Publications

• Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene.

  Lee C.A., Saier M.H. Jr.

  The nucleotide sequence of the mtlA gene, which codes for the mannitol-specific Enzyme II of the Escherichia coli phosphotransferase system, is presented. From the gene sequence, the primary translation product is predicted to consist of 637 amino acids (Mr = 67,893). This result is compared to th ... >> More

  The nucleotide sequence of the mtlA gene, which codes for the mannitol-specific Enzyme II of the Escherichia coli phosphotransferase system, is presented. From the gene sequence, the primary translation product is predicted to consist of 637 amino acids (Mr = 67,893). This result is compared to the amino acid composition and molecular weight of the purified mannitol Enzyme II protein. The hydrophobic and hydrophilic properties of the enzyme were evaluated along its amino acid sequence using a computer program (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). The computer analysis predicts that the NH2-terminal half of the enzyme resides within the membrane, whereas the COOH-terminal half of the enzyme has the properties of a soluble protein. The possible functions of such a protein structure are discussed. RNA mapping has identified the promoter and mRNA start point for the mtl operon. << Less

  J. Biol. Chem. 258:10761-10767(1983) [PubMed] [EuropePMC]

• Substrate and phospholipid specificity of the purified mannitol permease of Escherichia coli.

  Jacobson G.R., Tanney L.E., Kelly D.M., Palman K.B., Corn S.B.

  D-Mannitol is transported and phosphorylated by a specific enzyme II of the phosphotransferase system of Escherichia coli. This protein was purified previously in detergent solution and has been partially characterized. As one approach in understanding the structure and mechanism of this enzyme/pe ... >> More

  D-Mannitol is transported and phosphorylated by a specific enzyme II of the phosphotransferase system of Escherichia coli. This protein was purified previously in detergent solution and has been partially characterized. As one approach in understanding the structure and mechanism of this enzyme/permease, we have tested a number of sugar alcohols and their derivatives as substrates and/or inhibitors of this protein. Our results show that the mannitol permease is highly, but not absolutely, specific for D-mannitol. Compounds accepted by the enzyme include those with substitutions in the C-2(= C-5) position of the carbon backbone of the natural substrate as well as D-mannonic acid, one heptitol and one pentitol. All of these compounds were both inhibitors and substrates for the mannitol permease except for D-mannoheptitol, which was an inhibitor but was not phosphorylated by the enzyme. No compound examined, however, exhibited an affinity for the enzyme as high as that for its natural substrate. We have also investigated the phospholipid requirements of the mannitol permease using phospholipids purified from E coli. The purified protein was significantly activated by phosphatidylethanolamine, but little activation was observed with phosphatidylglycerol or cardiolipin. These observations partially delineate requirements for interaction of sugar alcohols and phospholipids with the mannitol permease. They suggest approaches for the design of specific active site probes for the protein, and strategies for stabilizing the enzyme's activity in vitro. << Less

  J. Cell. Biochem. 23:231-240(1983) [PubMed] [EuropePMC]

• Relation between the oligomerization state and the transport and phosphorylation function of the Escherichia coli mannitol transport protein: interaction between mannitol-specific enzyme II monomers studied by complementation of inactive site-directed mutants.

  Boer H., ten Hoeve-Duurkens R.H., Robillard G.T.

  Previous experiments with the mannitol-specific enzyme II of Escherichia coli, EIImtl, have demonstrated that (1) the enzyme is a dimer, (2) the dimer is necessary for maximum activity, and (3) phosphoryl groups could be transferred between EIImtl subunits [van Weeghel et al. (1991) Biochemistry 3 ... >> More

  Previous experiments with the mannitol-specific enzyme II of Escherichia coli, EIImtl, have demonstrated that (1) the enzyme is a dimer, (2) the dimer is necessary for maximum activity, and (3) phosphoryl groups could be transferred between EIImtl subunits [van Weeghel et al. (1991) Biochemistry 30, 1768-1773; Weng et al. (1992) J. Biol. Chem. 267, 19529-19535; Weng & Jacobson (1993) Biochemistry 32, 11211-11216; Stolz et al. (1993) J. Biol. Chem. 268, 27094-27099]. The experiments in this article address the mechanistic role of the dimer. They indicate that the A, B, and C domains of EIImtl preferentially interact within the same subunit. Site-directed mutants in each of the three domains of EIImtl were used to study phosphoryl group transfer by the EIImtl dimer in vitro and mannitol transport in vivo. The C domain mutant, EIImtl-G196D, which was unable to bind mannitol, and the separated C domain, IICmtl, which was unable to phosphorylate mannitol, formed a heterodimer which was capable of mannitol phosphorylation in vitro and mannitol transport in vivo. The rates of phosphorylation were approximately 10-fold lower in heterodimers containing two inactive subunits relative to the rates in heterodimers containing one inactive and one wild type subunit; phosphoryl group transfer through one subunit is kinetically preferred to intersubunit transfer. Heterodimers formed in vivo between one wild type EIImtl subunit and the CB domain double mutant, EIImtl-G196D/C384S, transported mannitol as rapidly as wild type EIImtl alone; the presence of the inactive double mutant subunit did not reduce the transport rate. Thus, only one active A, B, and C domain in the dimer is sufficient for transport and phosphorylation activity, and if all three domains are situated on the same subunit, maximum rates are achieved. << Less

  Biochemistry 35:12901-12908(1996) [PubMed] [EuropePMC]

• Cytoplasmic phosphorylating domain of the mannitol-specific transport protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli: overexpression, purification, and functional complementation with the mannitol binding domain.

  van Weeghel R.P., Meyer G., Pas H.H., Keck W., Robillard G.T.

