Papers - Miyamoto, Kenji

Fusion then fission: splitting and reassembly of an artificial fusion-protein nanocage

Ohara N., Kawakami N., Arai R., Adachi N., Ikeda A., Senda T., Miyamoto K.

Chemical Communications 60 ( 34 ) 4605 - 4608 2024.04

ISSN  13597345

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A split-protein system is a simple approach to introduce new termini which are useful as modification sites in protein engineering, but has been adapted mainly for monomeric proteins. Here we demonstrate the design of split subunits of the 60-mer artificial fusion-protein nanocage TIP60. The subunit fragments successfully reformed the cage structure in the same manner as prior to splitting. One of the newly introduced terminals at the interior surface can be modified using a tag peptide and green fluorescent protein. Therefore, the termini could serve as a versatile modification site for incorporating a wide variety of functional peptides and proteins.

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Thermally Reversible Gel-Sol Transition of Hydrogels via Dissociation and Association of an Artificial Protein Nanocage

Nasu E., Kawakami N., Takamura S., Hotta A., Arai R., Miyamoto K.

Biomacromolecules 25 ( 4 ) 2358 - 2366 2024.04

ISSN  15257797

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Oligomeric protein nanocages often disassemble into their subunits and reassemble by external stimuli. Thus, using these nanocages as cross-linkers for hydrogel network structures is a promising approach to allow hydrogels to undergo stimuli-responsive gel-sol transitions or self-healing. Here, we report hydrogels that show a reversible gel-sol transition resulting from the heat-induced dissociation and reassociation of protein nanocages. The hydrogel contained the 60-mer artificial protein nanocage, TIP60, as a supramolecular cross-linker for polyethylene glycol network structures. The hydrogel showed a gel-to-sol transition upon heating at a temperature above the melting point of TIP60 and immediately returned to a gel state upon cooling to room temperature. During the heating and cooling treatment of the hydrogel, small-angle X-ray scattering analysis suggested the dissociation and reassociation of TIP60. Furthermore, we demonstrated redox-responsive cargo release from TIP60 in the hydrogel. These results showed the potential of TIP60 as a component of multi-stimuli-responsive hydrogels.

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Alcanivorax bacteria as important polypropylene degraders in mesopelagic environments

Koike H., Miyamoto K., Teramoto M.

Applied and Environmental Microbiology 89 ( 12 )  2023.12

ISSN  00992240

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Plastics are causing serious problems in the sea and settling even to abyssal depths. Polypropylene (PP) is the second most common plastic product and thus would constitute a large fraction of plastics in the sea. The biodegradation of PP has not been clearly shown. In this study, Alcanivorax bacteria (mainly Alcanivorax borkumensis) were indicated to be enriched most abundantly on liquid PP and on its structurally similar branched alkane, pristane, in mesopelagic water. An Alcanivorax isolate probably of A. borkumensis showed the highest liquid PP-degrading activity among the isolates. These results indicate that Alcanivorax bacteria could be major degraders of PP in mesopelagic environments. Alcanivorax bacteria did not use liquid PP as the sole carbon and energy source. Short PP was preferentially degraded, and PP of all lengths appeared to be degraded. Liquid PP was degraded more efficientlyat 10°C than at 20°C, and correspondingly, higher concentrations of another carbon source were required at 10°C. Regarding the degradation of solid PP, an initial degradation sign, oxidation, was detected, but a weight loss of at least 1% was not detected. By using this study as a model, various PP-degrading microbes would start to be clarified.

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Column-free purification of an artificial protein nanocage, TIP60

Nasu E., Kawakami N., Ohara N., Hayashi K., Miyamoto K.

Protein Expression and Purification 205 2023.05

ISSN  10465928

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Protein nanocages, which have inner cavities and surface pores, are attractive materials for various applications, such as in catalysts and medicine. Recently, we produced an artificial protein nanocage, TIP60, and demonstrated its potential as a stimuli-responsive nanocarrier. In the present study, we report a simple purification method for TIP60 that can replace time-consuming and costly affinity chromatography purification. TIP60, which has an anionic surface charge, aggregated at mildly acidic pH and redissolved at neutral pH, maintaining its cage structure. This pH-responsive reversible precipitation allowed us to purify TIP60 from soluble fractions of the E. coli cell lysate by controlling the pH. Compared with conventional Ni-NTA column purification, the pH-responsive precipitation method provided purified TIP60 with similar purity (∼80%) and higher yield. This precipitation purification method should facilitate the large-scale investigation and practical use of TIP60 nanocages.

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Hydrophobization of a TIP60 Protein Nanocage for the Encapsulation of Hydrophobic Compounds

Yamashita M., Kawakami N., Miyamoto K.

