Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.     

A Nitrile Glucoside and Biflavones from the Leaves of Campylospermum excavatum (Ochnaceae)

The study of the MeOH extract of the leaves of Campylospermum excavatum led to the isolation of a nitrile glucoside, named campyloside C ( 1 ) and an original derivative of ochnaflavone, 7‐O‐methylochnaflavone ( 2 ), along with three known biflavonoids, amentoflavone, sequoiaflavone, and sotetsuflavone ( 3 – 5 ). The linkage site of the sub‐units of 2 was confirmed by chemical correlation, after semi‐synthesis of a trimethoxylated derivative of ochnaflavone ( 2a ). The structures of these compounds as well as their relative and absolute configurations were assigned by 1D‐ and 2D‐NMR experiments, HR‐ESI‐MS and Electronic Circular Dichroism (ECD) calculations. A low‐pass J filter HMBC experiment was performed in order to define the configuration of the double bond of 1 . All of the biflavonoids were evaluated against protozoan parasites. Amentoflavone moderately inhibited the promastigote form of Leishmania infantum.     

A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1

The 2.9 A crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a beta strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.     

BioCaster: detecting public health rumors with a Web-based text mining system

SUMMARY: BioCaster is an ontology-based text mining system for detecting and tracking the distribution of infectious disease outbreaks from linguistic signals on the Web. The system continuously analyzes documents reported from over 1700 RSS feeds, classifies them for topical relevance and plots them onto a Google map using geocoded information. The background knowledge for bridging the gap between Layman's terms and formal-coding systems is contained in the freely available BioCaster ontology which includes information in eight languages focused on the epidemiological role of pathogens as well as geographical locations with their latitudes/longitudes. The system consists of four main stages: topic classification, named entity recognition (NER), disease/location detection and event recognition. Higher order event analysis is used to detect more precisely specified warning signals that can then be notified to registered users via email alerts. Evaluation of the system for topic recognition and entity identification is conducted on a gold standard corpus of annotated news articles. AVAILABILITY: The BioCaster map and ontology are freely available via a web portal at http://www.biocaster.org.     

The stable, functional core of DdrA from Deinococcus radiodurans R1 does not restore radioresistance in vivo

DdrA protein binds to and protects 3′ DNA ends and is essential for preserving the genome integrity of Deinococcus radiodurans following treatment by gamma radiation in an environment lacking nutrients. Limited proteolysis was used to identify a stable and functional protein core, designated DdrA157, consisting of the first 157 residues of the protein. In vitro, the biochemical differences between wild-type and mutant proteins were modest. DdrA exhibits a strong bias in binding DNA with 3′ extensions but not with 5′ extensions. The mutant DdrA157 exhibited a greater affinity for 5′ DNA ends but still bound to 3′ ends more readily. However, when we replaced the wild-type ddrA gene with the mutant gene for ddrA157, the resulting D. radiodurans strain became almost as sensitive to gamma radiation as the ddrA knockout strain. These results suggest that while the stable protein core DdrA157 is functional for DNA binding and protection assays in vitro, the carboxyl terminus is required for important functions in vivo. The C terminus may therefore be required for protein or DNA interactions or possibly as a regulatory region for DNA binding or activities not yet identified.     

Nitrile glucosides and serotobenine from Campylospermum glaucum and Ouratea turnarea

Two nitrile glucosides (1S,3S,4S,5R)-4-benzoyloxy-2-cyanomethylene-3,5-dihydroxycyclohexyl-1-O-beta-glucopyranoside (campyloside A) and (1S,3S,4S,5R)-5-benzoyloxy-2-cyanomethylene-3-hydroxy-4-(2-pyrrolcarboxyloxy)cyclohexyl-1-O-beta-glucopyranoside (campyloside B) were isolated from the stem roots of Campylospermum glaucum, whereas serotobenine was isolated from Ouratea turnarea. The structure elucidations were based on spectroscopic evidence. The biological assays of compounds and crude extract of plant species showed good antimicrobial activity of crude extracts against Gram-positive cocci.     

Methyl-branched poly(hydroxyalkanoate) biosynthesis from 13-methyltetradecanoic acid and mixed isostearic acid isomer substrates

Nonylphenol, the most abundant environmental pollutant with endocrine disrupting activity, is also toxic to plants and microorganisms, but its actual impact in the field is unknown. In this study, diversity of culturable soil microfungal and plant communities was assessed in a disused industrial estate, at three sites featuring different nonylphenol pollution. Although soil microfungal assemblages varied widely among the sites, no significant correlation was found with point pollutant concentrations, thus suggesting indirect effects of soil contamination on microfungal assemblages. The potential of indigenous fungi and plants to remove nonylphenol was assessed in mesocosm experiments. Poplar plants and a fungal consortium consisting of the most abundant strains in the nonylphenol-polluted soil samples were tested alone or in combination for their ability to reduce, under greenhouse conditions, nonylphenol levels either in a sterile, artificially contaminated sand substrate, or in two non-sterile soils from the original industrial area. Introduction of indigenous fungi consistently reduced nonylphenol levels in all substrates, up to ca. 70% depletion, whereas introduction of the plant proved to be effective only with high initial pollutant levels. In native non-sterile soil, nonylphenol depletion following fungal inoculation correlated with biostimulation of indigenous fungi, suggesting positive interactions between introduced and resident fungi. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hec1 Contributes to Mitotic Centrosomal Microtubule Growth for Proper Spindle Assembly through Interaction with Hice1

Previous studies have stipulated Hec1 as a conserved kinetochore component critical for mitotic control in part by directly binding to kinetochore fibers of the mitotic spindle and by recruiting spindle assembly checkpoint proteins Mad1 and Mad2. Hec1 has also been reported to localize to centrosomes, but its function there has yet to be elucidated. Here, we show that Hec1 specifically colocalizes with Hice1, a previously characterized centrosomal microtubule-binding protein, at the spindle pole region during mitosis. In addition, the C-terminal region of Hec1 directly binds to the coiled-coil domain 1 of Hice1. Depletion of Hice1 by small interfering RNA (siRNA) reduced levels of Hec1 in the cell, preferentially at centrosomes and spindle pole vicinity. Reduction of de novo microtubule nucleation from mitotic centrosomes can be observed in cells treated with Hec1 or Hice1 siRNA. Consistently, neutralization of Hec1 or Hice1 by specific antibodies impaired microtubule aster formation from purified mitotic centrosomes in vitro. Last, disruption of the Hec1/Hice1 interaction by overexpressing Hice1ΔCoil1, a mutant defective in Hec1 interaction, elicited abnormal spindle morphology often detected in Hec1 and Hice1 deficient cells. Together, the results suggest that Hec1, through cooperation with Hice1, contributes to centrosome-directed microtubule growth to facilitate establishing a proper mitotic spindle.