Papers - Sasaki, Eita

Self-Assembly of Proteinaceous Multishell Structures Mediated by a Supercharged Protein

E Sasaki, D Hilvert

The Journal of Physical Chemistry B (AMER CHEMICAL SOC)  120 ( 26 ) 6089 - 6095 2016.07

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  1520-6106

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Engineered variants of the capsid-forming enzyme lumazine synthase can exploit electrostatic interactions to encapsulate complementarily charged guest macromolecules. Here we investigate the effect of ionic strength and cargo molecules on assembly of AaLS-13, a negatively supercharged lumazine synthase protein cage, and we show that multishell structures are produced upon mixing the capsid core with free capsomers and a positively supercharged variant of the green fluorescent protein GFP(+36). The assembly process is mediated by favorable electrostatic interactions between the negatively charged capsid shells/capsomers and GFP(+36) molecules, and it is therefore strongly dependent on ionic strength. The mechanism of formation of these assemblages is likely similar to the assembly of multishell structures of some virus-like particles, where outer shells organize as nonicosahedral structures with larger radii of curvature than the templating inner shell. In contrast to the viral multishell structures, the positively charged mediator was found to be essential for the assembly of multilayered structures of different shapes and sizes constituted of AaLS-13 capsomers. This mediator-bridging approach may be widely applicable to create protein-based hierarchical nanostructures for various nanotechnology applications such as drug delivery and bioimaging.

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Insights into Complex Oxidation during BE-7585A Biosynthesis: Structural Determination and Analysis of the Polyketide Monooxygenase BexE

David R. Jackson, Xia Yu, Guojung Wang, Avinash B. Patel, Jordi Calveras, Jesus F. Barajas, Eita Sasaki, Mikko Metsa-Ketela, Hung-wen Liu, Juergen Rohr, Shiou-Chuan Tsai

ACS chemical biology (AMER CHEMICAL SOC)  11 ( 4 ) 1137 - 1147 2016.04

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1554-8929

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Cores of aromatic polyketides are essential for their biological activities. Most type II polyketide synthases (PKSs) biosynthesize these core structures involving the minimal PKS, a PKS-associated ketoreductase (KR) and aromatases/cyclases (ARO/CYCs). Oxygenases (OXYs) are rarely involved. BE-7585A is an anticancer polyketide with an angucyclic core. C-13 isotope labeling experiments suggest that its angucyclic core may arise from an oxidative rearrangement of a linear anthracyclinone. Here, we present the crystal structure and functional analysis of BexE, the oxygenase proposed to catalyze this key oxidative rearrangement step that generates the angucyclinone framework. Biochemical assays using various linear anthracyclinone model compounds combined with docking simulations narrowed down the substrate of BexE to be an immediate precursor of aklaviketone, possibly 12-deoxy-aklaviketone. The structural analysis, docking simulations, and biochemical assays provide insights into the role of BexE in BE-7585A biosynthesis and lay the groundwork for engineering such framework-modifying enzymes in type II PKSs.

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Structural and functional analysis of two di-domain aromatase/cyclases from type II polyketide synthases

Grace Caldara-Festin, David R. Jackson, Jesus F. Barajas, Timothy R. Valentic, Avinash B. Patel, Stephanie Aguilar, MyChi Nguyen, Michael Vo, Avinash Khanna, Eita Sasaki, Hung-wen Liu, Shiou-Chuan Tsai

Proceedings of the National Academy of Sciences (NATL ACAD SCIENCES)  112 ( 50 ) E6844 - E6851 2015.12

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0027-8424

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Aromatic polyketides make up a large class of natural products with diverse bioactivity. During biosynthesis, linear poly-beta-ketone intermediates are regiospecifically cyclized, yielding molecules with defined cyclization patterns that are crucial for polyketide bioactivity. The aromatase/cyclases (ARO/CYCs) are responsible for regiospecific cyclization of bacterial polyketides. The two most common cyclization patterns are C7-C12 and C9-C14 cyclizations. We have previously characterized three monodomain ARO/CYCs: ZhuI, TcmN, and WhiE. The last remaining uncharacterized class of ARO/CYCs is the di-domain ARO/CYCs, which catalyze C7-C12 cyclization and/or aromatization. Di-domain ARO/CYCs can further be separated into two subclasses: "nonreducing" ARO/CYCs, which act on nonreduced poly-beta-ketones, and "reducing" ARO/CYCs, which act on cyclized C9 reduced poly-beta-ketones. For years, the functional role of each domain in cyclization and aromatization for di-domain ARO/CYCs has remained a mystery. Here we present what is to our knowledge the first structural and functional analysis, along with an in-depth comparison, of the nonreducing (StfQ) and reducing (BexL) di-domain ARO/CYCs. This work completes the structural and functional characterization of mono-and di-domain ARO/CYCs in bacterial type II polyketide synthases and lays the groundwork for engineered biosynthesis of new bioactive polyketides.

