Engineering and characterization of the isolated C-terminal domain of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase.
5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly (CTD(GG)) and gly-ser-ser-gly (CTD(GSSG)) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), 15N-HSQC, and 31P-NMR revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.
Department of Applied Polar Science, Korea Polar Research Institute, Incheon 406-840, Korea.
Hak Jun
HJ
Kim
Hyun Woo
HW
Kim
Sung-Ho
SH
Kang
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010759:Phosphorus Isotopes / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D004791:Enzyme Inhibitors; D009587:Nitrogen Isotopes; D010759:Phosphorus Isotopes; D012765:Shikimic Acid; C042786:shikimic acid-3-phosphate; D010728:Phosphoenolpyruvate; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase