Transporters of glucose and other carbohydrates in bacteria.

Glucose arguably is the most important energy carrier, carbon source for metabolites and building block for biopolymers in all kingdoms of life. The proper function of animal organs and tissues depends on the continuous supply of glucose from the bloodstream. Most animals can resorb only a small number of monosaccharides, mostly glucose, galactose and fructose, while all other sugars oligosaccharides and dietary fibers are degraded and metabolized by the microbiota of the lower intestine. Bacteria, in contrast, are omnivorous. They can import and metabolize structurally different sugars and, as a consortium of different species, utilize almost any sugar, sugar derivative and oligosaccharide occurring in nature. Bacteria have membrane transport systems for the uptake of sugars against steep concentration gradients energized by ATP, the proton motive force and the high energy glycolytic intermediate phosphoenolpyruvate (PEP). Different uptake mechanisms and the broad range of overlapping substrate specificities allow bacteria to quickly adapt to and colonize changing environments. Here, we review the structures and mechanisms of bacterial representatives of (i) ATP-dependent cassette (ABC) transporters, (ii) major facilitator (MFS) superfamily proton symporters, (iii) sodium solute symporters (SSS) and (iv) enzyme II integral membrane subunits of the bacterial PEP-dependent phosphotransferase system (PTS). We give a short overview on the distribution of transporter genes and their phylogenetic relationship in different bacterial species. Some sugar transporters are hijacked for import of bacteriophage DNA and antibacterial toxins (bacteriocins) and they facilitate the penetration of polar antibiotics. Finally, we describe how the expression and activity of certain sugar transporters are controlled in response to the availability of sugars and how the presence and uptake of sugars may affect pathogenicity and host-microbiota interactions.

Carbohydrate Transport by Group Translocation: The Bacterial Phosphoenolpyruvate: Sugar Phosphotransferase System.

The Bacterial Phosphoenolpyruvate (PEP) : Sugar Phosphotransferase System (PTS) mediates the uptake and phosphorylation of carbohydrates, and controls the carbon- and nitrogen metabolism in response to the availability of sugars. PTS occur in eubacteria and in a few archaebacteria but not in animals and plants. All PTS comprise two cytoplasmic phosphotransferase proteins (EI and HPr) and a species-dependent, variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC, IID). EI and HPr transfer phosphorylgroups from PEP to the IIA units. Cytoplasmic IIA and IIB units sequentially transfer phosphates to the sugar, which is transported by the IIC and IICIID integral membrane protein complexes. Phosphorylation by IIB and translocation by IIC(IID) are tightly coupled. The IIC(IID) sugar transporters of the PTS are in the focus of this review. There are four structurally different PTS transporter superfamilies (glucose, glucitol, ascorbate, mannose) . Crystal structures are available for transporters of two superfamilies: bcIICmal (MalT, 5IWS, 6BVG) and bcIICchb (ChbC, 3QNQ) of B. subtilis from the glucose family, and IICasc (UlaA, 4RP9, 5ZOV) of E. coli from the ascorbate superfamily . They are homodimers and each protomer has an independent transport pathway which functions by an elevator-type alternating-access mechanism. bcIICmal and bcIICchb have the same fold, IICasc has a completely different fold. Biochemical and biophysical data accumulated in the past with the transporters for mannitol (IICBAmtl) and glucose (IICBglc) are reviewed and discussed in the context of the bcIICmal crystal structures. The transporters of the mannose superfamily are dimers of protomers consisting of a IIC and a IID protein chain. The crystal structure is not known and the topology difficult to predict. Biochemical data indicate that the IICIID complex employs a different transport mechanism . Species specific IICIID serve as a gateway for the penetration of bacteriophage lambda DNA across, and insertion of class IIa bacteriocins into the inner membrane. PTS transporters are inserted into the membrane by SecYEG translocon and have specific lipid requirements. Immunoelectron- and fluorescence microscopy indicate a non-random distribution and supramolecular complexes of PTS proteins.

Phosphoenolpyruvate: sugar phosphotransferase system from the hyperthermophilic Thermoanaerobacter tengcongensis.

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.

Structure and function of the glucose PTS transporter from Escherichia coli.

The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12Å resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies.

Crystal structure of enzyme I of the phosphoenolpyruvate sugar phosphotransferase system in the dephosphorylated state.

The bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system mediates sugar uptake and controls the carbon metabolism in response to carbohydrate availability. Enzyme I (EI), the first component of the phosphotransferase system, consists of an N-terminal protein binding domain (EIN) and a C-terminal PEP binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here we report the 2.4 A crystal structure of the homodimeric EI from Staphylococcus aureus. EIN consists of the helical hairpin HPr binding subdomain and the phosphorylatable betaalpha phospho-histidine (P-His) domain. EIC folds into an (betaalpha)(8) barrel. The dimer interface of EIC buries 1833 A(2) of accessible surface per monomer and contains two Ca(2+) binding sites per dimer. The structures of the S. aureus and Escherichia coli EI domains (Teplyakov, A., Lim, K., Zhu, P. P., Kapadia, G., Chen, C. C., Schwartz, J., Howard, A., Reddy, P. T., Peterkofsky, A., and Herzberg, O. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16218-16223) are very similar. The orientation of the domains relative to each other, however, is different. In the present structure the P-His domain is docked to the HPr binding domain in an orientation appropriate for in-line transfer of the phosphate to the active site histidine of the acceptor HPr. In the E. coli structure the phospho-His of the P-His domain projects into the PEP binding site of EIC. In the S. aureus structure the crystallographic temperature factors are lower for the HPr binding domain in contact with the P-His domain and higher for EIC. In the E. coli structure it is the reverse.

Expression, purification, crystallization and preliminary X-ray analysis of the EIICGlc domain of the Escherichia coli glucose transporter.

The glucose-import system of Escherichia coli consists of a hydrophilic EIIA(Glc) subunit and a transmembrane EIICB(Glc) subunit. EIICB(Glc) (UniProt P69786) contains two domains: the transmembrane EIIC(Glc) domain (40.6 kDa) and the cytoplasmic EIIB(Glc) domain (8.0 kDa), which are fused by a linker that is strongly conserved among its orthologues. The EIICB(Glc) subunit can be split within this motif by trypsin. Here, the crystallization of the tryptic EIIC(Glc) domain is described. A complete data set was collected to 4.5 A resolution at 100 K.

The mannose phosphotransferase system (Man-PTS) - Mannose transporter and receptor for bacteriocins and bacteriophages.

Mannose transporters constitute a superfamily (Man-PTS) of the Phosphoenolpyruvate Carbohydrate Phosphotransferase System (PTS). The membrane complexes are homotrimers of protomers consisting of two subunits, IIC and IID. The two subunits without recognizable sequence similarity assume the same fold, and in the protomer are structurally related by a two fold pseudosymmetry axis parallel to membrane-plane (Liu et al. (2019) Cell Research 29 680). Two reentrant loops and two transmembrane helices of each subunit together form the N-terminal transport domain. Two three-helix bundles, one of each subunit, form the scaffold domain. The protomer is stabilized by a helix swap between these bundles. The two C-terminal helices of IIC mediate the interprotomer contacts. PTS occur in bacteria and archaea but not in eukaryotes. Man-PTS are abundant in Gram-positive bacteria living on carbohydrate rich mucosal surfaces. A subgroup of IICIID complexes serve as receptors for class IIa bacteriocins and as channel for the penetration of bacteriophage lambda DNA across the inner membrane. Some Man-PTS are associated with host-pathogen and -symbiont processes.

Crystal structure of the nucleotide-binding subunit DhaL of the Escherichia coli dihydroxyacetone kinase.

Dihydroxyacetone (Dha) kinases are a family of sequence-related enzymes that utilize either ATP or phosphoenolpyruvate (PEP) as source of high energy phosphate. The PEP-dependent Dha kinase of Escherichia coli consists of three subunits. DhaK and DhaL are homologous to the Dha and nucleotide-binding domains of the ATP-dependent kinase of Citrobacter freundii. The DhaM subunit is a multiphosphorylprotein of the PEP:sugar phosphotransferase system (PTS). DhaL contains a tightly bound ADP as coenzyme that gets transiently phosphorylated in the double displacement of phosphate between DhaM and Dha. Here we report the 2.6A crystal structure of the E.coli DhaL subunit. DhaL folds into an eight-helix barrel of regular up-down topology with a hydrophobic core made up of eight interlocked aromatic residues and a molecule of ADP bound at the narrower end of the barrel. The alpha and beta phosphates of ADP are complexed by two Mg2+ and by a hydrogen bond to the imidazole ring of an invariant histidine. The Mg ions in turn are coordinated by three gamma-carboxyl groups of invariant aspartate residues. Water molecules complete the octahedral coordination sphere. The nucleotide is capped by an alpha-helical segment connecting helices 7 and 8 of the barrel. DhaL and the nucleotide-binding domain of the C.freundii kinase assume the same fold but display strongly different surface potentials. The latter observation and biochemical data indicate that the domains of the C.freundii Dha kinase constitute one cooperative unit and are not randomly interacting and independent like the subunits of the E.coli enzyme.

Crystal structure of the phosphoenolpyruvate-binding enzyme I-domain from the Thermoanaerobacter tengcongensis PEP: sugar phosphotransferase system (PTS).

Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal protein-binding domain (EIN) and a C-terminal PEP-binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here, we report the 1.82A crystal structure of the homodimeric EIC domain from Thermoanaerobacter tengcongensis, a saccharolytic eubacterium that grows optimally at 75 degrees C. EIC folds into a (betaalpha)(8) barrel with three large helical insertions between beta2/alpha2, beta3/alpha3 and beta6/alpha6. The large amphipathic dimer interface buries 3750A(2) of accessible surface area per monomer. A comparison with pyruvate phosphate dikinase (PPDK) reveals that the active-site residues in the empty PEP-binding site of EIC and in the liganded PEP-binding site of PPDK have almost identical conformations, pointing to a rigid structure of the active site. In silico models of EIC in complex with the Z and E-isomers of chloro-PEP provide a rational explanation for their difference as substrates and inhibitors of EI. The EIC domain exhibits 54% amino acid sequence identity with Escherichia coli and 60% with Bacillus subtilis EIC, has the same amino acid composition but contains additional salt-bridges and a more complex salt-bridge network than the homology model of E.coli EIC. The easy crystallization of EIC suggests that T.tengcongensis can serve as source for stable homologs of mesophilic proteins that are too labile for crystallization.

Crystal structure of the IIB(Sor) domain of the sorbose permease from Klebsiella pneumoniae solved to 1.75A resolution.

The phosphoenolpyruvate transferase system (PTS) is the major pathway by which bacteria import hexose sugars across the plasma membrane. The PTS transfers a phosphoryl group sequentially via several components from the glycolytic intermediate phosphoenolpyruvate (PEP) to the translocated sugar. It is comprised of the two general proteins enzyme I and HPr, and a sugar-specific enzyme II complex. Sugar translocation is through the membrane domain of the enzyme II complex. The enzyme II complex can belong to one of six families based upon sequence similarity, with the sorbose transporter from Klebsiella pneumoniae a member of the mannose family.The structure of the IIB(Sor) domain was solved to 1.75A resolution by molecular replacement. It has a central core of seven parallel beta-strands surrounded by a total of six alpha-helices. Three helices cover the front face, one the back face with the remaining two capping the central beta-sheet at the top and bottom. The catalytic His15 residue is situated on the surface-exposed loop between strand 1 and helix 1. In addition to the features previously observed in the homologous IIB(Lev) domain from Bacillus subtilis we see new features in the IIB(Sor) structure. First, the catalytic His15 side-chain is fixed in a specific conformation by forming a short hydrogen bond with Asp10, which in turn makes a salt-bridge with Arg8. Second, as observed in other phosphoproteins, an arginine residue (Arg12) is well poised to stabilize a phosphoryl group on His15. Third, we see an Asp/His pair reminiscent of that observed in the IIA(Man) domain from Escherichia coli. Finally, docking of IIA(Man) to IIB(Sor) shows that Arg12 in its current conformation is well positioned to assist the subsequent transfer of the phosphoryl group onto the sugar in line with previous mutagenesis studies.