ChemPlusChem (ChemPlusChem)  88 ( 3 )  2023.03

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Encapsulation of hydrophobic molecules in protein-based nanocages is a promising approach for dispersing these molecules in water. Here, we report a chemical modification approach to produce a protein nanocage with a hydrophobic interior surface based on our previously developed nanocage, TIP60. The large pores of TIP60 act as tunnels for small molecules, allowing modification of the interior surface by hydrophobic compounds without nanocage disassembly. We used four different hydrophobic compounds for modification. The largest modification group tested, pyrene, resulted in a modified TIP60 that could encapsulate aromatic photosensitizer zinc phthalocyanine (ZnPC) more efficiently than the other modification compounds. The encapsulated ZnPC generated singlet oxygen upon light activation in the aqueous phase, whereas ZnPC alone formed inert aggregates under the same experimental conditions. Given that chemical modification allows a wider diversity of modifications than mutagenesis, this approach could be used to develop more suitable nanocages for encapsulating hydrophobic molecules of interest.

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Reversible Assembly of an Artificial Protein Nanocage Using Alkaline Earth Metal Ions

Ohara N., Kawakami N., Arai R., Adachi N., Moriya T., Kawasaki M., Miyamoto K.

Journal of the American Chemical Society (Journal of the American Chemical Society)  145 ( 1 ) 216 - 223 2023.01

ISSN  00027863

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Protein nanocages are of increasing interest for use as drug capsules, but the encapsulation and release of drug molecules at appropriate times require the reversible association and dissociation of the nanocages. One promising approach to addressing this challenge is the design of metal-dependent associating proteins. Such designed proteins typically have Cys or His residues at the protein surface for connecting the associating proteins through metal-ion coordination. However, Cys and His residues favor interactions with soft and borderline metal ions, such as Au+ and Zn2+, classified by the hard and soft acids and bases concept, restricting the types of metal ions available to drive association. Here, we show the alkaline earth (AE) metal-dependent association of the recently designed artificial protein nanocage TIP60, which is composed of 60-mer fusion proteins. The introduction of a Glu (hard base) mutation to the fusion protein (K67E mutant) prevented the formation of the 60-mer but formed the expected cage structure in the presence of Ca, Sr, or Ba ions (hard acids). Cryogenic electron microscopy (cryo-EM) analysis indicated a Ba ion at the interface of the subunits. Furthermore, we demonstrated the encapsulation and release of single-stranded DNA molecules using this system. Our results provide insights into the design of AE metal-dependent association and dissociation mechanisms for proteins.

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Generation of Fusarium oxysporum-suppressive soil with non-soil carriers using a multiple-parallel-mineralization technique

Meeboon J., Ando A., Ogawa J., Miyamoto K., Kato Y., Shinohara M.

Scientific Reports 12 ( 1 )  2022.12

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Disease-suppressive soils exist worldwide. However, the disease-suppression mechanism is unknown, and it’s unclear how to produce such soils. The microbiota that develop in a multiple-parallel-mineralization system (MPM) can increase nutrient production efficiency and decrease root disease in hydroponic systems. Artificial media inoculated with MPM microorganisms can degrade organic matter to produce inorganic nutrients similarly to natural soil, but it’s unknown whether they can also suppress pathogen growth. Here, we produced an artificial medium that inhibited root disease similarly to disease-suppressive soils. Microbial MPM culture solution was inoculated into non-soil carriers (rockwool, rice husk charcoal, and vermiculite) to test whether it could suppress growth of Fusarium oxysporum f. sp. lactucae J. C. Hubb. & Gerik. We inoculated F. oxysporum f. sp. conglutinans (Wollenweber) Snyder et Hansen strain Cong:11 and F. oxysporum f. sp. lactucae J. C. Hubb. & Gerik into artificial media sown each with Arabidopsis thaliana (L.) Heynh. and Lactuca sativa L. var. capitata supplemented with MPM culture microbes. The MPM microorganisms suppressed F. oxysporum f. sp. lactucae J. C. Hubb. & Gerik growth and prevented plant disease. Thus, MPM-inoculated non-soil carriers that can generate inorganic nutrients from organic matter may also suppress disease in the absence of natural soil. Our study shows novel creation of a disease-suppressive effect in non-soil media using the microbial community from MPM culture solution.

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Icosahedral 60-meric porous structure of designed supramolecular protein nanoparticle TIP60

Obata J., Kawakami N., Tsutsumi A., Nasu E., Miyamoto K., Kikkawa M., Arai R.

Chemical Communications 57 ( 79 ) 10226 - 10229 2021.10

ISSN  13597345

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Supramolecular protein nanoparticles and nanocages have potential in a broad range of applications. Recently, we developed a uniform supramolecular protein nanoparticle, TIP60, symmmetrically self-assembled from fusion proteins of a pentameric Sm-like protein and a dimeric MyoX-coil domain. Herein, we report the icosahedral 60-meric structure of TIP60 solved using single-particle cryo-electron microscopy. Interestingly, the structure revealed 20 regular-triangle-like pores on the surface. TIP60 and its mutants have many modifiable sites on their exterior and interior surfaces. The TIP60 architecture will be useful in the development of biomedical and biochemical nanoparticles/nanocages for future applications.

