Thermoanaerobacter tengcongensis is a thermophilic eubacterium that has a phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) of 22 proteins. The general PTS proteins, enzyme I and HPr, and the transporters for N-acetylglucosamine (EIICB(GlcNAc)) and fructose (EIIBC(Fru)) have thermal unfolding transitions at ∼90 °C and a temperature optimum for in vitro sugar phosphotransferase activity of 65 °C. The phosphocysteine of a EIICB(GlcNAc) mutant is unusually stable at room temperature with a t(1/2) of 60 h. The PEP binding C-terminal domain of enzyme I (EIC) forms a metastable covalent adduct with PEP at 65 °C. Crystallization of this adduct afforded the 1.68 Å resolution structure of EIC with a molecule of pyruvate in the active site. We also report the 1.83 Å crystal structure of the EIC-PEP complex. The comparison of the two structures with the apo form and with full-length EI shows differences between the active site side chain conformations of the PEP and pyruvate states but not between the pyruvate and apo states. In the presence of PEP, Arg465 forms a salt bridge with the phosphate moiety while Glu504 forms salt bridges with Arg186 and Arg195 of the N-terminal domain of enzyme I (EIN), which stabilizes a conformation appropriate for the in-line transfer of the phosphoryl moiety from PEP to His191. After transfer, Arg465 swings 4.8 Å away to form an alternative salt bridge with the carboxylate of Glu504. Glu504 loses the grip of Arg186 and Arg195, and the EIN domain can swing away to hand on the phosphoryl group to the phosphoryl carrier protein HPr.

Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EII Glc ) of the Escherichia coli phosphoenolpyruvate-sugar phosphotransferase system (PTS). 2¢,3¢-Epoxypropyl b-D-glucopyranoside (3a) is an inhibitor and also a pseudosubstrate. Five analogues are inhibitors of nonvectorial Glc phosphorylation by EII Glc but not pseudosubstrates. They are selective for EII Glc as demonstrated by comparison with EII Man, another Glc-specific but structurally different transporter. 3a is the only analogue that inhibits EII Glc by binding to the highaffinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter. The most potent inhibitor in vitro, methyl 6,7-anhydro-D,L-glycero-a-Dgluco-heptopyranoside (1d), preferentially interacts with the low-affinity cytoplasmic site but only weakly inhibits Glc uptake. Binding and/or phosphorylation from the cytoplasmic side of EII Glc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site. EII Glc is rapidly inactivated by the 6-O-bromoacetyl esters of methyl a-D-glucopyranoside (1a) and methyl a-D-manno- ) of diverse specificity and structure are components of the bacterial phosphoenolpyruvate-sugar phosphotransferase system (PTS) [4]. The PTS in addition comprises a number of proteins that act as allosteric regulators of enzymes and/ or transcription factors.The PTS transporters are homodimers, as indicated by cross-linking, ultracentrifugation, gel filtration, interallelic complementation and cryo-electron crystallography [5][6][7][8][9]. One protomer comprises three (or four) functional units, IIA, IIB and IIC(IID), which occur either as protein subunits or as domains in polypeptide chains. IIA and IIB sequentially transfer phosphoryl groups from HPr to the transported sugars. IIC contains the major determinants for sugar recognition and translocation, as inferred from binding studies [10] and the substrate selectivity of a chimeric EII GlcNAc/Glc [11]. EI, HPr and IIA are phosphorylated at His, whereas IIB domains are phosphorylated at Cys421 in EII Glc and at His175 in EII Man. EII Glc is specific for Glc, but EII Man has a broader substrate specificity for Glc, Man, and other derivatives of Glc altered at the C-2 carbon. Both transporters phosphorylate their hexose substrates at OH-6. In spite of their overlapping substrate specificity and analogous mechanism of action, EII Glc and EII Man do not share amino-acid sequence similarity, and, as judged by the known X-ray structures of their cytoplasmic domains, also assume completely different folds (for a review see [12]). The topology of the membrane-spanning units IIC Glc and IIC Man -IID Man are also different, as judged by the characterization of protein fusions between C-terminally truncated IIC(D) domains with alkaline phosphatase and b-galactosidase [13,14]. Whereas the sites of EII phosphorylation are known and easily recognized from the ...