Biophysical characterization of the enzyme I of the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system.
The first protein in the bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system is the homodimeric 60-kDa enzyme I (EI), which autophosphorylates in the presence of PEP and Mg2+. The conformational stability and structure of the EI from Streptomyces coelicolor, EI(sc), were explored in the absence and in the presence of its effectors by using several biophysical probes (namely, fluorescence, far-ultraviolet circular dichroism, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry) and computational approaches. The structure of EI(sc) was obtained by homology modeling of the isolated N- and C-terminal domains of other EI proteins. The experimental results indicate that at physiological pH, the dimeric EI(sc) had a well-folded structure; however, at low pH, EI(sc) showed a partially unfolded state with the features of a molten globule, as suggested by fluorescence, far-ultraviolet circular dichroism, FTIR, and 8-anilino-1-naphthalene-sulfonic acid binding. The thermal stability of EI(sc), in the absence of PEP and Mg2+, was maximal at pH 7. The presence of PEP and Mg2+ did not change substantially the secondary structure of the protein, as indicated by FTIR measurements. However, quenching experiments and proteolysis patterns suggest conformational changes in the presence of PEP; furthermore, the thermal stability of EI(sc) was modified depending on the effector added. Our approach suggests that thermodynamical analysis might reveal subtle conformational changes.
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Alicante, Spain.
Estefanía
E
Hurtado-Gómez
Gregorio
G
Fernández-Ballester
Harald
H
Nothaft
Javier
J
Gómez
Fritz
F
Titgemeyer
José Luis
JL
Neira
D004789:Enzyme Activation; D004795:Enzyme Stability; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000032:analysis* / Q000737:chemistry*; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000032:analysis* / Q000737:chemistry*; D011487:Protein Conformation; D048372:Streptomyces coelicolor / Q000201:enzymology*; D013379:Substrate Specificity
D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase