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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

DOI： 10.1039/C7CY02263H

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

DOI： 10.1039/c5dt00797f

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

X-ray crystallography has revolutionized our understanding of biological macromolecules by elucidating their three-dimensional structures. However, the use of X-rays in this technique raises concerns about potential damage to the protein crystals, which results in a quality degradation of the diffraction data even at very low temperatures. Since such damage can occur on the micro- to millisecond timescale, a development in its real-time measurement has been expected. Here, we introduce diffracted X-ray blinking (DXB), which was originally proposed as a method to analyze the intensity fluctuations of diffraction of crystalline particles, to small-angle X-ray scattering (SAXS) of a lysozyme single-crystal. This novel technique, called the small-angle X-ray blinking (SAXB) method, analyzes the fluctuation in SAXS intensity reflecting the domain fluctuation in the protein crystal caused by the X-ray irradiation, which could be correlated with the X-ray-induced damage on the crystal. There was no change in the protein crystal’s domain dynamics between the first and second X-ray exposures at 95K, each of which lasted 0.7 s. On the other hand, its dynamics at 295K increased remarkably. The SAXB method further showed a dramatic increase in domain fluctuations with an increasing dose of X-ray radiation, indicating the significance of this method.

DOI： 10.3390/ijms242316640

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acscatal.3c01158

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

ABSTRACT

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the solution nuclear magnetic resonance structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

DOI： 10.1093/nar/gkac1082

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Language：English   Publishing type：Research paper (scientific journal)  

Tetraphenylporphyrin (TPP) is a symmetrically substituted synthetic porphyrin whose properties can be readily modified, providing it with significant advantages over naturally occurring porphyrins. Herein, we report the first example of a stable complex between a native biomolecule, the haemoprotein HasA, and TPP as well as its derivatives. The X-ray crystal structures of nine different HasA-TPP complexes were solved at high resolutions. HasA capturing TPP derivatives was also demonstrated to inhibit growth of the opportunistic pathogen Pseudomonas aeruginosa. Mutant variants of HasA binding FeTPP were shown to possess a different mode of coordination, permitting the cyclopropanation of styrene.

DOI： 10.1002/cbic.202200095

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

Trimethylation of histone H3 at K9 by the lysine methyltransferase, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) plays a pivotal role in silencing tissue-specific genes and retrotransposable elements. In mammalian cells, SETDB1 undergoes monoubiquitination in the insertion region of the SET domain in an E3 ubiquitin ligase-independent manner. This ubiquitination has been shown to enhance the histone H3-K9 methyltransferase activity of SETDB1; however, the molecular mechanism underlying SETDB1 activation by ubiquitination is unknown. In this study, we developed an E. coli ubiquitination plasmid for the preparation of ubiquitinated SETDB1. Western blotting and mutational analyses showed that coexpression of the SET domain of SETDB1 with the proteins encoded by the ubiquitination plasmid led to site-specific monoubiquitination of the SET domain at K867. An in vitro histone H3 methylation assay demonstrated that the ubiquitinated SET domain of SETDB1 acquired enzymatic activity. Taken together, these findings demonstrate successful preparation of the active form of SETDB1 with the E. coli ubiquitination system, which will aid biochemical and structural studies of ubiquitinated SETDB1.

DOI： 10.1093/jb/mvab087

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

Highly effective dipeptidic decoy molecules, which stimulate the direct hydroxylation of benzene by wild-type cytochrome P4SOBM3, were successfully developed through a rationally designed screening method. Extensive synthesis and step-wise screening of over 600 dipeptide derivatives were performed for the efficient evolution of decoy molecules. In the presence of N-(3-cyclopentyl)propanoyl-L-pipecolyl-L-phenylalanine (3CPPA-Pip-Phe), one of the most effective decoy molecules discovered herein, the catalytic turnover frequency and total turnover number for benzene hydroxylation reached 405 min(-1) P450BM3(-1) and 54,500 P450BM3(-1), respectively. Furthermore, the decoy molecules developed in this work drastically accelerated the hydroxylation of other non-native substrates, such as anisole and toluene, as well as nonaromatic compounds, such as cyclohexane, propane, and ethane. Using N-enanthoyl-L-pipecolyl-L-phenylalanine (C7AM-Pip-Phe), the hydroxylation rate for ethane to ethanol reached 82.7 min(-1) P450BM3(-1).

DOI： 10.1021/acscatal.0c01951

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

We prepared enzyme-immobilized hydrogels and investigated the effects of the cross-linking density and polymer properties on their oxidation reaction rate. The oxidation rate of enzyme-immobilized hydrogels increased as the cross-linking density in the hydrogels increased. In addition, we controlled the oxidation rate using hydrogels exhibiting an appropriate interaction with a decoy molecule in the hydrogel.

DOI： 10.1039/D0CC01332C

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.

DOI： 10.1039/C8CC02760A

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

DOI： 10.1039/c6cy00630b

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Event date： 2022.6

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2022.5

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Event date： 2022.3

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Event date： 2021.11

Presentation type：Symposium, workshop panel (nominated)  

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Event date： 2016.9

Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Event date： 2015.6

Language：English   Presentation type：Oral presentation (invited, special)  

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Event date： 2023.11

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Event date： 2023.9

Language：English  

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Event date： 2023.1

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Event date： 2023.1

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2022.10

Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Event date： 2021.12

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Event date： 2018.3

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2016.6

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2016.3

Language：Japanese   Presentation type：Oral presentation (general)  

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