Luzodiol (4), a diterpene possessing a new carbon skeleton, and five new sesquiterpenes (5-9) of the snyderane class have been isolated from the red alga Laurencia luzonensis and their structures determined by spectroscopic analysis. The relative stereochemistry of the known luzonensol (3) was assigned by its conversion to palisadin B (10).
To obtain an enzyme for the production of chito-disaccharides (GlcN(2)) by converting endo-chitosanase to exo-chitosanase, we chose an endo-chitosanase from Bacillus circulans MH-K1 (Csn) as the candidate for protein engineering. Using molecular modeling, two peptides with five amino acids (PCLGG) and six amino acids (SRTCKP) were designed and inserted after the positions of D(115) and T(222) of Csn, respectively. The inserted fragments are expected to form loops that might protrude from opposite walls of the substrate-binding cleft, thus forming a 'roof' over the catalytic site that might alter the product specificity. The chimeric chitosanase (Chim-Csn) and wild-type chitosanase (WT-Csn) were both over-expressed in Escherichia coli and purified nearly to homogeneity. The products formed from chitosan were analyzed by ESI-MS (electrospray ionization-mass spectrometry). A mixture of GlcN(2), GlcN(3) and GlcN(4) was obtained with WT-Csn, whereas Chim-Csn formed, with a smaller catalytic rate (3% of WT-Csn activity), GlcN(2) as the dominant product. Measurements of viscosity showed that, with similar amounts of enzyme activity, Chim-Csn catalyzed the hydrolysis of chitosan with a smaller rate of viscosity decrease than WT-Csn. The results indicate that, on inserting two surface loops, the endo-type chitosanase was converted into an exo-type chitosanase, which to our knowledge is the first chitosanase that releases GlcN(2) from chitosan as the dominant product.
Laminaripentaose-producing β-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of β-1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to >90% homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residues--Asp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.
Epidermal growth factor (EGF)-mediated activation of EGF receptors (EGFRs) has become an important target in drug development due to the implication of EGFR-mediated cellular signaling in cancer development. While various in vitro approaches are developed for monitoring EGF-EGFR interactions, they have several limitations. Herein, we describe a live cell-based sensor system that can be used to monitor the interaction of EGF and EGFR as well as the subsequent signaling events. The design of the EGF-detecting sensor cells is based on the split-intein-mediated conditional protein trans-cleavage reaction (CPC). CPC is triggered by the presence of the target (EGF) to activate a signal peptide that translocates the fluorescent cargo to the target cellular location (mitochondria). The developed sensor cell demonstrated excellent sensitivity with a fast response time. It was also successfully used to detect an agonist and antagonist of EGFR (transforming growth factor-α and Cetuximab, respectively), demonstrating excellent specificity and capability of screening the analytes based on their function. The usage of sensor cells was then expanded from merely detecting the presence of target to monitoring the target-mediated signaling cascade, by exploiting previously developed Ca2+-detecting sensor cells. These sensor cells provide a useful platform for monitoring EGF-EGFR interaction, for screening EGFR effectors, and for studying downstream cellular signaling cascades.
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