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Nanopore-Controlled Dual-Surface Modifications on Artificial Protein Nanocages as Nanocarriers

Nasu E., Kawakami N., Miyamoto K.

ACS Applied Nano Materials (ACS Applied Nano Materials)  4 ( 3 ) 2434 - 2439 2021.03

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Chemical modification of the interior and exterior surfaces of protein nanocages holds promise for various applications such as cosmetics, pharmaceuticals, and catalysts. However, dual-surface modification of these surfaces using different chemicals remains challenging, particularly when the same substituents, such as cysteine thiols, are modified. We recently produced an artificial protein nanocage called TIP60 that has 20 large surface pores. Chemical modification of cysteine residues introduced in the interior surface of TIP60 showed that these pores allow the passage of small molecules from the outside environment to the inside of the nanocage. In this study, we found that the surface pores on TIP60 function as size-dependent molecular filters. Modification experiments using different-sized polymers containing maleimide groups, which specifically react with thiols, showed that macromolecules with diameters larger than that of the pores could not penetrate into the inner cavity. This molecular size discrimination by the pores prompted us to perform stepwise dual-surface functionalization of a double mutant of TIP60 presenting cysteine residues on the interior and exterior surfaces. This was achieved by modifying the exterior cysteine residues with a polymer containing a maleimide group that cannot penetrate to the inside of the nanocage, followed by modification of the interior cysteine residues using thiol-containing small molecules. Dual-functionalized TIP60 released internal small molecules in a redox-responsive manner. This simple approach for dual-surface modification would make TIP60 a useful nanocarrier for a broad range of applications including drug-delivery and molecular filtration systems.

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Apoptosis-inducing activity and antiproliferative effect of Paeoniflorigenone from moutan cortex

Ying Huang,Osamu Ohno, Kiyotake Suenaga, Kenji Miyamoto,

Bioscience, Biotechnology, and Biochemistry  2017

Joint Work, Accepted

5-keto-4-deoxy-D-Glucarate Dehydratase/Decarboxylase: Key role in the oxidative pathway towards α-keto-glutarate

André Pick, Barbara Beer, Risa Hemmi, Rena Momma, Jochen Schmid, Kenji Miyamoto and Volker Sieber

BMC Biotechnology 16 ( 1 )  2016.12

Joint Work, Accepted

Response to Comment on “A bacterium that degrades and assimilates poly(ethylene terephthalate)

S. Yoshida, K. Hiraga, T. Takehana, I. Taniguchi, H. Yamaji, Y. Maeda, K. Toyohara, K. Miyamoto, Y. Kimura and K. Oda

Science 353   759 2016.07

Joint Work, Accepted

Discovery of a poly(ethylene terephthalate) assimilation

S. Yoshida, K. Hiraga, T. Takehana, I. Taniguchi, H. Yamaji, Y. Maeda, K. Toyohara, K. Miyamoto, Y. Kimura and K. Oda

Science 351 ( 6278 ) 1196 - 1199 2016.03

Research paper (scientific journal), Joint Work, Accepted

Semiempirical QM/MM calculations reveal a step-wise proton transfer and an unusual thiolate pocket in the mechanism of the unique arylpropionate racemase AMDase G74C

F. Busch, J. Enoki, N. Hülsemann, K. Miyamoto, M. Bocola and R. Kourist

Catalysis Science & Technology  2016.03

Research paper (scientific journal), Accepted

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Arylmalonate decarboxylase-catalyzed asymmetric synthesis of both enantiomers of optically pure flurbiprofen

S. K. Gaßmeyer, J. Wetzig, C. Mügge, M. Assmann, J. Enoki, L. Hilterhaus, R. Zuhse, K. Miyamoto, A. Liese and R. Kourist

ChemCatChem 8   916 - 921 2016.01

Research paper (scientific journal), Joint Work, Accepted

ポリエチレンテレフタレート(PET)分解菌の発見、解析とその応用への展望

平賀和三、宮本憲二、吉田昭介、小田耕平

バイオサイエンストインダストリー 74   492 - 496 2016

Joint Work

Arylmalonate decarboxylase – a highly selective bacterial biocatalyst with unkown function

K. Miyamoto and R. Kourist

Appl. Microbiol. Biotechnol., 100   8621 - 8631 2016

Joint Work, Accepted

Improved enantioselectivity of thermostable esterase ST0071 from archaeon Sulforobus tokodaii by site-saturation mutagenesis

M. Ozaki, N. Kawakami, H. Ohta and K. Miyamoto

Biocatal. Biotransformation  2016

Joint Work, Accepted

Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance

S. Yoshida, J. Enoki, R. Kourist and K. Miyamoto

Biosci. Biotechnol. Biochem. 79 ( 12 ) 1965 - 1971 2015.12

Research paper (scientific journal), Accepted

Modulating the catalytic activity and the substrate specificity of alcohol dehydrogenases using cyclic ethers

N. Kawakami, Y. Hara and K. Miyamoto

Catal. Sci. Technol. 5 ( 8 ) 3922 - 2925 2015.08

Research paper (scientific journal), Accepted