Biophysical characterization of the enzyme I of the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system

Abstract

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, EIsc, 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 EIsc 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 EIsc had a well-folded structure; however, at low pH, EIsc 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 EIsc, in the absence of PEP and Mg2+, was maximal at pH 7. The presence of PEP and Mg 2+ 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 EIsc was modified depending on the effector added. Our approach suggests that thermodynamical analysis might reveal subtle conformational changes. © 2006 by the Biophysical Society.