Biomedical subjects

S Roseman

Publications and source records attributed to S Roseman.

At least 37 records · Page 2Linked to original sources

PubMed1996

The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of a periplasmic beta-N-acetylglucosaminidase.

We have described some steps in chitin catabolism by Vibrio furnissii, and proposed that chitin oligosaccharides are hydrolyzed in the periplasmic space to GlcNAc and (GlcNAc)2. Since (GlcNAc)2 is an important inducer in the cascade, it must resist hydrolysis in the periplasm. Known V. furnissii periplasmic hydrolases comprise an endoenzyme (Keyhani, N. O. and Roseman, S. (1996) J. Biol. Chem. 271, 33414-33424), and the beta-N-acetylglucosaminidase, ExoI, reported here. ExoI was isolated from a recombinant strain of Escherichia coli, and hydrolyzes aryl-beta-GlcNAc, aryl-beta-GalNAc, and chitin oligosaccharides. No other beta-GlcNAc glycosides were cleaved. The pH optimum was 7.0 for (GlcNAc)n, n = 3-6, but 5.8 for (GlcNAc)2. At the pH of sea water (8.0-8.3), the enzymatic activity with (GlcNAc)2 is virtually undetectable. These results explain the stability of (GlcNAc)2 in the periplasmic space. The cloned beta-GlcNAcidase gene, exoI, encodes a 69,377-kDa protein (611 amino acids); the predicted N-terminal 20 amino acid residues matched those of the isolated protein. The protein amino acid sequence displays significant homologies to the alpha- and beta-chains of human hexosaminidase despite their marked differences in substrate specificities and pH optima.

Acetylglucosaminidase↗

PubMed1996

Molecular cloning and characterization of a novel beta-N-acetyl-D-glucosaminidase from Vibrio furnissii.

The accompanying papers (Keyhani, N. O., and Roseman, S. (1996) J. Biol. Chem. 271, 33414-33424; Keyhani, N. O., and Roseman, S. (1996) J. Biol. Chem. 271, 33425-33432) describe two unique beta-N-acetylglucosaminidases from Vibrio furnissii. A third, ExoII, is reported here. The gene, exoII, was cloned into Escherichia coli, sequenced, and ExoII purified to apparent homogeneity (36 kDa). The molecular weight and N-terminal 16 amino acids of the protein conform to the predicted sequence. ExoII exhibited unique substrate specificity. It rapidly cleaved p-nitrophenyl and 4-methylumbelliferyl beta-GlcNAc, was slightly active with p-nitrophenyl-beta-GalNAc, and was inactive with all other GlcNAc derivatives tested, including N,N'-diacetylchitobiose and (GlcNAc)n, n = 3-6. Unlike GlcNAc (Ki, 210 microM), (GlcNAc)n are poor inhibitors of ExoII. The predicted protein sequence is unique among beta-N-acetylglucosaminidases excepting Cht60, recently cloned from a marine Alteromonas (Tsujibo, H., Fujimoto, K., Tanno, H., Miyamoto, K., Imada, C., Okami, Y., and Inamori, Y. (1994) Gene (Amst.) 146, 111-115). Cht60, a chitobiase, is 26.9% identical to ExoII in a 182-amino acid overlap, but the two enzymes differ in substrate specificity and other properties. ExoII shares similarity with five bacterial and yeast beta-glucosidases, up to 44% identity in the 25-amino acid catalytic domain. By analogy, ExoII may play a role in signal transduction between invertebrate hosts and V. furnissii.

Acetylglucosamine↗

PubMed1996

Rate and equilibrium constants for phosphoryltransfer between active site histidines of Escherichia coli HPr and the signal transducing protein IIIGlc.

