EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF ENZYME IIA(GLC) OF THE PHOSPHOENOLPYRUVATE-SUGAR PHOSPHOTRANSFERASE SYSTEM OF MYCOPLASMA-CAPRICOLUM

The gene encoding enzyme IIA(glc) (EIIA) of the phosphoenolpyruvate:su gar phosphotransferase system of Mycoplasma capricolum was cloned into a regulated expression vector. The purified protein product of the ov erexpressed gene was characterized as an active phosphoacceptor from H Pr with a higher pI than previously described EIIAs. M. capricolum EII A was unreactive with antibodies directed against the corresponding pr oteins from either Gram-positive or Gram-negative bacteria. Enzyme IIA (glc) behaved as a homogeneous, monomeric species of 16 700 M-r in ana lytical ultracentrifugation. The circular dichroism far-UV spectrum of EIIA reflects a low alpha-helical content and predominantly beta-shee t structural content: temperature-induced changes in ellipticity at 20 5 nm showed that the protein undergoes reversible, two-state thermal u nfolding with T-m = 70.0 +/- 0.3 degrees C and a van't Hoff Delta H of 90 kcal/mol. Enzyme I (64 600 M-r) from M. capricolum exhibited a mon omer-dimer-tetramer association at 4 and 20 degrees C with dimerizatio n constants of log K-A = 5.6 and 5.1 [M-1], respectively, in sedimenta tion equilibrium experiments. A new vector, capable of introducing an N-terminal His tag on a protein, was developed in order to generate hi ghly purified heat-stable protein (HPr). No significant interaction of EIIA with HPr was detected by gel-filtration chromatography, intrinsi c tryptophanyl residue fluorescence changes, titration calorimetry, bi omolecular interaction, or sedimentation equilibrium studies. While Es cherichia coli EIIA inhibits Gram-negative glycerol kinase activity, t he M. capricolum EIIA has no effect on the homologous glycerol kinase. The probable regulator of sugar transport systems, HPr(Ser) kinase, w as demonstrated in extracts of M. capricolum and Mycoplasma genitalium . Gene mapping studies demonstrated that, in contrast to the clustered arrangement of genes encoding HPr and enzyme I in E. coli, these gene s are located diametrically opposite in the M. capricolum chromosome.