Communicated by Ada Yonath, Weizmann Institute of Science, Rehovot, Israel, September 7, 2006 (received for review July 24, 2006)

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 (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg²⁺ and PEP, followed by oxalate, an EI 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-189P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an -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 EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.