The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) plays a central role in catabolizing many sugars; regulation is effected by phosphorylation of PTS proteins. In Escherichia coli, the phosphoryltransfer sequence for glucose uptake is: PEP --> Enzyme I(His191) --> HPr(His15) --> IIIGlc(His90) --> IIGlc(Cys421) --> glucose. A rapid quench method has now been developed for determining the rate and equilibrium constants of these reactions. The method was validated by control experiments, and gave the following results for phosphoryltransfer between the following protein pairs. For phospho-HPr/IIIGlc (and HPr/phospho-IIIGlc), k1 = 6.1 x 10(7) M-1 s-1, k-1 = 4.7 x 10(7); for the mutant H75QIIIGlc in place of IIIGlc, k1 = 2.8 x 10(5) M-1 s-1, k-1 = 2.3 x 10(5). The derived Keq values agreed with the Keq obtained without use of the rapid quench apparatus. Keq for both reactions is 1-1.5. The rate of phosphoryltransfer between HPr and wild type IIIGlc is close to a diffusion-controlled process, while the reactions involving the mutant H75QIIIGlc are 200-fold slower. These rate differences are explained by an hypothesis for the mechanism of phosphoryltransfer between HPr and IIIGlc based on the structures of mutant and wild type proteins (see Pelton et al. (Pelton, J. G., Torchia, D. A., Remington, S. J., Murphy, K. P., Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33446-33456)).

Antigens, Bacterial↗

PubMed1996

Structures of active site histidine mutants of IIIGlc, a major signal-transducing protein in Escherichia coli. Effects on the mechanisms of regulation and phosphoryl transfer.

IIIGlc (also called IIAGlc), a major signal-transducing protein in Escherichia coli, is also a phosphorylcarrier in glucose uptake. The crystal and NMR structures of IIIGlc show that His90, the phosphoryl acceptor, adjoins His75 in the active site. Glutamine was substituted for His-, giving H75QIIIGlc and H90QIIIGlc, respectively (Presper, K. A., Wong, C.-Y., Liu, L., Meadow, N. D., and Roseman, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4052-4055), but the mutants showed unexpected properties. H90QIIIGlc loses regulatory functions of IIIGlc, and the phosphoryltransfer rates between HPr/H75QIIIGlc are 200-fold less than HPr/IIIGlc (Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33440-33445). X-ray crystallography, differential scanning calorimetry, and NMR have now been used to determine the structures of the mutants (phospho-H75QIIIGlc was studied by NMR). The three methods gave completely consistent results. Except for the His to Gln substitutions, the only significant structural changes were in a few hydrogen bonds. H90QIIIGlc contains two structured water molecules (to Gln90), which could explain its inability to regulate glycerol kinase. Phospho-IIIGlc contains a chymotrypsin-like, hydrogen bond network (Thr73-His75-O--phosphoryl), whereas phospho-H75QIIIGlc contains only one bond (Gln75-O--phosphoryl). Hydrogen bonds play an essential role in a proposed mechanism for the phosphoryltransfer reaction.

Binding Sites↗

PubMed1996

Sugar transport by the marine chitinolytic bacterium Vibrio furnissii. Molecular cloning and analysis of the glucose and N-acetylglucosamine permeases.

Chitin catabolism by the marine bacterium Vibrio furnissii involves chemotaxis to and transport of N-acetyl-D-glucosamine (GlcNAc) and D-glucose. We report the properties of the respective permeases that complemented E. coli Glc- Man- mutants. Although the V. furnissii Glc-specific permease (55,941 Da) shares 38% identity with E. coli IIGlc (ptsG), it is 67% identical to MalX of the E. coli maltose operon (Reidl, J., and Boos, W. (1991) J. Bacteriol. 173, 4862-4876). An adjacent open reading frame encodes a protein with 52% identity to E. coli MalY. Glc phosphorylation requires only V. furnissii MalX and the accessory phosphoenolpyruvate:glycose phosphotransferase system proteins. The V. furnissii equivalent of IIGlc was not found in the 25,000 transformants screened. The GlcNAc/Glc-specific permease (52,894 Da) shares 47% identity with the N-terminal, hydrophobic domain of E. coli IINag, but is unique among IINag proteins in that it lacks the C-terminal domain and thus requires IIIGlc for sugar fermentation in vivo and phosphorylation in vitro. While there are similarities between the phosphoenolpyruvate:glycose phosphotransferase system of V. furnissii and enteric bacteria, the differences may be important for survival of V. furnissii in the marine environment.

Amino Acid Sequence↗

PubMed1996

Sugar transport by the marine chitinolytic bacterium Vibrio furnissii. Molecular cloning and analysis of the mannose/glucose permease.

We have previously reported that the chitin catabolic cascade in Vibrio furnissii involves multiple signal transducing systems, and that mono- and disaccharide chemoreceptors/transporters are essential components of some of these systems. This and the accompanying papers (Bouma, C. L., and Roseman, S. (1996) J. Biol. Chem 271, 33457-33467; Keyhani, N. O., Wang, L.-X., Lee, Y. C., and Roseman, S. (1996) J. Biol. Chem. 271, 33409-33413) describe some of the sugar transporters. A 13-kilobase pair fragment of V. furnissii DNA was found to impart a Glc+, Man+ phenotype to Escherichia coli ptsG ptsM mutants, and encodes the mannose transporter, ptsM, of the phosphoenolpyruvate:glycose phosphotransferase system. Unlike the E. coli mannose permease, V. furnissii IIMan is inactive with GlcNAc and Fru, and is encoded by four genes rather than three. The gene order is manXYZW, where the product of manY corresponds to IIPMan, manZ to the mannose receptor IIBMan, and manX and manW to the single E. coli gene, manX (which encodes IIIMan, viz. IIAMan). Thus, in V. furnissii, the E. coli manX equivalent comprises two genes, which are separated in the genome by two other genes of the ptsM complex. Two additional open reading frames were detected in the V. furnissii DNA fragment. One encodes a GlcNAc-6-P deacetylase, and the other is similar to aldolase.

Amidohydrolases↗

PubMed1996

The N-terminal domain of Escherichia coli enzyme I of the phosphoenolpyruvate/glycose phosphotransferase system: molecular cloning and characterization.

The bacterial phosphoenolpyruvate/glycose phosphotransferase system (PTS) comprises a group of proteins that catalyze the transfer of the phosphoryl group from phosphoenolpyruvate (PEP) to sugars concomitant with their translocation. The first two steps of the phosphotransfer sequence are PEP <--> Enzyme I (EI) <--> HPr (the histidine-containing phosphocarrier protein). We have proposed that many functions of the PTS are regulated by EI, which undergoes a monomer/dimer transition. EI monomer (63.5 kDa) comprises two major domains: a flexible C-terminal domain (EI-C) and a protease-resistant, structurally stable N-terminal domain (EI-N) containing the active site His. Trypsin treatment of Salmonella typhimurium EI yielded EI-N, designated EI-N(t). Homogeneous recombinant Escherichia coli EI-N [i.e., EI-N(r)], has now been prepared in quantity, shows the expected thermodynamic unfolding properties and, similarly to EI-N(t), is phosphorylated by phospho-HPr, but not by PEP. In addition, binding of EI-N(r) to HPr was studied by isothermal titration calorimetry: K/a = 1.4 x 10(5) M(-1) and delta H = +8.8 kcal x mol(-1). Both values are comparable to those for HPr binding to intact EI. Fluorescence anisotropy [dansyl-EI-N(r)] and gel filtration of EI-N(r) show that it does not dimerize. These results emphasize the role of EI-C in dimerization and the regulation of intact EI.

Bacterial Proteins↗

PubMed1996

Active site phosphorylation of enzyme I of the bacterial phosphotransferase system by an ATP-dependent kinase.

Enzyme I (EI) of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) is autocatalytically phosphorylated by P-enolpyruvate. We report here an ATP-dependent kinase (EI-K) from Escherichia coli that reversibly phosphorylates EI at its active site histidine; ATP and EI-K can therefore replace P-enolpyruvate. EI-K contains a bound cofactor that is lost during purification with concomitant loss of activity. NAD+ and NADP+ substitute for the cofactor and restore activity to the apoenzyme, whereas their analogues are inactive. The pyridine nucleotides do not activate EI-K by covalent modification (e.g. ADP-ribosylation), but must be present during the kinase reaction. NADH and NADPH are potent inhibitors of EI-K at all stages of purity, and enzyme activity in a mixture of NAD+ and NADH depends on the ratio of the two pyridine nucleotides. Inhibition is observed with reduced beta-NMN and alpha-NADH, but neither is as effective as beta-NADH. The reverse reaction, the transfer of the phosphoryl moiety from phospho-EI to ADP, also requires NAD+ or NADP+. In the absence of NAD+ or NADH, [32P]phospho-EI is hydrolyzed to 32Pi, suggesting that EI-K can act as a phospho-EI phosphatase. EI kinase may serve as a link between PTS-driven sugar transport and the electron transport chain.

