IN-VIVO AND IN-VITRO COMPLEMENTATION OF THE N-TERMINAL DOMAIN OF ENZYME-I OF THE ESCHERICHIA-COLI PHOSPHOTRANSFERASE SYSTEM BY THE CLONED C-TERMINAL DOMAIN

Enzyme I (EI) is the first protein in the phosphoryl transfer sequence from phosphoenolpyruvate (PEP) to sugar in carbohydrate uptake via th e bacterial PEP:glycose phosphotransferase system. The EI monomer/dime r transition may regulate the phosphotransferase system because only t he EI dimer is autophosphorylated by PEP, We previously showed that th e EI monomer comprises two major domains: (i) a compact, protease-resi stant N-terminal domain (EI-N), containing the active site His, and (i i) a flexible, protease-sensitive C-terminal domain (EI-C), which is r equired for EI dimerization, EI-N interacts with the second protein, H Pr, and phospho-HPr, but EI-N neither dimerizes nor is phosphorylated by PEP, We report here the molecular cloning and some properties of EI -C. EI-C is rapidly proteolyzed in vivo. Therefore, two different over expression vectors encoding fusion proteins were constructed. Fusion X a contains MalE (the maltose-binding protein), the four-amino acid seq uence required by protease factor;Ya, followed by EI-C, Fusion G conta ins His-Tyr between MalE and EI-C and is cleaved by the protease genen ase. Homogenous EI-C was isolated from fusion G, [P-32]PEP phosphoryla ted EI-N when supplemented with EI-C, fusion Xa, or fusion G. EI-C may act catalytically. Complementation was also demonstrated in vivo. An Escherichia coli ptsI deletion grew on mannitol as the sole source of carbon after it was transformed with tno compatible vectors; one vecto r encoded EI-N and the other encoded fusion Xa or fusion G. The molecu lar details underlying important properties of EI can now be studied.