  The cytoplasmic C-terminal domain, residues 348-637, and the membrane-bound N-terminal domain, residues 1-347, of EIImtl have been subcloned and expressed in Escherichia coli. The N-terminal domain, IICmtl, contains the mannitol binding site, and the C-terminal domain, IIBAmtl, contains the activi ... >> More

  The cytoplasmic C-terminal domain, residues 348-637, and the membrane-bound N-terminal domain, residues 1-347, of EIImtl have been subcloned and expressed in Escherichia coli. The N-terminal domain, IICmtl, contains the mannitol binding site, and the C-terminal domain, IIBAmtl, contains the activity-linked phosphorylation sites, His-554 and Cys-384. Overexpression of the BA domain was achieved by a translational in-frame fusion of the gene with the cro ATG start codon, downstream of the strong PR promoter of phage lambda. The domain has been purified and characterized in in vitro complementation assays. It possessed no mannitol phosphorylation activity itself but was able to restore the phosphoenolpyruvate-dependent phosphorylation activity of two EIImtl phosphorylation site mutants, lacking His-554 or Cys-384. The complementary N-terminal domain was also expressed. Membranes possessing IICmtl were unable to phosphorylate mannitol at the expense of phosphoenolpyruvate. However, when the membranes were combined with the purified C-terminal domain, mannitol phosphorylation activity was restored. Mannitol transport and phosphorylation were also restored in vivo when the two plasmids encoding the N- and C-terminal domains were expressed in the same cell. These data demonstrate the existence of structurally and functionally distinct domains in EIImtl: a cytoplasmic domain with phosphorylating activity and a membrane-bound N-terminal domain which, in the presence of the cytoplasmic domain, is able to actively transport and phosphorylate mannitol. The ability to separate, overproduce, and purify structurally stable, enzymatically active domains opens the way for 3D structural studies as well as complete kinetic analysis of the activities of the individual domains and their interactions. << Less

  Biochemistry 30:9478-9485(1991) [PubMed] [EuropePMC]

• Functional reconstitution of the purified phosphoenolpyruvate-dependent mannitol-specific transport system of Escherichia coli in phospholipid vesicles: coupling between transport and phosphorylation.

  Elferink M.G., Driessen A.J., Robillard G.T.

  Purified mannitol-specific enzyme II (EII) from Escherichia coli was reconstituted into phospholipid vesicles with the aid of a detergent-dialysis procedure followed by a freeze-thaw sonication step. The orientation of EII in the proteoliposomes was random. The cytoplasmic moiety of the inverted E ... >> More

  Purified mannitol-specific enzyme II (EII) from Escherichia coli was reconstituted into phospholipid vesicles with the aid of a detergent-dialysis procedure followed by a freeze-thaw sonication step. The orientation of EII in the proteoliposomes was random. The cytoplasmic moiety of the inverted EII could be removed with trypsin without effecting the integrity of the liposomal membrane. This enabled us to study the two different EII orientations independently. The population of inverted EII molecules was monitored by measuring active extrusion of mannitol after the addition of phosphoenolpyruvate, EI, and histidine-containing phosphocarrier protein (HPr) at the outside of the vesicles. The population of correctly oriented EII molecules was monitored by measuring active uptake of mannitol with internal phosphoenolpyruvate, EI, and HPr. A low rate of facilitated diffusion of mannitol via the unphosphorylated carrier could be measured. On the other hand, a high phosphorylation activity without translocation was observed at the outside of the liposomes. The kinetics of the phosphoenolpyruvate-dependent transport reaction and the nonvectorial phosphorylation reaction were compared. Transport of mannitol into the liposomes via the correctly oriented EII molecules occurred with a high affinity (Km, lower than 10 microM) and with a relatively low Vmax. Phosphorylation at the outside of the liposomes catalyzed by the inverted EII molecules occurred with a low affinity (Km of about 66 microM), while the maximal velocity was about 10 times faster than the transport reaction. The latter observation is kinetic proof for the lack of strict coupling between transport and phosphorylation in these enzymes. << Less

  J. Bacteriol. 172:7119-7125(1990) [PubMed] [EuropePMC]

• Purification of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

  Jacobson G.R., Lee C.A., Saier M.H. Jr.

  The inducible, mannitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system has been purified approximately 230-fold from Escherichia coli membranes. The enzyme, initially solubilized with deoxycholate, was first subjected to hydrophobic chromatography on hexyl agarose an ... >> More

  The inducible, mannitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system has been purified approximately 230-fold from Escherichia coli membranes. The enzyme, initially solubilized with deoxycholate, was first subjected to hydrophobic chromatography on hexyl agarose and then purified by several ion exchange steps in the presence of the nonionic detergent, Lubrol PX. The purified protein appears homogeneous by several criteria and probably consists of a single kind of polypeptide chain with a molecular weight of 60,000 (+/-5%). In addition to catalyzing phosphoenolpyruvate-dependent phosphorylation of mannitol in the presence of the soluble enzymes of the phosphotransferase system, the purified Enzyme II also catalyzes mannitol 1-phosphate:mannitol transphosphorylation in the absence of these components. A number of other physical and catalytic properties of the enzyme are described. The availability of a stable, homogeneous Enzyme II should be invaluable for studying the mechanism of sugar translocation and phosphorylation catalyzed by the bacterial phosphotransferase system. << Less

  J. Biol. Chem. 254:249-252(1979) [PubMed] [EuropePMC]