Binding Sites↗

PubMed1996

A single amino acid change in Escherichia coli glycerol kinase abolishes glucose control of glycerol utilization in vivo.

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in glucose control of glycerol metabolism. Its catalytic activity is inhibited allosterically by the glycolytic intermediate, fructose 1,6-biphosphate, and by the phosphotransferase system phosphocarrier protein, IIIGlc (also known as IIAGlc). These inhibitors provide mechanisms by which glucose blocks glycerol utilization in vivo. We report here the cloning and sequencing of the glpK22 gene isolated from E. C. C. Lin strain 43, a strain that shows the loss of glucose control of glycerol utilization. DNA sequencing shows a single missense mutation that translates to the amino acid change Gly-304 to Ser (G-304-S) in glycerol kinase. The effects of this substitution on the functional and physical properties of the purified mutant enzyme were determined. Neither of the allosteric ligands inhibits it under conditions that produce strong inhibition of the wild-type enzyme, which is sufficient to explain the phenotype of strain 43. However, IIIGlc activates the mutant enzyme, which could not be predicted from the phenotype. In the wild-type enzyme, G-304 is located 1.3 nm from the active site and 2.5 nm from the IIIGlc binding site (M. Feese, D. W. Pettigrew, N. D. Meadow, S. Roseman, and S. J. Remington, Proc. Natl. Acad. Sci. USA 91:3544-3548, 1994). It is located in the same region as amino acid substitutions in the related protein DnaK which alter its catalytic and regulatory properties and which are postulated to interfere with a domain closure motion (A. S. Kamath-Loeb, C. Z. Lu, W.-C. Suh, M. A. Lonetto, and C. A. Gross, J. Biol. Chem. 270:30051-30059, 1995). The global effect of the G-304-S substitution on the conformation and catalytic and regulatory properties of glycerol kinase is consistent with a role for the domain closure motion in the molecular mechanism for glucose control of glycerol utilization.

Allosteric Regulation↗

PubMed1994

Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer equilibrium by fluorescence anisotropy.

Enzyme I (EI), the first protein of the bacterial phosphotransferase system (PTS), exists in a monomer/dimer (M/D) equilibrium. We have proposed that the two species are functionally different and that their interconversion may regulate sugar transport via the PTS. The C-terminal Cys of Escherichia coli EI was reacted with pyrene maleimide (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995), and the pyrene conjugate used to characterize the M/D equilibrium by fluorescence anisotropy. The properties of unlabeled and pyrene-labeled EI are indistinguishable. Values for the apparent association constant, K'eq, and the steady-state anisotropy of the monomer and the dimer were obtained under a variety of conditions. K'eq increases 23-fold, from 0.45 x 10(5) to 10.7 x 10(5) M-1, as the temperature increases from 6 to 30 degrees C; the association appears to be entropically driven. Under all conditions tested, the K'eq for phospho-EI is 6-12-fold less than for dephospho-EI. For phospho-EI, PEP and Mg2+ induce a 240-fold increase of K'eq when both ligands are present. Based on these data, EI was preincubated under conditions that change K'eq, and the initial activities of the different species were determined at 37 degrees C in a PTS sugar phosphorylation assay with PEP as the phosphoryl donor. The initial rate depends on the M/D ratio; it is maximal when EI is 100% dimer, and zero when EI is 100% monomer. In the latter case, the rate gradually increases in the assay mixture. The results have important implications for how the PTS regulates sugar transport and other physiological phenomena.

Biological Transport↗

PubMed1994

Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer transition kinetics by fluorescence anisotropy.

Enzyme I of the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) exists in a monomer/dimer (M/D) equilibrium. These two forms are functionally different, and their interconversion may be a means of regulating the PTS. The M/D equilibrium was studied by fluorescence anisotropy of a pyrene derivative (Chauvin, F., Brand, L., and Roseman, S. (1994) J. Biol. Chem. 269, 20263-20269). In this paper, the kinetics of the transition is investigated. The following apparent rate constants were found for the M/D transition of phospho-Enzyme I in the presence of Mg2+ and PEP at 6 degrees C: k*A = 3.4 x 10(3) M-1 s-1 and k*D = 1.04 x 10(3) s-1. The association rate is especially slow, 2-3 orders of magnitude slower than the average dimerization rate determined for other proteins. Furthermore, the rate of quaternary structure changes matches that of enzymatic activity changes, as well as that of tertiary structure changes (Chauvin, F., Toptygin, D., Roseman, S., and Brand, L. (1992) Biophys. Chem. 44, 163-173). Finally, the effect of two ligands is shown; PEP increases the relaxation rate by 3-fold at 23 degrees C, and Mg2+ addition causes a 4-fold increase in the relaxation rate.

