Immobilization of Baeyer-Villiger monooxygenase from acetone grown Fusarium sp.

OBJECTIVE: A novel biocatalyst for Baeyer-Villiger oxidations is necessary for pharmaceutical and chemical industries, so this study aims to find a Baeyer-Villiger monooxygenase (BVMO) and to improve its stability by immobilization. RESULTS: Acetone, the simplest ketone, was selected as the only carbon source for the screening of microorganisms with a BVMO. A eukaryote, Fusarium sp. NBRC 109816, with a BVMO (FBVMO), was isolated from a soil sample. FBVMO was overexpressed in E. coli and successfully immobilized by the organic-inorganic nanocrystal formation method. The immobilization improved the thermostability of FBVMO. Substrate specificity investigation revealed that both free and immobilized FBVMO were found to show catalytic activities not only for Baeyer-Villiger oxidation of ketones to esters but also for oxidation of sulfides to sulfoxides. Furthermore, a preparative scale reaction using immobilized FBVMO was successfully conducted. CONCLUSIONS: FBVMO was discovered from an environmental sample, overexpressed in E. coli, and immobilized by the organic-inorganic nanocrystal formation method. The immobilization successfully improved its thermostability.

Trapping and Releasing of Oxygen in Liquid by Metal-Organic Framework with Light and Heat.

Nanoporous materials can adsorb small molecules into their nanospaces. However, the trapping of light gas molecules dissolved in solvents suffers from low concentration and poor adsorption affinity. Here, the reversible trapping and releasing of dissolved oxygen are shown through integrating photosensitization and chemical capturing abilities into a metal-organic framework (MOF), MOMF-1. 9,10-Di(4-pyridyl)anthracene (dpa) ligands in MOMF-1 generates singlet oxygen from triplet oxygen under photoirradiation without additional photosensitizers, and successively reacts with it to produce anthracene endoperoxide, forming MOMF-2, which is proved crystallographically. The reverse reaction also proceeds quantitatively by heating MOMF-2. Moreover, MOMF-1 exhibits excellent water resistance, and completely removes oxygen of ppm order concentrations in water. The new material shown in this report allows controlling of the amount of dissolved oxygen, which can be applicable in various fields relating to numerous oxidation phenomena.

Synthesis and Evaluation of Fuligocandin B Derivatives with Activity for Overcoming TRAIL Resistance.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling pathway induces apoptosis in cancer cells but not in normal cells. Therefore, this pathway has attracted attention regarding possible clinical treatment of cancer. However, many cancer cells demonstrate TRAIL resistance. To overcome this problem, small molecules that sensitize cancer cells to TRAIL are desired. Heterocyclic derivatives of the natural product, fuligocandin B (2), with activity for overcoming TRAIL resistance were synthesized, and their activity was evaluated. Of the synthetic molecules, the quinoline derivative (10g) showed potent activity against TRAIL-resistant gastric adenocarcinoma cells. After a docking study of the target protein valosin-containing protein, 7'-amino fuligocandin B (10m) was designed and synthesized. Compound 10m also showed good activity for overcoming TRAIL resistance. 10m produced a 49.7% difference in viability with TRAIL at 30 µM compared to without TRAIL. This activity was better than that of fuligocandin B (2).

Valosin-containing Protein is a Target of 5'-l Fuligocandin B and Enhances TRAIL Resistance in Cancer Cells.

Fuligocandin B (2) is a novel natural product that can overcome TRAIL resistance. We synthesized enatiomerically pure fuligocandin B (2) and its derivative 5'-I fuligocandin B (4), and found that the latter had an improved biological activity against the human gastric cancer cell line, AGS. We attached a biotin linker and photoactivatable aryl diazirine group to 5'-I fuligocandin B (4), and employed a pull-down assay to identify valosin-containing protein (VCP/p97), an AAA ATPase, as a 5'-I fuligocandin B (4) target protein. Knock-down of VCP by siRNA enhanced sensitivity to TRAIL in AGS cells. In addition, 4 enhanced CHOP and DR5 protein expression, and overall intracellular levels of ubiquitinated protein. These data suggest that endoplasmic reticulum stress caused through VCP inhibition by 4 increases CHOP-mediated DR5 up-regulation, which enhances TRAIL-induced cell death in AGS cells. To the best of our knowledge, this is the first example to show a relationship between VCP and TRAIL-resistance-overcoming activity in cancer cells.

Essential roles of PIKfyve and PTEN on phagosomal phosphatidylinositol 3-phosphate dynamics.

PtdIns(3)P (phosphatidylinositol 3-phosphate) is a signaling molecule important for phagosome maturation. The major role of Vps34 in production of phagosomal PtdIns(3)P has been indicated. However, the fate of the newly generated PtdIns(3)P has not been well described. Here we show that elimination of PtdIns(3)P from phagosomal membrane was significantly delayed in RAW264.7 macrophages lacking PTEN or PIKfyve. In the PTEN-deficient cells treated with a PIKfyve inhibitor, degradation of PtdIns(3)P was almost lost, indicating that PTEN and PIKfyve are two major players in phagosomal PtdIns(3)P metabolism.