Papers - Sasaki, Eita

Biosynthetic studies of thiosugar-containing natural products, BE-7585A and Lincomycin A

Eita Sasaki

 2011

Doctoral thesis, Single Work, Lead author, Last author, Corresponding author

Mechanistic studies of the biosynthesis of 2-thiosugar: evidence for the formation of an enzyme-bound 2-ketohexose intermediate in BexX-catalyzed reaction

E Sasaki, H Liu

Journal of the American Chemical Society (AMER CHEMICAL SOC)  132 ( 44 ) 15544 - 15546 2010.11

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  0002-7863

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The first mechanistic insight into 2-thiosugar production in an angucycline-type antibiotic, BE-7585A, is reported. D-Glucose 6-phosphate was identified as the substrate for the putative thiosugar biosynthetic protein, BexX, by trapping the covalently bonded enzyme-substrate intermediate. The site-specific modification at K110 residue was determined by mutagenesis studies and LC-MS/MS analysis. A key intermediate carrying a keto functionality was confirmed to exist in the enzyme-substrate complex. These results suggest that the sulfur insertion mechanism in 2-thiosugar biosynthesis shares similarities with that for thiamin biosynthesis.

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A Biosynthetic Pathway for BE-7585A, a 2-Thlosugar-Containing Angucycline-Type Natural Product

Eita Sasaki, Yasushi Ogasawara, Hung-wen Liu

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY (AMER CHEMICAL SOC)  132 ( 21 ) 7405 - 7417 2010.06

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  0002-7863

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Sulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been studied. BE-7585A produced by Amycolatopsis orientalis subsp. vinearia BA-07585 has a 2-thiosugar and is a member of the angucycline class of compounds. We report herein the results of our initial efforts to study the biosynthesis of BE-7585A. Spectroscopic analyses verified the structure of BE-7585A, which is closely related to rhodonocardin A. Feeding experiments using (13)C-labeled acetate were carried out to confirm that the angucycline core is indeed polyketide-derived. The results indicated an unusual manner of angular tetracyclic ring construction, perhaps via a Baeyer-Villiger type rearrangement. Subsequent cloning and sequencing led to the identification of the bex gene cluster spanning similar to 30 kbp. A total of 28 open reading frames, which are likely involved in BE-7585A formation, were identified in the cluster. In view of the presence of a homologue of a thiazole synthase gene (thiG), bexX, in the bex cluster, the mechanism of sulfur incorporation into the 2-thiosugar moiety could resemble that found in thiamin biosynthesis. A glycosyltransferase homologue, BexG2, was heterologously expressed in Escherichia coli. The purified enzyme successfully catalyzed the coupling of 2-thioglucose 6-phoshate and UDP-glucose to produce 2-thiotrehalose 6-phosphate, which is the precursor of the disaccharide unit in BE-7585A. On the basis of these genetic and biochemical experiments, a biosynthetic pathway for BE-7585A can now be proposed. The combined results set the stage for future biochemical studies of 2-thiosugar biosynthesis and BE-7585A assembly.

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Molecular Design Strategies for Near-Infrared Ratiometric Fluorescent Probes Based on the Unique Spectral Properties of Aminocyanines

Kazuki Kiyose, Sakiko Aizawa, Eita Sasaki, Hirotatsu Kojima, Kenjiro Hanaoka, Takuya Terai, Yasuteru Urano, Tetsuo Nagano

CHEMISTRY-A EUROPEAN JOURNAL (WILEY-V C H VERLAG GMBH)  15 ( 36 ) 9191 - 9200 2009

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0947-6539

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In spite of the wide availability of various near-infrared (NIR) fluorophores as labeling reagents, there are few functional NIR fluorescent probes for which change in the absorption and/or fluorescence spectra upon specific reaction with biomolecules is seen. The widely used photoinduced electron-transfer mechanism is unsuitable for NIR fluorophores, such as tricarbocyanines, because their long excitation wavelength results in a small singlet ex-citation energy. We have reported the unique spectral properties of amine-substituted tricarbocyanines, which were utilized to develop two design strategies. One approach was based on control of the absorption wavelength by using the difference in electron-donating ability before and after a specific reaction with a biomolecule, and the other approach was based on control of the fluorescence intensity by modulating the Forster resonance energy-transfer efficiency through a change in the overlap integral that arises from the change in absorption under acidic conditions. These strategies were validated by obtaining tricarbocyanine-based ratiometric NIR fluorescent probes for esterase and for pH level.

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Development of near-infrared fluorescent probes for nitric oxide and zinc ion

Hirotatsu Kojima, Kazuki Kiyose, Eita Sasaki, Hiroaki Nishimatsu, Yasunobu Hirata, Tetsuo Nagano

IMAGING, MANIPULATION, AND ANALYSIS OF BIOMOLECULES, CELLS, AND TISSUES V (SPIE-INT SOC OPTICAL ENGINEERING)  6441 2007

Research paper (international conference proceedings), Joint Work, Accepted,  ISSN  0277-786X

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In fluorescence imaging studies of biological mechanisms, cyanine dyes have been employed as fluorescent labels. In particular, tricarbocyanines have the advantage that light at their emission and absorption maxima in the near-infrared (NIR) region around 650-900 nm can penetrate deeply into tissues. We successfully developed two types of cyanine dyes whose fluorescence properties change upon specific reaction with nitric oxide (NO) or zinc ion. The mechanism of fluorescence modulation of the NO probes involves photoinduced electron transfer, and the fluorescent intensity can change at the same wavelengths. We synthesized a series of amine-substituted tricarbocyanines in order to examine the correlation between the electron-donating ability of the amine and the fluorescence peak wavelength. We found that changing the electron-donating ability of the amine substituent altered the absorption and emission wavelengths. Then, we synthesized dipicolylcyanine (DIPCY), consisting of tricarbocyanine as a fluorophore and dipicolylethylenediamine as a heavy metal chelator, and investigated its response to various heavy metal ions. DIPCY can work as a ratiometric fluorescent sensor for zinc ion. This fluorescence modulation of amine-substituted tricarbocyanines should be applicable to dual-wavelength measurement of various biomolecules or enzyme activities. Thus, we have established two mechanisms for modulating the fluorescence properties of cyanines.

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Highly sensitive near-infrared fluorescent probes for nitric oxide and their application to isolated organs

E Sasaki, H Kojima, H Nishimatsu, Y Urano, K Kikuchi, Y Hirata, T Nagano

Journal of the American Chemical Society (AMER CHEMICAL SOC)  127 ( 11 ) 3684 - 3685 2005.03

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  0002-7863

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