Biological Transport↗

PubMed1994

Cation-promoted association of a regulatory and target protein is controlled by protein phosphorylation.

A central question in molecular biology concerns the means by which a regulatory protein recognizes different targets. IIIGlc, the glucose-specific phosphocarrier protein of the bacterial phosphotransferase system, is also the central regulatory element of the PTS. Binding of unphosphorylated IIIGlc inhibits several non-PTS proteins, but there is little or no sequence similarity between IIIGlc binding sites on different target proteins. The crystal structure of Escherichia coli IIIGlc bound to one of its regulatory targets, glycerol kinase, has been refined at 2.6-A resolution in the presence of products, adenosine diphosphate and glycerol 3-phosphate. Structural and kinetic analyses show that the complex of IIIGlc with glycerol kinase creates an intermolecular Zn(II) binding site with ligation identical to that of the zinc peptidase thermolysin. The zinc is coordinated by the two active-site histidines of IIIGlc, a glutamate of glycerol kinase, and a water molecule. Zn(II) at 0.01 and 0.1 mM decreases the Ki of IIIGlc for glycerol kinase by factors of about 15 and 60, respectively. The phosphorylation of one of the histidines of IIIGlc, in its alternative role as phosphocarrier, provides an elegant means of controlling the cation-enhanced protein-protein regulatory interaction. The need for the target protein to supply only one metal ligand may account for the lack of sequence similarity among the regulatory targets of IIIGlc.

Binding Sites↗

PubMed1993

Chemotaxis of the marine bacterium Vibrio furnissii to sugars. A potential mechanism for initiating the chitin catabolic cascade.

Immense quantities of chitin are catabolized by marine bacteria, and this process involves at least three signal transduction systems in Vibrio furnissii. One system, chemotaxis to chitin oligosaccharides, is probably used to colonize chitin particles. But how do the first few cells find this highly insoluble polysaccharide? The following hypothesis is proposed to answer this question: the bacteria respond to soluble chemo-attractants in exudates from injured organisms. Virtually all chitin-producing organisms also contain glucose and/or trehalose, often at high concentrations such as trehalose in insect hemolymph. Chemotaxis of V. furnissii was therefore studied with a variety of sugars. Fructose, ribose, and glycerol are catabolites but not attractants. The cells exhibit weak constitutive taxis to Glc and GlcNAc. After induction, they show a weak response to galactose but are strongly attracted to the following substrates of the phosphoenolpyruvate:glycose phosphotransferase system (PTS): GlcNAc, trehalose, glucose, sucrose, mannose, and mannitol. There is a rough qualitative but no quantitative correlation between the rate of phosphorylation and the chemotactic response to PTS sugars. Trehalose is especially noteworthy because it is phosphorylated at a very rapid rate by uninduced cells but is not an attractant until the cells are induced. We suggest that unidentified inducible factors link the PTS to chemotaxis.

Carbohydrate Metabolism↗

PubMed1993

Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol kinase.

The phosphocarrier protein IIIGlc is an integral component of the bacterial phosphotransferase (PTS) system. Unphosphorylated IIIGlc inhibits non-PTS carbohydrate transport systems by binding to diverse target proteins. The crystal structure at 2.6 A resolution of one of the targets, glycerol kinase (GK), in complex with unphosphorylated IIIGlc, glycerol, and adenosine diphosphate was determined. GK contains a region that is topologically identical to the adenosine triphosphate binding domains of hexokinase, the 70-kD heat shock cognate, and actin. IIIGlc binds far from the catalytic site of GK, indicating that long-range conformational changes mediate the inhibition of GK by IIIGlc. GK and IIIGlc are bound by hydrophobic and electrostatic interactions, with only one hydrogen bond involving an uncharged group. The phosphorylation site of IIIGlc, His90, is buried in a hydrophobic environment formed by the active site region of IIIGlc and a 3(10) helix of GK, suggesting that phosphorylation prevents IIIGlc binding to GK by directly disrupting protein-protein interactions.

