Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose

Wild-type strains of Escherichia coli are normally unable to metabolize cellobiose. However, cellobiose-positive (Cel+) mutants arise upon prolonged incubation on media containing cellobiose as the sole carbon source. We show that the Cel+ derivatives carry two classes of mutations that act concertedly to alter the regulation of the chb operon involved in the utilization of N,N'-diacetylchitobiose. These consist of mutations that abrogate negative regulation by the repressor NagC as well as single base-pair changes in the transcriptional regulator chbR that translate into single-amino-acid substitutions. Introduction of chbR from two Cel+ mutants resulted in activation of transcription from the chb promoter at a higher level in the presence of cellobiose, in reporter strains carrying disruptions of the chromosomal chbR and nagC. These transformants also showed a Cel+ phenotype on MacConkey cellobiose medium, suggesting that the wild-type permease and phospho-β-glucosidase, upon induction, could recognize, transport and cleave cellobiose respectively. This was confirmed by expressing the wild-type genes encoding the permease and phospho-β-glucosidase under a heterologous promoter. Biochemical characterization of one of the chbR mutants, chbRN238S, showed that the mutant regulator makes stronger contact with the target DNA sequence within the chb promoter and has enhanced recognition of cellobiose 6-phosphate as an inducer compared with the wild-type regulator

Solution structures and thermodynamics of cis-trans X-Pro conformers of a novel single disulfide conopeptide

710-728The conopeptide Mo1853 (MW = 1853 Da) consists of 17 residues and a single disulfide bond. Structural studies using homonuclear solution NMR methods (2D 1H,1H DQF-COSY, TOCSY, NOESY and ROESY spectra) revealed that Mo1853 exists as two equally populated cis and trans X–Pro peptide bond conformers which are in slow exchange regime, compared to the chemical shift time scale. Temperature dependence of chemical shifts was measured and using coalescence temperature of two amide protons, the rate of exchange and the free energy of activation for the conformational exchange were determined to be 59 Hz and ≈ 67.2 kJ mol−1, respectively, at 318 K. Additional evidence for this conformational equilibrium was also observed as exchange correlation peaks in the 2D-NOESY and ROESY spectra. Tertiary structures of both the cis (PDB ID 8K3N) and trans (PDB ID 8K3M) conformers were determined using distance restraints, backbone dihedral angle restraints, the disulfide bond restraint and the cis or trans conformation of the X–Pro peptide bond. The trans conformer of Mo1853 is stabilized by hydrogen bonds while the cis conformer seems to be stabilized predominantly by hydrophobic interactions. This was further corroborated by the fact that at lower temperatures, the hydrophobic interactions became weaker reducing the population of the cis conformer with respect to that of the trans conformer. The cis and trans X–Pro peptide bond conformational exchange could be another means to enhance the structural variability of the conopeptides and could have significance in the synergistic functional response caused by the cone snail venom peptides

Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose

Wild-type strains of Escherichia coli are normally unable to metabolize cellobiose. However, cellobiosepositive $(Cel^+)$ mutants arise upon prolonged incubation on media containing cellobiose as the sole carbon source. We show that the $Cel^+$ derivatives carry two classes of mutations that act concertedly to alter the regulation of the chb operon involved in the utilization of $N,N^{\prime}$-diacetylchitobiose. These consist of mutations that abrogate negative regulation by the repressor NagC as well as single base-pair changes in the transcriptional regulator chbR that translate into singleamino-acid substitutions. Introduction of chbR from two $Cel^+$ mutants resulted in activation of transcription from the chb promoter at a higher level in the presence of cellobiose, in reporter strains carrying disruptions of the chromosomal chbR and nagC. These transformants also showed a $Cel^+$ phenotype on MacConkey cellobiose medium, suggesting that the wild-type permease and phospho-\beta-glucosidase, upon induction, could recognize, transport and cleave cellobiose respectively. This was confirmed by expressing the wild-type genes encoding the permease and phospho-\beta-glucosidase under a heterologous promoter. Biochemical characterization of one of the chbR mutants, chbRN238S, showed that the mutant regulator makes stronger contact with the target DNA sequence within the chb promoter and has enhanced recognition of cellobiose 6-phosphate as an inducer compared with the wild-type regulator

A Disulfide Stabilized beta-Sandwich Defines the Structure of a New Cysteine Framework M-Superfamily Conotoxin

The structure of a new cysteine framework (-C-CC-C-C-C) ``M''-superfamily conotoxin, Mo3964, shows it to have a beta-sandwich structure that is stabilized by inter-sheet cross disulfide bonds. Mo3964 decreases outward K+ currents in rat dorsal root ganglion neurons and increases the reversal potential of the Na(V)1.2 channels. The structure of Mo3964 (PDB ID: 2MW7) is constructed from the disulfide connectivity pattern, i.e., 1-3, 2-5, and 4-6, that is hitherto undescribed for the ``M''-superfamily conotoxins. The tertiary structural fold has not been described for any of the known conus peptides. NOE (549), dihedral angle (84), and hydrogen bond (28) restraints, obtained by measurement of (h3)J(NC') scalar couplings, were used as input for structure calculation. The ensemble of structures showed a backbone root mean square deviation of 0.68 +/- 0.18 angstrom, with 87% and 13% of the backbone dihedral (phi, psi) angles lying in the most favored and additional allowed regions of the Ramachandran map. The conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules

A Disulfide Stabilized β‑Sandwich Defines the Structure of a New Cysteine Framework M‑Superfamily Conotoxin

The structure of a new cysteine framework (−CCCCCC−) “M”-superfamily conotoxin, Mo3964, shows it to have a β-sandwich structure that is stabilized by inter-sheet cross disulfide bonds. Mo3964 decreases outward K⁺ currents in rat dorsal root ganglion neurons and increases the reversal potential of the Na_V1.2 channels. The structure of Mo3964 (PDB ID: 2MW7) is constructed from the disulfide connectivity pattern, i.e., 1-3, 2-5, and 4-6, that is hitherto undescribed for the “M”-superfamily conotoxins. The tertiary structural fold has not been described for any of the known <i>conus</i> peptides. NOE (549), dihedral angle (84), and hydrogen bond (28) restraints, obtained by measurement of ^h3<i>J</i>_NC′ scalar couplings, were used as input for structure calculation. The ensemble of structures showed a backbone root mean square deviation of 0.68 ± 0.18 Å, with 87% and 13% of the backbone dihedral (ϕ, ψ) angles lying in the most favored and additional allowed regions of the Ramachandran map. The conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules