What question did this study set out to answer?

To determine the catalytic mechanism and oxygen atom transfer pathway of Escherichia coli KDO 8-P synthase during the condensation of phosphoenolpyruvate with D-arabinose 5-phosphate.

June 1, 1995Journal of Biological ChemistryOpen Access

Overproduction and One-step Purification of Escherichia coli 3-Deoxy-D-manno-octulosonic Acid 8-Phosphate Synthase and Oxygen Transfer Studies during Catalysis Using Isotopic-shifted Heteronuclear NMR

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To determine the catalytic mechanism and oxygen atom transfer pathway of Escherichia coli KDO 8-P synthase during the condensation of phosphoenolpyruvate with D-arabinose 5-phosphate.
Amplified the E. coli kdsA gene via PCR and subcloned it into the pT7-7 vector to express KDO 8-P synthase for single-step purification.
Synthesized [2-13C,18O]phosphoenolpyruvate and followed the fate of the enolic oxygen during enzyme catalysis using 13C and 31P heteronuclear NMR spectroscopy.
Conducted enzymatic reactions in 2H2 18O water and evaluated nonenzymatic 18O incorporation into the C-2 position of [2-13C]KDO 8-P alongside mass spectral analysis of inorganic phosphate.
The single-step expression and purification protocol produced 200 mg of homogeneous KDO 8-P synthase per liter of cell culture.
The enolic oxygen atom of phosphoenolpyruvate transferred quantitatively to the released inorganic phosphate byproduct.
The C-2 oxygen of the resulting KDO 8-P sugar was derived directly from the aqueous solvent rather than from phosphoenolpyruvate.

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Implication

In vitro biochemical study demonstrates oxygen transfer mechanisms in Escherichia coli KDO 8-P synthase, indicating solvent water supplies the C-2 oxygen of KDO 8-P.

Cite This Study

Dotson et al. (1995) studied this question.

synapsesocial.com/papers/6a9bf4072c9fe64af70269bb https://doi.org/10.1074/jbc.270.23.13698

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Implication

In vitro biochemical study demonstrates oxygen transfer mechanisms in Escherichia coli KDO 8-P synthase, indicating solvent water supplies the C-2 oxygen of KDO 8-P.

Overproduction and One-step Purification of Escherichia coli 3-Deoxy-D-manno-octulosonic Acid 8-Phosphate Synthase and Oxygen Transfer Studies during Catalysis Using Isotopic-shifted Heteronuclear NMR

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Discussion

Implication

Cite This Study

Overproduction and One-step Purification of Escherichia coli 3-Deoxy-D-manno-octulosonic Acid 8-Phosphate Synthase and Oxygen Transfer Studies during Catalysis Using Isotopic-shifted Heteronuclear NMR

Key Points

Discussion

Implication

Cite This Study

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