Adenosine Diphosphate↗

PubMed1993

Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques.

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein. Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.

Binding Sites↗

PubMed1992

NAD+ and NADH regulate an ATP-dependent kinase that phosphorylates enzyme I of the Escherichia coli phosphotransferase system.

Crude extracts of Escherichia coli contain a protein kinase, EI-K, that phosphorylates enzyme I (EI) of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). Phosphorylation occurs at the active site histidine residue. The activity of EI-K was lost during purification. However, kinase activity was restored by adding NAD+ or NADP+.NADH reversed NAD+ activation of the kinase, and the level of EI-K activity was dependent on the NAD+/NADH ratio. Although crude preparations of EI-K showed no NAD+ requirement, they were completely inhibited by NADH, either in the assay mixture or when the enzyme was pretreated and the NADH was removed prior to the assay. NAD+ restored full activity to the NADH-pretreated inactive fractions. The results suggest that EI-K contains a bound cofactor that is lost during purification and that may be analogous to NAD+. EI-K activity may serve to link some of the diverse functions of the PTS, such as sugar transport, to the metabolic state of the cell.

Adenosine Triphosphate↗

PubMed1992

Evidence for two promoters upstream of the pts operon: regulation by the cAMP receptor protein regulatory complex.

Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP).cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo. Promoter-reporter gene fusion studies identified two CRP.cAMP-dependent promoters (the previously identified P1 and another promoter, P0) upstream of ptsH. The crr promoters (P2) within ptsI may be negatively regulated by CRP.cAMP.

Chloramphenicol O-Acetyltransferase↗

PubMed1992

Structural comparison of phosphorylated and unphosphorylated forms of IIIGlc, a signal-transducing protein from Escherichia coli, using three-dimensional NMR techniques.

The 18.1-kDa protein IIIGlc from Escherichia coli acts as both a phosphocarrier protein in the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and as a signal-transducing protein with respect to the uptake of non-PTS sugars. Phosphorylation of IIIGlc at the N epsilon (N3) position of His-90 was effected through a regeneration system that included MgCl2, DTT, excess PEP, and catalytic amounts of Enzyme I and HPr. NH, 15N, and 13C alpha signal assignments for P-IIIGlc were made through comparison of 15N-1H correlation spectra (HSQC) of uniformly 15N-labeled preparations of phosphorylated and unphosphorylated protein and through analysis of three-dimensional triple-resonance HNCA spectra of P-IIIGlc uniformly labeled with both 15N and 13C. Backbone and side-chain 1H and 13C beta signals were assigned using 3D heteronuclear HCCH-COSY and HCCH-TOCSY spectra of P-IIIGlc. Using this approach, the assignments were made without reference to nuclear Overhauser effect data or assumptions regarding protein structure. The majority of NH, 15N, H alpha, and 13C alpha chemical shifts measured for P-IIIGlc were identical to those obtained for the unphosphorylated protein [Pelton, J. G., Torchia, D. A., Meadow, N. D., Wong, C.-Y., & Roseman, S. (1991) Biochemistry 30, 10043]. Those signals that exhibited shifts corresponded to residues within four segments (1) Leu-87-Gly-100, (2) Val-36-Val-46, (3) His-75-Ser-78, and (4) Ala-131-Val-138. These four segments are in close proximity to the active site residues His-75 and His-90 in the unphosphorylated protein [Worthylake, D., Meadow, N. D., Roseman, S., Liao, D., Hertzberg, O., & Remington, S.J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10382], and the chemical shift data provide strong evidence that if any structural changes accompany phosphorylation, they are confined to residues in these four segments. This conclusion is confirmed by comparing NOEs observed in 3D 15N/13C NOESY-HMQC spectra of the two forms of the protein. No NOE differences are seen for residues having the same chemical shifts in IIIGlc and P-IIIGlc. Furthermore, with the exception of residues Ala-76, Asp-94, and Val-96, the NOEs of residues (in the four segments) which exhibited chemical shift differences also had the same NOEs in IIIGlc and P-IIIGlc. In the case of residues Ala-76, Asp-94, and Val-96, minor differences in NOEs, corresponding to interproton distances changes of less than 1.5 A, were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