The structure of enzyme IIAlactose from Lactococcus lactis reveals a new fold and points to possible interactions of a multicomponent system

Abstract

Enzyme IIAlactose is the first representative of the family of lactose/cellobiose-specific enzymes IIA for which a three-dimensional structure has been determined. Some of its structural features, like the presence of two histidine residues at the active site, seem to be common to all enzymes no overall structural homology is observed to any PTS proteins or to any other proteins in the Protein Data Bank. Enzyme IIAlactose shows surface complementarity to the phosphorylated form of HPr and several energetically favourable interactions between the two molecules can be predicted.

Isolation and Characterization of IIAChb, a Soluble Protein of the Enzyme II Complex Required for the Transport/Phosphorylation ofN,N′-Diacetylchitobiose in Escherichia coli

“…Temperatures such as 37°C could not be used because of hydrolysis of the phosphoprotein over the prolonged times required for equilibrium sedimentation experiments. (10). Control experiments (data not shown) established that 2 mM EDTA had no effect on the thermal denaturation curves of IIA Chb and phospho-IIA Chb and that EDTA completely reversed the divalent cation effects discussed below including the marked effects on thermal denaturation of the proteins.…”

“…For the studies described below, preparations of phospho-IIA Chb were used only where there was no detectable unphosphorylated protein (i.e., Ͻ5%). As reported in the accompanying paper, both IIA Chb and phospho- (8,10). A characteristic feature of the phosphohistidinyl linkage in phosphorylated PTS proteins is the sensitivity of this bond to acid, hydroxylamine, and pyridine and its stability to alkali (20).…”

Three-dimensional Solution Structure of the Cytoplasmic B Domain of the Mannitol Transporter IIMannitol of the Escherichia coli Phosphotransferase System

“…The selected clone was verified by DNA sequencing. The IIB mtl -fusion protein was expressed at 37°C in E. coli BL-21(DE3) cells in minimal medium using 15 NH 4 Cl and [ 13 C 6 ]glucose as the sole nitrogen and carbon sources, respectively.…”

“…Backbone / torsion angle restraints were derived from backbone chemical shifts using the program TALOS (41). Side-chain torsion angle restraints were derived from 3 J heteronuclear couplings coupled with analysis of a short mixing time ( m ϭ 35 ms), threedimensional, 13 C-separated NOE spectrum and a three-dimensional 15 N-separated rotating frame Overhauser effect spectrum. The structures were calculated using well established procedures from the experimental restraints (42)(43)(44)(45) by simulated annealing in torsion angle space (46) using the program Xplor-NIH (47).…”

“…Crystal and NMR structures of the N-terminal domain of enzyme I (EIN) (5,6) and HPr (7-11) have been determined. Crystal and/or NMR structures from a variety of species have been solved for representatives of the IIA domains from all four classes (12)(13)(14)(15)(16) and the IIB domains from three of the four classes (17)(18)(19)(20)(21)(22), the exception being IIB Mtl . In addition, four cytoplasmic protein-protein complexes of the PTS have been solved by NMR: EIN-HPr (23), IIA Glc -HPr (24), IIA Glc -IIB Glc (22), and IIA Mtl -HPr (25).…”