% glycerol, with 0.002 ml of well solution containing 17-19% PEG 4000, 0.1 M HEPES, pH 6.8-7.2, 200-350 mM Na-acetate, and 5-7% 2-propanol, 3-4 weeks, 42°C, method optimization, crystals are soaked in 0.1 mM ligand solution, X-ray diffraction structure determination and analysis at 2.2-2.5 A resolution	Pseudomonas aeruginosa

Crystallization (Comment)

Organism

purified recombinant His-tagged enzyme in complex with diphosphate, substrate analogue AMPPNP, and inhibitors acetyl-CoA and CoA, sitting drop vapor diffusion method, mixing of 0.002 ml of protein in 25 mM HEPES, pH 7.0, 300 mM NaCl, and 2% glycerol, with 0.002 ml of well solution containing 17-19% PEG 4000, 0.1 M HEPES, pH 6.8-7.2, 200-350 mM Na-acetate, and 5-7% 2-propanol, 3-4 weeks, 42°C, method optimization, crystals are soaked in 0.1 mM ligand solution, X-ray diffraction structure determination and analysis at 2.2-2.5 A resolution

Pseudomonas aeruginosa

Inhibitors

Inhibitors	Comment	Organism
acetyl-CoA	allosteric inhibitor	Pseudomonas aeruginosa
CoA	the enzyme is allosteric in nature and regulated by coenzyme A through feedback inhibition, PPAT forms a ternary complex with diphosphate and coenzyme A	Pseudomonas aeruginosa
EDTA	-	Pseudomonas aeruginosa
additional information	transition of PPAT in Pseudomonas aeruginosa from substrate binding to inhibitory states is triggered by an arginine switch	Pseudomonas aeruginosa

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	the enzyme is allosteric in nature	Pseudomonas aeruginosa
0.191	-	ATP	pH 7.0, 25°C, recombinant enzyme	Pseudomonas aeruginosa

Metals/Ions

Metals/Ions	Comment	Organism
Ca2+	activates, 90% activity compared to Mg2+	Pseudomonas aeruginosa
Mg2+	activates about 2.5fold, Mg2+ is essential for enzymatic activity of PPAT	Pseudomonas aeruginosa
Mn2+	activates, 80% activity compared to Mg2+	Pseudomonas aeruginosa
Zn2+	activates, 70% activity compared to Mg2+	Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + pantetheine 4'-phosphate	Pseudomonas aeruginosa	-	diphosphate + 3'-dephospho-CoA	-	r

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas aeruginosa	Q9I6D1	gene coaD	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and ultrafiltration	Pseudomonas aeruginosa

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + pantetheine 4'-phosphate	-	Pseudomonas aeruginosa	diphosphate + 3'-dephospho-CoA	-	r

Subunits

Subunits	Comment	Organism
More	the enzyme exists as monomer, dimer, and/or trimer depending on the pH value at pH 5.8-7.0, SDS-PAGE and gel filtration, overview	Pseudomonas aeruginosa

Synonyms

Synonyms	Comment	Organism
phosphopantetheine adenylyltransferase	-	Pseudomonas aeruginosa
PPAT	-	Pseudomonas aeruginosa

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at, forward and reverse reactions	Pseudomonas aeruginosa

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at, forward and reverse reactions	Pseudomonas aeruginosa

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.0084	-	acetyl-CoA	pH 7.0, 5°C, recombinant enzyme	Pseudomonas aeruginosa

General Information

General Information	Comment	Organism
additional information	the enzyme is allosteric in nature and regulated by coenzyme A through feedback inhibition. Structure-function analysis, and analysis of catalytic, allosteric and inhibitory mechanisms involved in regulation of PPAT. Changes in side chains R90 and D94 are responsible for transition between catalytic and allosteric inhibitory states. Diphosphate binds in close vicinity of ATP and produces a 10 A flip in the adenine ring of coenzyme A moiety. Transition of PPAT in Pseudomonas aeruginosa from substrate binding to inhibitory states is triggered by an arginine switch	Pseudomonas aeruginosa