14 research outputs found
Structure and function of the Ts2631 endolysin of <i>Thermus scotoductus</i> phage vB_Tsc2631 with unique N-terminal extension used for peptidoglycan binding
Abstract To escape from hosts after completing their life cycle, bacteriophages often use endolysins, which degrade bacterial peptidoglycan. While mesophilic phages have been extensively studied, their thermophilic counterparts are not well characterized. Here, we present a detailed analysis of the structure and function of Ts2631 endolysin from thermophilic phage vB_Tsc2631, which is a zinc-dependent amidase. The active site of Ts2631 consists of His30, Tyr58, His131 and Cys139, which are involved in Zn2+ coordination and catalysis. We found that the active site residues are necessary for lysis yet not crucial for peptidoglycan binding. To elucidate residues involved in the enzyme interaction with peptidoglycan, we tested single-residue substitution variants and identified Tyr60 and Lys70 as essential residues. Moreover, substitution of Cys80, abrogating disulfide bridge formation, inactivates Ts2631, as do substitutions of His31, Thr32 and Asn85 residues. The endolysin contains a positively charged N-terminal extension of 20 residues that can protrude from the remainder of the enzyme and is crucial for peptidoglycan binding. We show that the deletion of 20 residues from the N-terminus abolished the bacteriolytic activity of the enzyme. Because Ts2631 exhibits intrinsic antibacterial activity and unusual thermal stability, it is perfectly suited as a scaffold for the development of antimicrobial agents
Crystal growth, structure and characterization of diglycine zinc dipicrate: Centrosymmetric crystal exhibiting second harmonic generation efficiency
<p></p> <p>Single crystals of diglycine zinc dipicrate (DZD) were grown by the slow evaporation solution growth technique from ethanol at room temperature. The structure is elucidated by single crystal XRD analysis and it belongs to the triclinic system with centrosymmetric space group P<sub>ī</sub>. The crystallinity of the material was confirmed by powder X-ray diffraction analysis. The functional groups present in the molecule are identified by FT-IR analysis and the band gap energy is estimated using diffuse reflectance data by the application of Kubelka–Munk algorithm. Investigation of the intermolecular interactions and crystal packing <i>via</i> Hirshfeld surface analysis, based on single-crystal XRD, reveals the close contacts associated with molecular interactions. Fingerprint plots of the Hirshfeld surfaces were used to locate and analyze the percentage of hydrogen-bonding interactions.The data are analysed by combined results of single crystal XRD and electronic structure calculations. Theoretical calculations done by density functional theory(DFT) gives an idea about structure-function relationship. The second harmonic generation efficiency (SHG) is estimated using the Kurtz and Perry technique and it reveals second order nonlinearity. The observation of a centrosymmetric crystal exbiting NLO character is rationalized.</p
Crystal growth, structure and characterization of diglycine zinc dipicrate: Centrosymmetric crystal exhibiting second harmonic generation efficiency
Prescreening of Nicotine Hapten Linkers in Vitro To Select Hapten-Conjugate Vaccine Candidates for Pharmacokinetic Evaluation in Vivo
Since
the demonstration of nicotine vaccines as a possible therapeutic
intervention for the effects of tobacco smoke, extensive effort has
been made to enhance nicotine specific immunity. Linker modifications
of nicotine haptens have been a focal point for improving the immunogenicity
of nicotine, in which the evaluation of these modifications usually
relies on in vivo animal models, such as mice, rats or nonhuman primates.
Here, we present two in vitro screening strategies to estimate and
predict the immunogenic potential of our newly designed nicotine haptens.
One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA)
to profile the interactions of nicotine haptens or hapten-protein
conjugates with nicotine specific antibodies, both polyclonal and
monoclonal. Another relies on computational modeling of the interactions
between haptens and amino acid residues near the conjugation site
of the carrier protein to infer linker-carrier protein conjugation
effect on antinicotine antibody response. Using these two in vitro
methods, we ranked the haptens with different linkers for their potential
as viable vaccine candidates. The ELISA-based hapten ranking was in
an agreement with the results obtained by in vivo nicotine pharmacokinetic
analysis. A correlation was found between the average binding affinity
(IC<sub>50</sub>) of the haptens to an anti-Nic monoclonal antibody
and the average brain nicotine concentration in the immunized mice.
The computational modeling of hapten and carrier protein interactions
helps exclude conjugates with strong linker-carrier conjugation effects
and low in vivo efficacy. The simplicity of these in vitro screening
strategies should facilitate the selection and development of more
effective nicotine conjugate vaccines. In addition, these data highlight
a previously under-appreciated contribution of linkers and hapten-protein
conjugations to conjugate vaccine immunogenicity by virtue of their
inclusion in the epitope that binds and activates B cells
NADH-dependent lactate dehydrogenase from Alcaligenes eutrophus H16 reduces 2-oxoadipate to 2-hydroxyadipate
Adipic acid is an important monomer for the production of nylon-6,6 polyamide. One novel biological route for the synthesis of adipic acid, which combines the lysine synthetic pathway and glutaconic acid production pathway, has been suggested, but this route has suffered from the lack of an efficient 2-oxoadipate reductase connecting the two pathways or converting 2-oxoadipate to 2-hydroxyadipate. In this study, we report that the lactate dehydrogenase of Alcaligenes eutrophus H16 is a promising catalyst for this reaction. The lactate dehydrogenase gene (Ae-ldhO) was cloned, expressed in Escherichia coli, purified, and characterized. The recombinant enzyme, having a molecular weight of 36.7 kDa, exhibited broad substrate specificity for various 2-oxoacids. NADH was the preferred coenzyme over NADPH for all 2-oxoacids tested. The maximum specific activity of Ae-LdhO on 2-oxoadipate was 454.5 +/- 20.1 U/mg protein at pH 7.0 and 30a"integral. The K (m) values for 2-oxoadipic acid and NADH were 0.32 +/- 0.02 and 0.09 +/- 0.002 mM, respectively. The activity of Ae-LdhO was enhanced in the presence of some metal ions, such as Mg2+, Co2+ or Ni2+, whereas it was completely inhibited by Hg2+, Ag+, Cu2+ and DTT
Surface Behavior of Hydrated Guanidinium and Ammonium Ions : A Comparative Study by Photoelectron Spectroscopy and Molecular Dynamics
Through the combination of surface sensitive photoelectron spectroscopy and molecular dynamics simulation, the relative surface propensities of guanidinium and ammonium ions in aqueous solution are characterized. The fact that the N Is binding energies differ between these two species was exploited to monitor their relative surface concentration through their respective photoemission intensities. Aqueous solutions of ammonium and guanidinium chloride, and mixtures of these salts, have been studied in a wide concentration range, and it is found that the guanidinium ion has a greater propensity to reside at the aqueous surface than the ammonium ion. A large portion of the relative excess of guanidinium ions in the surface region of the mixed solutions can be explained by replacement of ammonium ions by guanidinium ions in the surface region in combination with a strong salting-out effect of guanidinium by ammonium ions at increased concentrations. This interpretation is supported by molecular dynamics simulations, which reproduce the experimental trends very well. The simulations suggest that the relatively higher surface propensity of guanidinium compared with ammonium ions is due to the ease of dehydration of the faces of the almost planar guanidinium ion, which allows it to approach the water-vapor interface oriented parallel to it