Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1

The allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

This work reports the evolutionary relationships, amplified expression, functional characterisation and purification of the putative allantoin transport protein, PucI, from Bacillus subtilis. Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between PucI and membrane proteins of the nucleobase-cation-symport-1 family of secondary active transporters. These include the sodium-coupled hydantoin transport protein, Mhp1, from Microbacterium liquefaciens and related proteins from bacteria, fungi and plants. Membrane topology predictions for PucI were consistent with twelve putative transmembrane-spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. The pucI gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions are described for optimal amplified expression of the PucI(His6) protein in Escherichia coli to a level of about 5% in inner membranes. Initial rates of inducible PucI-mediated uptake of 14C-allantoin into energised Escherichia coli whole cells conformed to Michaelis-Menten kinetics with an apparent affinity (Kmapp) of 24 ± 3M, therefore confirming that PucI is a medium affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that PucI recognises some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His6) was solubilised from inner membranes using n-dodecyl-β-D-maltoside and purified. The isolated protein comprised a substantial proportion of α-helix secondary structure, consistent with the predictions, and a three-dimensional model was therefore constructed on a template of the Mhp1 structure, which aided localisation of the potential ligand binding site in PucI

Microbial transformations of selenite by methane-oxidizing bacteria

Abstract Methane oxidizing bacteria are well known for their role in the global methane cycle and their potential for microbial transformation of wide range of hydrocarbon and chlorinated hydrocarbon pollution. Recently, it has also emerged that methane-oxidizing bacteria interact with inorganic pollutants in the environment. Here we report what we believe to be the first study of the interaction of pure strains of methane-oxidizing bacteria with selenite. Results indicate that the commonly used laboratory model strains of methane oxidizing bacteria, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b are both able to reduce the toxic selenite (SeO32-) but not selenate (SeO42-) to red spherical nanoparticulate elemental selenium (Se0), which was characterised via EDX and EXAFS. The cultures also produced volatile selenium-containing species, which suggests that both strains may have an additional activity that can either transform Se0 or selenite into volatile methylated forms of selenium. Transmission electron microscopy (TEM) measurements and experiments with the cell fractions: cytoplasm, cell wall and cell membrane show that the nanoparticles are formed mainly on the cell wall. Collectively these results are promising for the use of methane-oxidizing bacteria for bioremediation or suggest possible uses in the production of selenium nanoparticles for biotechnology

Efficiency of Purine Utilization by Helicobacter pylori: Roles for Adenosine Deaminase and a NupC Homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase

Modeling violations of the race model inequality in bimodal paradigms: co-activation from decision and non-decision components

The redundant-signals paradigm (RSP) is designed to investigate response behavior in perceptual tasks in which response-relevant targets are defined by either one or two features, or modalities. The common finding is that responses are speeded for redundantly compared to singly defined targets. This redundant-signals effect (RSE) can be accounted for by race models if the response times do not violate the race model inequality (RMI). When there are violations of the RMI, race models are effectively excluded as a viable account of the RSE. The common alternative is provided by co-activation accounts, which assume that redundant target signals are integrated at some processing stage. However, “co-activation” has mostly been only indirectly inferred and the accounts have only rarely been explicitly modeled; if they were modeled, the RSE has typically been assumed to have a decisional locus. Yet, there are also indications in the literature that the RSE might originate, at least in part, at a non-decisional or motor stage. In the present study, using a distribution analysis of sequential-sampling models (ex-Wald and Ratcliff Diffusion model), the locus of the RSE was investigated for two bimodal (audio-visual) detection tasks that strongly violated the RMI, indicative of substantial co-activation. Three model variants assuming different loci of the RSE were fitted to the quantile reaction time proportions: a decision, a non-decision, and a combined variant both to vincentized group as well as individual data. The results suggest that for the two bimodal detection tasks, co-activation has a shared decisional and non-decisional locus. These findings point to the possibility that the mechanisms underlying the RSE depend on the specifics (task, stimulus, conditions, etc.) of the experimental paradigm

The Effects of Oral Consumption of Selenium Nanoparticles on Chemotactic and Respiratory Burst Activities of Neutrophils in Comparison with Sodium Selenite in Sheep

The present study was designed to compare the effects of nano-selenium and of sodium selenite on the chemotactic and respiratory burst activities of neutrophils in sheep. Fifteen sheep were randomly divided into three groups. Groups 1 and 2 received selenium nanoparticles (1 mg/kg) or sodium selenite (1 mg/kg) orally, respectively, for ten consecutive days, and the third group was considered as the control. To determine the chemotactic and respiratory burst activities of the neutrophils, the leading front assay and the NBT test were used on heparinized blood samples that were collected at different intervals (days 0, 10th, 20th, and 30th). The results obtained showed that the chemotactic activities in groups 1 and 2 increased significantly on the 10th, 20th, and 30th day, compared to day 0, and on the 20th day in comparison with the 10th day, while in group 2, there was a significant decrease on the 30th day compared to the 20th day. The chemotactic activities in group 1 were significantly higher than in group 2 on the 10th day and in the control group on the 10th, 20th, and 30th day, but the chemotactic activities in group 2 were significantly higher than those in the control group only on the 20th day. On the 30th day into the experiment, the respiratory bursts in groups 1 and 2 were significantly stronger in comparison with those at day 0. Overall, nano-selenium increased the chemotactic and respiratory burst activities more significantly than sodium selenite, which is suggestive of a stronger stimulatory effect of the Se nanoparticles on intracellular activities