Structure of Phosphorylated Enzyme I, the Phosphoenolpyruvate: Sugar Phosphotransferase System Sugar Translocation Signal Protein

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg²⁺dependent phosphorylation of enzyme I (El) by PEP. Crystals of Escherichia coli El were obtained by mixing the protein with Mg²⁺ and PEP, followed by oxalate, an El inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg²⁺ and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189∼P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an α-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an El fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189∼P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.