Intein-mediated cyclization of a soluble and a membrane protein in vivo: function and stability.

Cyclized subunits of the E. coli glucose transporter were produced in vivo by intein mediated trans-splicing. IIA(Glc) is a beta-sandwich protein, IICB(Glc) spans the membrane eight times. Genes encoding the circularly permuted precursors U(Cdelta)-IIA(Glc)-U(Ndelta) and U(Cdelta)-IICB(Glc)-U(Ndelta) were assembled from DNA fragments encoding the 3' and 5' segments of the recA intein of M. tuberculosis and crr and ptsG of E. coli, respectively. A 20-residues long, Ala-Pro rich linker peptide and/or a histidine tag were used to join the native N- and C-termini in the cyclized proteins. The cyclized proteins complemented growth of glucose auxotrophic strains. Purified, cyclized IIA(Glc) and IICB(Glc) had 100 and 25%, respectively, of wild-type glucose phosphotransferase activity. They had an increased electrophoretic mobility, which decreased upon linearization of the proteins with chymotrypsin. Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding (75 vs. 70 degrees C; 1.52 vs. 1.05 M).

X-ray structures of the three Lactococcus lactis dihydroxyacetone kinase subunits and of a transient intersubunit complex.

Bacterial dihydroxyacetone (Dha) kinases do not exchange the ADP for ATP but utilize a subunit of the phosphoenolpyruvate carbohydrate phosphotransferase system for in situ rephosphorylation of a permanently bound ADP-cofactor. Here we report the 2.1-angstroms crystal structure of the transient complex between the phosphotransferase subunit DhaM of the phosphotransferase system and the nucleotide binding subunit DhaL of the Dha kinase of Lactococcus lactis, the 1.1-angstroms structure of the free DhaM dimer, and the 2.5-angstroms structure of the Dha-binding DhaK subunit. Conserved salt bridges and an edge-to-plane stacking contact between two tyrosines serve to orient DhaL relative to the DhaM dimer. The distance between the imidazole Nepsilon2 of the DhaM His-10 and the beta-phosphate oxygen of ADP, between which the gamma-phosphate is transferred, is 4.9 angstroms. An invariant arginine, which is essential for activity, is appropriately positioned to stabilize the gamma-phosphate in the transition state. The (betaalpha)4alpha fold of DhaM occurs a second time as a subfold in the DhaK subunit. By docking DhaL-ADP to this subfold, the nucleotide bound to DhaL and the C1-hydroxyl of Dha bound to DhaK are positioned for in-line transfer of phosphate.

Genetic requirements for growth of Escherichia coli K12 on methyl-alpha-D-glucopyranoside and the five alpha-D-glucosyl-D-fructose isomers of sucrose.

Strains of Escherichia coli K12, including MG-1655, accumulate methyl-alpha-D-glucopyranoside via the phosphoenolpyruvate-dependent glucose:phosphotransferase system (IICB(Glc)/IIA(Glc)). High concentrations of intracellular methyl-alpha-D-glucopyranoside 6-phosphate are toxic, and cell growth is prevented. However, transformation of E. coli MG-1655 with a plasmid (pAP1) encoding the gene aglB from Klebsiella pneumoniae resulted in excellent growth of the transformant MG-1655 (pAP1) on the glucose analog. AglB is an unusual NAD+/Mn2+-dependent phospho-alpha-glucosidase that promotes growth of MG-1655 (pAP1) by catalyzing the in vivo hydrolysis of methyl-alpha-D-glucopyranoside 6-phosphate to yield glucose 6-phosphate and methanol. When transformed with plasmid pAP2 encoding the K. pneumoniae genes aglB and aglA (an alpha-glucoside-specific transporter AglA (IICB(Agl))), strain MG-1655 (pAP2) metabolized a variety of other alpha-linked glucosides, including maltitol, isomaltose, and the following five isomers of sucrose: trehalulose alpha(1-->1), turanose alpha(1-->3), maltulose alpha(1-->4), leucrose alpha(1-->5), and palatinose alpha(1-->6). Remarkably, MG-1655 (pAP2) failed to metabolize sucrose alpha(1-->2). The E. coli K12 strain ZSC112L (ptsG::cat manXYZ nagE glk lac) can neither grow on glucose nor transport methyl-alpha-D-glucopyranoside. However, when transformed with pTSGH11 (encoding ptsG) or pAP2, this organism provided membranes that contained either the PtsG or AglA transporters, respectively. In vitro complementation of transporter-specific membranes with purified general phosphotransferase components showed that although PtsG and AglA recognized glucose and methyl-alpha-D-glucopyranoside, only AglA accepted other alpha-D-glucosides as substrates. Complementation experiments also revealed that IIA(Glc) was required for functional activity of both PtsG and AglA transporters. We conclude that AglA, AglB, and IIA(Glc) are necessary and sufficient for growth of E. coli K12 on methyl-alpha-D-glucoside and related alpha-D-glucopyranosides.

