sugar transport ͉ phosphorylation ͉ x-ray crystallography T he phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (1) catalyzes the synchronized uptake and phosphorylation of a number of carbohydrates in eubacteria (group translocation) (2, 3). With some variations, the PTS comprises three proteins. In the cytoplasm, PEP phosphorylates enzyme I (EI), which then transfers the phosphoryl group to the histidine phosphocarrier protein, HPr. From HPr, the phosphoryl group is transferred to various sugar-specific membrane associated transporters [enzyme II (EII)], each comprising two cytoplasmic domains, EIIA and EIIB, and an integral membrane domain EIIC. Within EII, EIIA accepts the phosphoryl group from HPr and donates it to EIIB, whereupon EIIC mediates sugar translocation. In addition to controlling sugar translocation, the phosphorylation state of PTS proteins is associated with regulation of metabolic pathways and signaling in bacterial cells (4-8).The Ϸ64-kDa EI is a homodimer, which is more tightly associated at the phosphorylated state than the unphosphorylated state (9-14). The phosphorylation by PEP requires Mg 2ϩ and targets the N atom of His-189 (numbering scheme of EI from Escherichia coli) (15). The dimer association rate constant is two to three orders of magnitude slower than typical rates measured for other dimeric proteins, suggesting that oligomerization is accompanied by major conformational rearrangements (13,16,17). The monomer-dimer equilibrium has been studied in vitro by various methods (18-21), and it has been proposed that the transition plays a regulatory role in the PEP:sugar phosphotransferase system. Yet, transient kinetic studies indicated that the EI dimer phosphorylates HPr without dissociating into monomers (17).Proteolytic cleavage of EI produces two domains (22, 23). The EI N-terminal domain (EIN, residues 1-230) contains the residue that transfers the phosphoryl group, 24) and the HPr-binding domain, whereas the EI C-terminal domain (EIC, residues 261-575) binds PEP in the presence of Mg 2ϩ (the PEP-binding domain) (22,25) and mediates dimerization (26,27). Site-directed mutagenesis showed that Cys-502, located on EIC, is essential for phosphorylation of His-189 by PEP (28). The structure of EIN from E. coli has been determined by x-ray crystallography (29) and NMR spectroscopy (30).

The bacterial phosphotransferase system (PTS) catalyzes the transport and phosphorylation of its sugar substrates. The protein‐kinase‐catalyzed phosphorylation of serine 46 in the phosphocarrier protein, HPr, inhibits PTS activity, but neither the mechanism of this inhibition nor its physiological significance is known. Site‐specific HPr mutants were constructed in which serine 46 was replaced by alanine (S46A), threonine (S46T), tyrosine (S46Y) or aspartate (S46D). The purified S46D protein exhibited markedly lower Vmax and higher Km values than the wild‐type, S46T or S46A protein for the phosphoryl transfer reactions involving HPr(His approximately P). Interactions of HPr with the enzymes catalyzing phosphoryl transfer to and from HPr regulated the kinase‐catalyzed reaction. These results establish the inhibitory effect of a negative charge at position 46 on PTS‐mediated phosphoryl transfer and suggest that HPr is phosphorylated on both histidyl and seryl residues by enzymes that recognize its tertiary rather than its primary structure. In vivo studies showed that a negative charge on residue 46 of HPr strongly inhibits PTS‐mediated sugar uptake, but that competition of two PTS permeases for HPr(His approximately P) is quantitatively more important to the regulation of PTS function than serine 46 phosphorylation.

An experiment was conducted to determine whether baby pigs develop hypersensitivity to dietary soybean proteins. Thirty-two pigs were orally infused with either dried skim milk (5 g/d; control) or soybean meal (48% CP; 5 g/d) from d 7 to 14 after birth. Sows were fed a corn-corn gluten meal-based diet supplemented with lysine and tryptophan to avoid exposure of pigs to soybean proteins. Pigs were weaned at 21 d of age and fed diets containing either soybean meal or milk proteins until d 56. One half of the pigs were killed at 28 d of age and the rest at 56 d of age. Segments of small intestine were collected, and intraepithelial lymphocytes were isolated. At 28 d of age, pigs fed diets containing soybean meal had lower (P less than .05) villus height (221 vs 298 microns) and rate of gain (86 vs 204 g/d) than control pigs did. Pigs fed a diet containing soybean meal had higher (P less than .05) immunoglobulin G (IgG) titers to soybean protein than did pigs fed a milk protein-based diet. Blood and intestinal lymphocytes collected on d 28 and 56 did not exhibit any proliferative response when cultured with purified soy proteins (2.5 or 5 microns/ml). Phytohemagglutinin- and pokeweed mitogen-induced lymphocyte proliferations were higher (P less than .05) at d 56 than at d 28, but there were no differences attributable to protein source. There were no differences (P greater than .05) in skin-fold thickness measurements following intradermal injection with soy or milk proteins. Decreased villus height and increased serum IgG titers to soybean proteins coinciding with inferior performance of early weaned pigs fed diets containing soybean meal indicate that conventionally processed, commercial soybean meal may retain some antigens that can cause transient hypersensitivity in piglets.

The crystal structure of the histidinecontaining phosphocarrier protein (HPr) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) from Bacillus subtilis has been determined at 2.0-A resolution and refined to a crystallographic residual error R-factor of 0.150. The secondary-structure folding topology of the molecule is that of an open-face (i-sandwich formed by four antiparallel (i-strands packed against three a-helices. The active-site histidine, His-15, caps the N terminus of the first helix, suggesting that the helix dipole plays a role in stabilizing the phosphorylated state of the histidine. A sulfate anion located between His-15 and the neighboring Arg-17 has been identified in the electron-density map. Association of this negatively charged species with the two key catalytic residues implies that the crystal structure resembles the phosphorylated state of the protein. A model of the phosphorylated form of the molecule is proposed, in which the negatively charged phosphoryl group interacts with two main-chain nitrogen atoms of the following helix and with the guanidinium group of Arg-17. It is also proposed that the phosphoryl transfer from HPr to the IIA domain of the glucose permease involves Arg-17 switching between two salt bridges: one with the phosphorylated histidyl of HPr and the other with two aspartyl residues associated with the active site of the IHA domain of glucose permease, which are accessible upon complex formation.The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) first described by Kundig et al. (1) transports some carbohydrates into the cell and simultaneously phosphorylates them to initiate their metabolism and prevent leakage from the cytoplasm. This system also plays a role in chemotaxis toward PTS sugars and in regulation of the uptake of several non-PTS sugars (for reviews, see refs. 2-5). The system consists of two general energy-coupling components [enzyme I and a histidine-containing phosphocarrier protein (HPr)] and of sugar-specific permeases (enzymes II). An enzyme II typically consists of three domains, IIA (also designated enzyme III or factor III), IIB, and IIC (6). A total of five phosphoryl group transfers occurs along the pathway: phosphoenolpyruvate (PEP) enzyme I --HPr ---hA -I