Open AccessIn 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
Author(s) -
Alexey Fomenkov,
Airat Valiakhmetov,
Ludwig Brand,
Saul Roseman
Publication year - 1998
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.95.15.8491
Subject(s) - pep group translocation , biochemistry , escherichia coli , fusion protein , biology , protease , complementation , dimer , microbiology and biotechnology , phosphotransferase , peptide sequence , c terminus , enzyme , chemistry , amino acid , recombinant dna , gene , organic chemistry , phenotype
Enzyme I (EI) is the first protein in the phosphoryl transfer sequence from phosphoenol 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 proteolyzedin 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 demonstratedin vivo . AnEscherichia 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.