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

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Abstract
Enzyme I (EI) is the first protein in the phosphoryl transfer sequence from phospho enol pyruvate (PEP) to sugar in carbohydrate uptake via the bacterial PEP:glycose phosphotransferase system. The EI monomer/dimer transition may regulate the phosphotransferase system because only the EI dimer is autophosphorylated by PEP. We previously showed that the EI monomer comprises two major domains: ( i ) a compact, protease-resistant N-terminal domain (EI-N), containing the active site His, and ( ii ) a flexible, protease-sensitive C-terminal domain (EI-C), which is required for EI dimerization. EI-N interacts with the second protein, HPr, 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 overexpression vectors encoding fusion proteins were constructed. Fusion Xa contains MalE (the maltose-binding protein), the four-amino acid sequence required by protease factor Xa, followed by EI-C. Fusion G contains His-Tyr between MalE and EI-C and is cleaved by the protease genenase. Homogenous EI-C was isolated from fusion G. [ 32 P]PEP phosphorylated 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 two compatible vectors; one vector encoded EI-N and the other encoded fusion Xa or fusion G. The molecular details underlying important properties of EI can now be studied.

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