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Co-opting sulphur-carrier proteins from primary metabolic pathways for 2-thiosugar biosynthesis

Eita Sasaki, Xuan Zhang, He G. Sun, Mei-Yeh Jade Lu, Tsung-lin Liu, Albert Ou, Jeng-yi Li, Yu-hsiang Chen, Steven E. Ealick, Hung-wen Liu

Nature (NATURE PUBLISHING GROUP)  510 ( 7505 ) 427 - 431 2014.06

Research paper (scientific journal), Joint Work, Lead author, Accepted,  ISSN  0028-0836

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Sulphur is an essential element for life and is ubiquitous in living systems(1,2). Yet how the sulphur atom is incorporated into many sulphur-containing secondary metabolites is poorly understood. For bond formation between carbon and sulphur in primary metabolites, the major ionic sulphur sources are the persulphide and thiocarboxylate groups on sulphur-carrier (donor) proteins(3,4). Each group is post-translationally generated through the action of a specific activating enzyme. In all reported bacterial cases, the gene encoding the enzyme that catalyses the carbon-sulphur bond formation reaction and that encoding the cognate sulphur-carrier protein exist in the same gene cluster(5). To study the production of the 2-thiosugar moiety in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action seem similar to those of ThiG, the enzyme that catalyses thiazole formation in thiamine biosynthesis(6,7). However, no gene encoding a sulphur-carrier protein could be located in the BE-7585A cluster. Subsequent genome sequencing uncovered a few genes encoding sulphur-carrier proteins that are probably involved in the biosynthesis of primary metabolites but only one activating enzyme gene in the A. orientalis genome. Further experiments showed that this activating enzyme can adenylate each of these sulphur carrier proteins and probably also catalyses the subsequent thiolation, through its rhodanese domain. A proper combination of these sulphur-delivery systems is effective for BexX-catalysed2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. This study is, to our knowledge, the first complete characterization of thiosugar formation in nature and also demonstrates the receptor promiscuity of the A. orientalis sulphur-delivery system. Our results also show that co-opting the sulphur-delivery machinery of primary metabolism for the biosynthesis of sulphur-containing natural products is probably a general strategy found in nature.

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In Vitro Characterization of LmbK and LmbO: Identification of GDP-D-erythro-alpha-D-gluco-octose as a Key Intermediate in Lincomycin A Biosynthesis

Chia-I Lin, Eita Sasaki, Aoshu Zhong, Hung-wen Liu

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY (AMER CHEMICAL SOC)  136 ( 3 ) 906 - 909 2014.01

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

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Lincomycin A is a clinically useful antibiotic isolated from Streptomyces lincolnensis. It contains an unusual methylmercapto-substituted octose, methylthiolincosamide (MTL). While it has been demonstrated that the C-8 backbone of MTL moiety is derived from D-fructose 6-phosphate and D-ribose 5-phosphate via a transaldol reaction catalyzed by LmbR, the subsequent enzymatic transformations leading to the MTL moiety remain elusive. Here, we report the identification of GDP-D-etythro-alpha-D-gluco-octose (GDP-D-alpha-D-octose) as a key intermediate in the MTL biosynthetic pathway. Our data show that the octose 1,8-bisphosphate intermediate is first converted to octose 1-phosphate by a phosphatase, LmbK. The subsequent conversion of the octose 1-phosphate to GDP-D-alpha-D-octose is catalyzed by the octose 1-phosphate guanylyltransferase, LmbO. These results provide significant insight into the lincomycin biosynthetic pathway, because the activated octose likely serves as the acceptor for the installation of the C1 sulfur appendage of MTL.

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Construction of the octose 8-phosphate intermediate in lincomycin A biosynthesis: characterization of the reactions catalyzed by LmbR and LmbN

E Sasaki, CI Lin, KY Lin, H Liu

Journal of the American Chemical Society (AMER CHEMICAL SOC)  134 ( 42 ) 17432 - 17435 2012.10

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

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Lincomycin A is a potent antimicrobial agent noted for its unusual C1 methylmercapto-substituted 8-carbon sugar. Despite its long clinical history for the treatment of Gram-positive infections, the biosynthesis of the C-8-sugar, methylthiolincosamide (MTL), is poorly understood. Here, we report our studies of the two initial enzymatic steps in the MTL biosynthetic pathway leading to the identification of D-erythro-D-gluco-octose 8-phosphate as a key intermediate. Our experiments demonstrate that this intermediate is formed via a transaldol reaction catalyzed by LmbR using D-fructose 6-phosphate or D-sedoheptulose 7-phosphate as the C-3 donor and D-ribose 5-phosphate as the C-5 acceptor. Subsequent 1,2-isomerization catalyzed by LmbN converts the resulting 2-keto C-8-sugar (octulose 8-phosphate) to octose 8-phosphate. These results provide, for the first time, in vitro evidence for the biosynthetic origin of the C-8 backbone of MTL.

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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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