Regulation of the Dha operon of Lactococcus lactis: a deviation from the rule followed by the Tetr family of transcription regulators.

Dihydroxyacetone (Dha) kinases are a novel family of kinases with signaling and metabolic functions. Here we report the x-ray structures of the transcriptional activator DhaS and the coactivator DhaQ and characterize their function. DhaQ is a paralog of the Dha binding Dha kinase subunit; DhaS belongs to the family of TetR repressors although, unlike all known members of this family, it is a transcriptional activator. DhaQ and DhaS form a stable complex that in the presence of Dha activates transcription of the Lactococcus lactis dha operon. Dha covalently binds to DhaQ through a hemiaminal bond with a histidine and thereby induces a conformational change, which is propagated to the surface via a cantilever-like structure. DhaS binding protects an inverted repeat whose sequence is GGACACATN6ATTTGTCC and renders two GC base pairs of the operator DNA hypersensitive to DNase I cleavage. The proximal half-site of the inverted repeat partially overlaps with the predicted -35 consensus sequence of the dha promoter.

From ATP as substrate to ADP as coenzyme: functional evolution of the nucleotide binding subunit of dihydroxyacetone kinases.

Dihydroxyacetone kinases are a family of sequence-related enzymes that utilize either ATP or a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) as a source of high energy phosphate. The PTS is a multicomponent system involved in carbohydrate uptake and control of carbon metabolism in bacteria. Phylogenetic analysis suggests that the PTS-dependent dihydroxyacetone kinases evolved from an ATP-dependent ancestor. Their nucleotide binding subunit, an eight-helix barrel of regular up-down topology, retains ADP as phosphorylation site for the double displacement of phosphate from a phospho-histidine of the PTS protein to dihydroxyacetone. ADP is bound essentially irreversibly with a t((1/2)) of 100 min. Complexation with ADP increases the thermal unfolding temperature of dihydroxyacetone L from 40 (apo-form) to 65 degrees C (holoenzyme). ADP assumes the same role as histidines, cysteines, and aspartic acids in histidine kinases and PTS proteins. This conversion of a substrate binding site into a cofactor binding site reflects a remarkable instance of parsimonious evolution.

Escherichia coli dihydroxyacetone kinase controls gene expression by binding to transcription factor DhaR.

Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize either ATP (in animals, plants, bacteria) or the bacterial phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) as a source of high-energy phosphate. The PTS-dependent kinase of Escherichia coli consists of three subunits: DhaK contains the Dha binding site, DhaL contains ADP as cofactor for the double displacement of phosphate from DhaM to Dha, and DhaM provides a phospho-histidine relay between the PTS and DhaL::ADP. DhaR is a transcription activator belonging to the AAA+ family of enhancer binding proteins. It stimulates transcription of the dhaKLM operon from a sigma70 promoter and autorepresses dhaR transcription. Genetic and biochemical studies indicate that the enzyme subunits DhaL and DhaK act antagonistically as coactivator and corepressor of the transcription activator by mutually exclusive binding to the sensing domain of DhaR. In the presence of Dha, DhaL is dephosphorylated and DhaL::ADP displaces DhaK and stimulates DhaR activity. In the absence of Dha, DhaL::ADP is converted by the PTS to DhaL::ATP, which does not bind to DhaR.

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes.

The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).

Enzyme I of the phosphotransferase system: induced-fit protonation of the reaction transition state by Cys-502.

Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal domain with the phosphorylation site (His-189) and a C-terminal domain with the PEP binding site. Here we use C3-substituted PEP analogues as substrates and inhibitors and the EI(C502A) mutant to characterize structure-activity relationships of the PEP binding site. EI(C502A) is 10 000 times less active than wild-type EI [EI(wt)] with PEP as the substrate, whereas the two forms are equally active with ZClPEP. Cys-502 acts as an acid-base catalyst which stereospecifically protonates the pyruvoyl enolate at C3. The electron-withdrawing chlorine of ZClPEP can compensate for the lack of Cys-502, and in this case, the released 3-Cl-enolate is protonated nonstereospecifically. Several PEP analogues were assayed as inhibitors and as substrates. The respective K(I)/K(m) ratios vary between 3 and 40 for EI(wt), but they are constant and around unity for EI(C502A). EI(wt) with PEP as the substrate is inhibited by oxalate, whereas EI(C502A) with ZClPEP is not. The different behavior of EI(wt) and EI(C502A) toward the PEP analogues and oxalate suggests that the PEP binding site of EI(wt) exists in a "closed" and an "open" form. The open to closed transition is triggered by the interaction of the substrate with Cys-502. The closed conformation is sterically disfavored by C3-modified substrate analogues such as ZClPEP and ZMePEP. If site closure does not occur as with EI(C502A) and bulky substrates, the transition state is stabilized by electron dispersion to the electron-withdrawing substituent at C3.

Effect of enzyme I of the bacterial phosphoenolpyruvate : sugar phosphotransferase system (PTS) on virulence in a murine model.

The phosphoenolpyruvate : sugar phosphotransferase system (PTS) catalyses translocation with concomitant phosphorylation of sugars and hexitols and it regulates metabolism in response to the availability of carbohydrates. The PTS forms an interface between energy and signal transduction and its inhibition is likely to have pleiotropic effects. It is present in about one-third of bacteria with fully sequenced genomes, including many common pathogens, but does not occur in eukaryotes. Enzyme I (ptsI) is the first component of the divergent protein phosphorylation cascade. ptsI deletions were constructed in Salmonella typhimurium, Staphylococcus aureus and Haemophilus influenzae and virulence of the mutants was characterized in an intraperitoneal mouse model. The log(attenuation) values were 2.3, 1.4 and 0.9 for the Sal. typhimurium, Sta. aureus and H. influenzae ptsI mutants, respectively. The degree of attenuation is correlated with the complexity of the respective PTS, which comprises approximately 40 components in Sal. typhimurium, but only 5 in H. influenzae.

Sugar recognition by the glucose and mannose permeases of Escherichia coli. Steady-state kinetics and inhibition studies.

The glucose (EII(Glc)) and mannose (EII(Man)) permeases of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Escherichia coli belong to structurally different families of PTS transporters. The sugar recognition mechanism of the two transporters is compared using as inhibitors and pseudosubstrates all possible monodeoxy analogues, monodeoxyfluoro analogues, and epimers of D-glucose. The analogues were tested as phosphoryl acceptors in vitro and as uptake inhibitors with intact cells. Both EII have a high K(m) of phosphorylation for glucose modified at C-4 and C-6, and these analogues also are weak inhibitors of uptake. Conversely, modifications at C-1 (and also at C-2 with EII(Man)) were well tolerated. OH-3 is proposed to interact with hydrogen bond donors on EII(Glc) and EII(Man), since only substitution by fluorine was tolerated. Glucose-6-aldehydes, which exist as gem-diols in aqueous solution, are potent and highly selective inhibitors of "nonvectorial" phosphorylation by EII(Glc) (K(I) 3-250 microM). These aldehydes are comparatively weak inhibitors of transport by EII(Glc) and of phosphorylation and transport by EII(Man). Both transporters display biphasic kinetics (with glucose and some analogues) but simple Michaelis-Menten kinetics with 3-fluoroglucose (and other analogues). Kinetic simulations of the phosphorylation activities measured with different substrates and inhibitors indicate that two independent activities are present at the cytoplasmic side of the transporter. A working model that accounts for the kinetic data is presented.