Enzyme IIB ^bcellobiose of the phosphoenol‐pyruvate‐dependent phosphotransferase system of Escherichia coli: Backbone assignment and secondary structure determined by three‐dimensional NMR spectroscopy

Abstract

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 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO 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 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-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.

Analytical Sedimentation of the IIAChb and IIBChb Proteins of the Escherichia coli N,N′-Diacetylchitobiose Phosphotransferase System

“…Chb have been described (19,20) Chb (C). Three cell loading concentrations were used in A and B (0.2, 0.25, and 0.3 optical density units at 230 nm), and two cell loading concentrations were used in C (0.2 and 0.25 optical density units at 230 nm).…”

The Transport/Phosphorylation ofN,N′-Diacetylchitobiose in Escherichia coli

“…The IIB Chb proteins were purified essentially as described (12). Two liters each of LB media supplemented with 100 g/ml ampicillin in three 6-liter flasks were inoculated with 40 ml (each) of an overnight culture of E. coli strain BL21 (DE3)⌬EI (containing a deletion in Enzyme I of the PTS) harboring the plasmid pET:chbB (or pET: Cys10SerchbB).…”

“…In the present studies, homogeneous phospho-IIB Chb was isolated and the phosphoryl linkage to Cys 10 (the only Cys in the protein) was established by 31 P NMR. A C10S mutant of IIB Chb (or IIB Cel ) has been crystallized, and both its crystal and solution structures have been determined (11,12). Apparently the Ser replacement was used because the Cys caused technical difficulties.…”

The Fructose Transporter of Bacillus subtilis Encoded by the Lev Operon Backbone Assignment and Secondary Structure of the IIBLev Subunit

“…As already observed with the IIA domains (Worthylake et al, 1991;Liao et al, 1991;Kroon et al, 1993;Nunn et al, 1996), the IIB subunits/domains from different families of PTS transporters have different superfolds. The 10-kDa IIB"IL domain of the E. coli glucose transporter is a split a/l sandwich (Eberstadt et al, 1996), while the 18-kDa IIBL" of B. subtilis and the IIB'" of E. coli (Ab et al, 1994) assume differently wound alp superfolds. Because all IIB domains have to dock to a IIA domain during the phosphoryltransfer reaction, their surfaces must be pairwise complementary in shape and chemistry.…”

“…The HAM"" domain of the E. coli mannose transporter assumes an a/p doubly wound superfold (Seip et al, 1994;Nunn et al, 1996) and the IIA domain of the mannitol transporter has an Q2a3 secondary structure (Kroon et al, 1993). The secondary-structure elements of the IIB domain of the cellobiose transporter appear to form an alp doubly wound superfold (Ab et al, 1994), and the 1IBG" domain of E. coli is a split ap sandwich but with a topology unrelated to HPr (Eberstadt et al, 1996). As shown in this report, the IIB subunit of the B. subtilis fructose transporter which has no sequence similarity with any of the above mentioned PTS proteins has an alp doubly wound superfold.…”