* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
With ATP; magnesium chloride; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid;SbnC (NIS synthetase); SbnE (NIS synthetase); SbnF (NIS synthetase); In water; at 20℃;pH 7.4;In the dark;
In Vitro Synthesis of Staphyloferrin BBioinformatic analyses of the predicted protein products from the staphyloferrin B biosynthesis operon identified three enzymes (SbnC, SbnE and SbnF) that belong to the NIS family of siderophore synthetase enzymes. These enzymes are thought to catalyze the ATP and Mg2+-dependent activation of carboxylate substrates, in a reaction that proceeds through an acyl-adenlyate intermediate that is then recognized by an amine substrate to yield an overall condensation reaction to an amide. To examine the activity of the SbnC, SbnE and SbnF enzymes, and to determine their role in staphyloferrin B synthesis, each was independently overexpressed in E. coli as a hexahistidine-tagged derivative, and subsequently purified using nickel-affinity chromatography. When the three synthetases were incubated together with staphyloferrin B components L-2,3-diaminopropionic acid (Dap), citrate, 1,2-diaminoethane (Dae), and alpha-ketoglutarate (alpha-KG), an ion corresponding to staphyloferrin B was not formed. Additional purified Sbn enzymes were added to the reaction. Notably, when SbnH, a putative PLP-dependent decarboxylase, was combined in reactions with the three synthetases and substrates, an ion corresponding to that of staphyloferrin B was produced. The staphyloferrin B ion was not produced when any of ATP, Mg2+, SbnC, SbnE, SbnF, SbnH, Dap, citrate or alpha-KG was omitted from the reaction (data not shown). Notably, staphyloferrin B synthesis could proceed without the addition of Dae in the reaction. ESI-MS/MS was used to confirm that staphyloferrin B produced in vitro was the same as that isolated from spent culture supernatants of iron-starved S. aureus.
With hydroxylamine; ATP; magnesium chloride; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid;SbnE (NIS synthetase); In water; at 20℃;pH 7.4;In the dark;
Substrate Selectivity (Hydroxanate Formation) AssaysAll reactions were performed in 300 muL volumes and the following were common to each reaction: 2.25 mM ATP, 15 mM MgCl2, 150 mM hydroxylamine, and 50 mM HEPES pH 7.4. To assess which enzyme utilized citrate as a substrate, 5 muM of SbnC, SbnE, or SbnF were incubated with 3 mM citrate and the common reaction components as described above. Similarly, to assess which enzyme utilized alpha-KG as a substrate, 5 muM of SbnC, SbnE, or SbnF were incubated with 3 mM alpha-KG and the common reaction components. To assess the potential recognition of citryl-Dae as a substrate by SbnF or SbnC, this intermediate was formed in overnight reactions containing 2.25 mM ATP, 15 mM MgCl2, 3 mM <strong>[68-04-2]sodium citrate</strong>, 50 mM Dae, 5 muM SbnE buffered in 50 mM HEPES pH 7.4. This reaction was then heat treated at 70 C. to deactivate SbnE and the reaction was then centrifuged at 14 500 rpm for 10 minutes to pellet precipitated enzyme. The supernatant which now contains the citryl-Dae intermediate was incubated with fresh 5 muM SbnF or SbnC, 2.25 mM ATP, and 150 mM hydroxylamine. To assess the potential recognition of citryl-Dap as a substrate for SbnF or SbnC, this intermediate was formed as described above except that Dae was replaced with 50 mM Dap instead. For each reaction, a duplicate control reaction was prepared in which the enzyme was previously inactivated by heating at 100 C. for 10 minutes. All reactions described above were incubated at room temperature in the dark for 1 hour before addition of 300 muL stopping solution which consisted of 10% (w/v) FeCl3 and 3.3% (w/v) trichloroacetic acid in 0.7 M HCl. Reactions were centrifuged at 20 000×g for 5 minutes to remove precipitate and the formation of ferric hydroxamate was detected spectrophotometrically at 540 nm. Relative absorbance values reported in this study were calculated by subtracting the absorbance of the control (boiled enzyme) reactions from the absorbance of experimental reactions.
2,3-diaminopropionyl-citryl-ethylene diamine[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With hydroxylamine; ATP; magnesium chloride; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid;SbnE (NIS synthetase); SbnH (NIS synthetase); SbnF (NIS synthetase); at 20℃;In the dark;Product distribution / selectivity;
Substrate Selectivity (Hydroxanate Formation) AssaysAll reactions were performed in 300 muL volumes and the following were common to each reaction: 2.25 mM ATP, 15 mM MgCl2, 150 mM hydroxylamine, and 50 mM HEPES pH 7.4. To assess which enzyme utilized citrate as a substrate, 5 muM of SbnC, SbnE, or SbnF were incubated with 3 mM citrate and the common reaction components as described above. Similarly, to assess which enzyme utilized alpha-KG as a substrate, 5 muM of SbnC, SbnE, or SbnF were incubated with 3 mM alpha-KG and the common reaction components. To assess the potential recognition of citryl-Dae as a substrate by SbnF or SbnC, this intermediate was formed in overnight reactions containing 2.25 mM ATP, 15 mM MgCl2, 3 mM <strong>[68-04-2]sodium citrate</strong>, 50 mM Dae, 5 muM SbnE buffered in 50 mM HEPES pH 7.4. This reaction was then heat treated at 70 C. to deactivate SbnE and the reaction was then centrifuged at 14 500 rpm for 10 minutes to pellet precipitated enzyme. The supernatant which now contains the citryl-Dae intermediate was incubated with fresh 5 muM SbnF or SbnC, 2.25 mM ATP, and 150 mM hydroxylamine. To assess the potential recognition of citryl-Dap as a substrate for SbnF or SbnC, this intermediate was formed as described above except that Dae was replaced with 50 mM Dap instead. For each reaction, a duplicate control reaction was prepared in which the enzyme was previously inactivated by heating at 100 C. for 10 minutes. All reactions described above were incubated at room temperature in the dark for 1 hour before addition of 300 muL stopping solution which consisted of 10% (w/v) FeCl3 and 3.3% (w/v) trichloroacetic acid in 0.7 M HCl. Reactions were centrifuged at 20 000×g for 5 minutes to remove precipitate and the formation of ferric hydroxamate was detected spectrophotometrically at 540 nm. Relative absorbance values reported in this study were calculated by subtracting the absorbance of the control (boiled enzyme) reactions from the absorbance of experimental reactions.
Chemistry
Pharmaceutical Intermediates
Inhibitors/Agonists
Material Science
For Research Only
120K+ Compounds
Competitive Price
1-2 Day Shipping
