Cloned (Comment) | Organism |
---|---|
gene coaD, recombinant His-tagged enzyme expression in Escherichia coli strain BL21(DE3) | 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 | Comment | Organism | Structure |
---|---|---|---|
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 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 | Comment | Organism | Structure |
---|---|---|---|
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 | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + pantetheine 4'-phosphate | Pseudomonas aeruginosa | - |
diphosphate + 3'-dephospho-CoA | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas aeruginosa | Q9I6D1 | gene coaD | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and ultrafiltration | Pseudomonas aeruginosa |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + pantetheine 4'-phosphate | - |
Pseudomonas aeruginosa | diphosphate + 3'-dephospho-CoA | - |
r |
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 | Comment | Organism |
---|---|---|
phosphopantetheine adenylyltransferase | - |
Pseudomonas aeruginosa |
PPAT | - |
Pseudomonas aeruginosa |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at, forward and reverse reactions | Pseudomonas aeruginosa |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at, forward and reverse reactions | Pseudomonas aeruginosa |
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 | 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 |