ENZYME-IIB(CELLOBIOSE) OF THE PHOSPHOENOL-PYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI - BACKBONE ASSIGNMENT AND SECONDARY STRUCTURE DETERMINED BY 3-DIMENSIONAL NMR-SPECTROSCOPY

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBCellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone H-1, (SN)-S-15, and C-13(alpha) resonances, and the HN(CA)CO and HNCO experiments, correlating backbone H-1, N-15 and (CO)-C-13 resonances. Heteronuclear H-1-NOE H-1-N-15 single quantum coherence (N-15-NOESY-HSQC) spectroscopy and heteronuclear H-1 total correlation H-1-N-15 single quantum coherence (N-15-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping C-13(alpha) and (CO)-C-13 frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional H-1 alpha-C-13 alpha-(CO)-C-13 experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1H alpha and C-13 alpha chemical shifts from their random coil values and H-1-NOE information from the N-15-NOESY-HSQC. These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices. In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand.