Abstract
N,N'-Diacetylchitobiose is transported/phosphorylated in Escherichia coli by the (GlcNAc)(2)-specific Enzyme II permease of the phosphoenolpyruvate:glycose phosphotransferase system. IIA(Chb), one protein of the Enzyme II complex, was cloned and purified to homogeneity. IIA(Chb) and phospho-IIA(Chb) form stable homodimers (). Phospho-IIA(Chb) behaves as a typical epsilon2-N (i.e. N-3) phospho-His protein. However, the rate constants for hydrolysis of phospho-IIA(Chb) at pH 8.0 unexpectedly increased 7-fold between 25 and 37 degrees C and increased approximately 4-fold with decreasing protein concentration at 37 degrees C (but not 25 degrees C). The data were explained by thermal denaturation studies using CD spectroscopy. IIA(Chb) and phospho-IIA(Chb) exhibit virtually identical spectra at 25 degrees C (approximately 80% alpha-helix), but phospho-IIA(Chb) loses about 30% of its helicity at 37 degrees C, whereas IIA(Chb) shows only a slight change. Furthermore, the T(m) for thermal denaturation of IIA(Chb) was 54 degrees C, only slightly affected by concentration, whereas the T(m) for phospho-IIA(Chb) was much lower, ranging from 40 to 46 degrees C, depending on concentration. In addition, divalent cations (Mg(2+), Cu(2+), and Ni(2+)) have a dramatic and differential effect on the structure, depending on the state of phosphorylation of the protein. Thus, phosphorylation destabilizes IIA(Chb) at 37 degrees C, potentially affecting the monomer/dimer transition, which correlates with its chemical instability at this temperature. The physiological consequences of this phenomenon are briefly considered.
Highlights
In Escherichia coli, the operon that encodes the proteins required for catabolism of the chitin disaccharide N,NЈ-diacetylchitobiose, (GlcNAc)2,1 was previously thought to be a “cryptic” cellobiose operon [6, 7]
We report [1, 5]2 that (GlcNAc)2 uptake is mediated by the phosphoenolpyruvate:glycose phosphotransferase system (PTS)
For some sugars the IIA, IIB, and IIC domains are encoded by a single polypeptide, for others IIBC forms a single membrane protein component with a separate soluble IIA protein
Summary
Buffers and reagents were of the highest purity commercially available. [32P]PEP was a kind gift from Dr N.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have