Probing the Subunit‐Subunit Interaction of the Tetramer of E. coli KDO8P Synthase by Electrospray Ionization Mass Spectrometry

Abstract

AbstractEscherichia coli 3‐Deoxy‐D‐manno‐octulosonate 8‐phosphate (KDO8P) synthase catalyzes the condensation reaction between D‐arabinose 5‐phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P and inorganic phosphate (Pi). This enzyme exists as a tetramer in solution, which is important for catalysis. Two different states of the enzyme were obtained: i) PEP‐bound and ii) PEP‐unbound. The effect of the substrates and products on the overall structure of KDO8P synthase in both PEP‐bound and unbound states was examined using electrospray ionization mass spectrometry. The analysis of our data showed that the complexes of the PEP‐unbound enzyme with PEP (or Pi) favored the formation of monomers, while the complexes with A5P (or KDO8P) mainly favored dimers. The PEP‐bound enzyme was found to exist in the monomer and dimer with a small amount of the tetramer, whereas the PEP‐unbound form primarily exists in the monomer and dimer, and no tetramer was observed, suggesting that the bound PEP have a role in stabilization of the tetrameric structure. Taken together, the results imply that the addition of the substrates or products to the unbound enzyme may alter the subunit‐subunit interactions and/or conformational change of the protein at the active site, and this study also demonstrates that the electrospray ionization mass spectrometric method may be a powerful tool in probing the subunit‐subunit interactions and/or conformational change of multi‐subunit protein upon binding to ligand.